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Dendritic Cell Therapy for Type 1 Diabetes

Permanent Link: http://ufdc.ufl.edu/UFE0024913/00001

Material Information

Title: Dendritic Cell Therapy for Type 1 Diabetes Role of Antigen Presentation in Immune Modulation
Physical Description: 1 online resource (117 p.)
Language: english
Creator: Lo-Dauer, Jeannette
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: antigen, cell, dendritic, determinant, diabetes, iddm, ignored, nod, peptide, proliferation, regulatory, t1d, therapy, type1
Immunology and Microbiology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Type 1 diabetes (T1D) is a metabolic disorder in which the body s immune system attacks its own insulin-producing pancreatic beta cells, resulting in insulin deficiency. This study investigates how dendritic cells (DC), an immune cell that identifies and presents foreign and self substances called antigens (Ag) to other immune cells, can be used as therapy to prevent T1D. DC therapy can help re-educate the immune system to respond properly to self Ag on beta cells. We find that DC presenting special portions of beta cell Ag can prevent T1D in an animal model of T1D. We also show that DC therapy results in Ag-independent changes in the immune system that help prevent autoimmunity. These findings will aid the understanding of how DC therapy modulates the immune response and guide the engineering of DC therapies for prevention of T1D and other Ag-based disorders.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jeannette Lo-Dauer.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Clare-Salzler, Michael J.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024913:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024913/00001

Material Information

Title: Dendritic Cell Therapy for Type 1 Diabetes Role of Antigen Presentation in Immune Modulation
Physical Description: 1 online resource (117 p.)
Language: english
Creator: Lo-Dauer, Jeannette
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: antigen, cell, dendritic, determinant, diabetes, iddm, ignored, nod, peptide, proliferation, regulatory, t1d, therapy, type1
Immunology and Microbiology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Type 1 diabetes (T1D) is a metabolic disorder in which the body s immune system attacks its own insulin-producing pancreatic beta cells, resulting in insulin deficiency. This study investigates how dendritic cells (DC), an immune cell that identifies and presents foreign and self substances called antigens (Ag) to other immune cells, can be used as therapy to prevent T1D. DC therapy can help re-educate the immune system to respond properly to self Ag on beta cells. We find that DC presenting special portions of beta cell Ag can prevent T1D in an animal model of T1D. We also show that DC therapy results in Ag-independent changes in the immune system that help prevent autoimmunity. These findings will aid the understanding of how DC therapy modulates the immune response and guide the engineering of DC therapies for prevention of T1D and other Ag-based disorders.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jeannette Lo-Dauer.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Clare-Salzler, Michael J.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024913:00001


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0e61419c51a4138801419ce59e070f4fb5df0244







DENDRITIC CELL THERAPY FOR TYPE 1 DIABETES:
ROLE OF ANTIGEN PRESENTATION IN IMMUNE MODULATION




















By

JEANNETTE LO-DAUER


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2009




































2009 Jeannette Lo-Dauer



































To Mom and Dad









ACKNOWLEDGEMENTS

I have been fortunate to have an extensive team of people whom I wish to thank for their

relentless support during the course of my graduate career. I would first like to extend my

gratitude for the immeasurable guidance provided to me by my mentor, Dr. Michael Clare-

Salzler. Mike has been instrumental in training me to think critically with a sound scientific

mind while feeding my curiosity in science with his vibrant and passionate discussions in

immunology. His enthusiasm and encouragement kept me inspired during times of challenge

and allowed me to emerge with renewed momentum. Mike has also been an admirable role

model both professionally and personally, demonstrating that in the development of a scientist's

career, there can be a graceful balance between academic responsibilities and family.

I also wish to thank my committee members, Dr. Mark Atkinson, Dr. Eric Sobel and Dr.

Jake Streit. These professors have provided me with insightful, unbiased advice and constructive

criticisms. Without their help, I could not have learned to examine and refine my own work

from a broader understanding of immunology. I especially want to thank Jake, who has been a

wonderful friend and motivator throughout my years as a developing scientist.

I would also like to express my gratitude to the members of my laboratory, past and

present, for their support and assistance during my graduate research. Dr. Changqing Xia has

been instrumental in educating me on dendritic cell biology as well as on basic principles of

immunology, and has served as a selfless and patient mentor. Dr. Ruihua Peng has been a

constant friend and endless supporter of me in countless ways. Both Ruihua and CQ have played

a prominent role in helping me develop the technical skills I needed to perform my work. I also

wish to thank Tolga Barker, Ed Paek, Dr. Michelle Rodriguez, Luke Smith, Dr. Yushi Qiu, Dr.

Ron Ferguson, and Dr. Sally Yuan for their kind support. My lab has been a home for me for the









past 4 years, not because of the time committed to research, but also because these friends have

provided a warm and nurturing environment that made it feel like home.

My graduate studies could not have been completed without the help of many support

staff. Fred Grant and Ronnie Middleton from the SPF Pathology Mouse Colony provided me

and my mice with unique attentive assistance and kind understanding. Dr. Dave Ostrov kindly

taught me about basic principles of antigen protein structure. Steve McClellan and Neal Benson

from the flow cytometry core provided service above and beyond what was expected and were

instrumental in my data collection. I especially would like to acknowledge the members of the

Atkinson lab who provided me with support, equipment and reagents when I most desperately

needed their help.

Finally, I wish to thank my family and friends. I could not have survived the demands of

graduate school without family and friends to laugh and celebrate with. My family: mom, dad,

my sister Paulette, you have been unstoppable sources of support for me. I cannot thank you

enough for all your sacrifices that allowed to me attain my goals. And to my love, my husband

Dan, you inspired and motivated me to achieve my professional ambitions, supported me as my

best friend and confidante, while always entertaining me with your irresistible charm and humor.

Of course, to my best friend Campbell, you are gone but never far from the heart. You

were my connection with all that is important in life and your spirit kept me going every day.









TABLE OF CONTENTS

page

A C K N O W L E D G E M E N T S ......... ................. ................................................................................. 4

L IST O F F IG U R E S ................................................................. 9

LIST OF ABBREVIATIONS ......... .................................. ............................. 10

A B S T R A C T ............................................................................................... ...... 12

CHAPTER

1 L IT E R A T U R E R E V IE W ........................................................................................................... 14

Type 1 D diabetes ....................... ....... ......................... ............... 14
Epidemiology: Incidence and Prevalance ............................................... ... ............ 14
Etiology: Genetic and Environm ental Factors................................................................ 14
Im m unopathology ............................... ........................ .................................... 15
Existing Therapies for Type 1 D iabetes ............... ........................................................... 16
N nutritional Intervention ......... .................... .................................................................... 17
G ene Therapy ............. .......................... .................... ................... ......... 17
Beta/Islet Cell Transplantion/Regeneration.................... ............. ............... 18
Im m u n e M odu nation ............... ................................................................. ................. 18
A ntigen-B asked T therapies ..................................................... ............................ ....... 19
Cellular Basis for Type 1 Diabetes Immunotherapy................. ........ .......... ............20
D endritic C ells ................................................ .............. .......... ...... 2 1
M odification to enhance function ......... ................. .......................... ............... 24
Current progress in clinical trials ........................................... ......................... 26
Regulatory T Cells ........ .......... ... ... .. ...... ......... .......... 26
Subclasses of regulatory T cells ..................... .... ..... ..................27
Role of dendritic cells in regulatory T cell development and maintenance .............28
A ntigen-Based D endritic Cell Therapy ........................................ ............... ............... 28
Type 1 D diabetes A utoantigens .......................... .......................... ...... .................. 29
Native Immunogenicity of Peptide Determinants ................................... ............... 30
Unknowns of Antigen-Based Dendritic Cell Therapy ............................................. 31
N O D M ou se M o del ......................................................................................... ...................... 32
Su m m ary an d R ationale .............................................................. .......................................... 33

2 ENGINEERING OF TOLEROGENIC DC ........................................ ........................... 34

In tro d u ctio n ................................................................................................................................. 3 4
M materials an d M eth od s................................................................ .......................................... 3 5
A nim als .............. ................................................ ............... 35
Bone Marrow-Derived Dendritic Cells: Isolation and Culture.......................................36
F low C ytom etry .................................................................. 37
Cytokine Analysis ................... ............. .. ...... .............. 37









R etin oic A cid ........................ ............................................................................................. 3 7
In vitro................................. ............. ............... 37
In vivo ................................................................ ..... ... 3 7
M ig ration Stu dies...................................................................................... ......... ........... 3 8
R results .................... ......... ............ .. .. ...... ....... .......... ............................... 38
Immature Dendritic Cells Express Lower Levels of T-cell Costimulatory Molecules
Relative to TNF-a semi-mature and LPS-Stimulated Mature DC.............................38
Immature and Semi-Mature Dendritic Cells Produce Lower Levels of Pro-
Inflammatory Cytokine IFN-y Relative to Mature Dendritic Cells.............................39
Immature Dendritic Cells Migrate from Injection Site to Draining Lymph Node..........40
Immature Dendritic Cells are Superior to Semi-Mature Dendritic Cells for
Induction of T olerance................. ...................... .......... .................................. 40
In vitro Retinoic Acid Stimulation Increases Expression of Maturation and Gut-
H om ing R eceptors on D endritic Cells............................................................ .......... 41
D discussion ............................... ............ ........... 42

3 ROLE OF ANTIGEN PEPTIDE IN DC THERAPY ................................ .... ............48

In tro d u ctio n ................................................................................................................................. 4 8
M materials and M eth od s................................................................ .......................................... 4 9
Animals .............................................................. 49
P e p tid e s ...................... .......................................................................................................... 4 9
D endritic Cell Peptide Pulsing.... ...................................................................... 50
H istology ................................ ............. ................... ............... 50
ELISA for Global Suppression Analysis ................................................ ... ............ 51
Statistical Analysis .............................. ...................... ............... 51
Results ............... ...... .................. ............................. .. ............... 51
Subdominant Determinant-Pulsed DC Therapy Protects Against T1D.........................51
Pancreatic Islet Survival is Enhanced in Subdominant Determinant-Pulsed DC
R recipients ...................................... ...... .................. ........................ 52
Ignored Determinant-Pulsed DC Therapy Confers Complete Protection Against
T1D But May Require Continuous Treatment........................................................... 53
T1D Specific Peptide-Pulsed DC Therapy Does Not Alter Natural Immunity to
Environm ental Challenge: K LH Study................................................... ... .................. 53
D discussion ............................... ............ ........... 54

4 ANTIGEN-INDEPENDENT EFFECTS OF DENDRITIC CELL THERAPY ...................60

In tro d u ctio n ................................................................................................................................. 6 0
M material an d M eth o d s .............................................................................. ............................. 6 1
M ic e .............................................................................................................. . ........... ...... 6 1
Proliferation A ssay ........................................ 61
F low C ytom etry .................................................................. 6 1
B rd U S tu d y .................................................................................................. ............... 6 2
H istology ................................................................ ......................... 62
R T-PC R and G ene A rray ................................................................... 63
R esu lts .................................... ................ ...................................... ........ ..... 6 3









Homeostatic Proliferation is Observed Following DC Therapy: Immediate and
Su stain ed E effects .................. ............................ .................. .. .... ................ 63
Homeostatic Proliferation Occurs in Healthy and Autoimmune Mouse Strains
F ollow ing D C T herapy ........................................................................... ......... .......... 64
Identification of Proliferating Cell Populations.......................................... .................. 64
DC Therapy Reprograms Cytokine Production of Proliferating T Lymphocytes ..........65
DC Therapy Results in Differential Gene Expression: Analysis of Cytokine and
Chemokine Genes ............................................................................................... 65
In Situ Observation of Homeostatic Proliferation ................................... ............... 67
D discussion ............................... ............ ........... 68

5 MODULATION OF REGULATORY T CELL POPULATION................ ..................... 82

In tro d u ctio n ................................................................................................................................. 8 2
M materials an d M eth od s................................................................ .......................................... 82
F low C ytom etry .................................................................. 82
Suppressor A ssay ...................................................... 83
R etin oic A cid T reatm ent ..................................................................................................... 83
R e su lts ......................................................... .. ... ..... ... ...................... 8 4
DC Therapy Results in Sustained Expansion of Regulatory T Cells ............................ 84
Regulatory T Cell Function is Enhanced Following DC Therapy ................................. 84
Retinoic Acid Increases Frequency of Regulatory T Cell Population ............................. 85
D discussion ............................... ............ ........... 85

CON CLU SION S .......................................................... ....... .......... ...... 90

L IST O F R E FE R E N C E S ............... ................................................................................................ 99

B IO G R A PH IC A L SK E T C H ........................................................................................................... 117









LIST OF FIGURES


Figure page

2-1 Characterization of DC by flow cytometry.. ................................... ............ .................. 45

2-2 Luminex analysis of cytokine production by DC subsets. .............................................45

2-3 M migration of iD C ......................................................................... ...................................... 46

2-4 Diabetes incidence in NOD mice following DC therapy.................................................46

2-5 Effect of retinoic acid on dendritic cell maturation.............. .............................................47

3-1 Kaplan-Meier survival curves of female NOD mice following PBS, unpulsed, and
SD -pu lsed D C th erap y .................................................... ................................................ 5 7

3-2 Histological assessment of islet number in NOD mice following DC treatment............ 57

3-3 Kaplan-Meier survival curves of female NOD mice following short-term PB S,
unpulsed, and ID-pulsed DC therapy................................................... 58

3-4 Kaplan-Meier survival curves of NOD mice following continuous PBS, unpulsed,
and ID-pulsed DC therapy ....................... ..................................... 58

3-5 Antibody response following KLH immunization in control and DC treated mice..........59

4-1 Homeostatic proliferation following DC therapy is immediate and sustained. ................73

4-2 Homeostatic proliferation is observed in healthy and autoimmune mouse models...........73

4-3 Identification of proliferating cell populations ..... .......... ......... .......................... 74

4-4 Cytokine profile of proliferating cell populations ......... ..............................................75

4-5 Gene array analysis of common cytokine and receptors gene expression........................76

4-6 Frequency of BrdU proliferation in organs. ......................................................... 80

4-7 In situ proliferation marked by BrdU incorporation. .............. ......................... ...... ........ 81

5-1 Assessment of regulatory T cell population following DC therapy.............................. 88

5 -2 Su p p re ssor cell a ssay ..................................................... ................................................. 8 9

5-3 Retinoic acid increases frequency of regulatory T cell population............................... 89









LIST OF ABBREVIATIONS


Ab antibody

Ag antigen

B6 C57/BL6

BM bone marrow

BrdU bromodeoxyuridine

CD cluster of differentiation

DC dendritic cell

DD dominant determinant

Dex dexamethasone

Foxp3 forkhead box P3

GAD glutamate decarboxylase

GMP good manufacturing practices

HLA human leukocyte antigen

HSC hematopoietic stem cell

ID ignored determinant

iDC immature dendritic cell

IL interleukin

IV intravenous

KLH keyhole limpet hemocyanin

LN lymph node

mDC mature dendritic cells

mLN mesenteric lymph node

MHC major histocompatibility complex

NOD non-obese diabetic









PALS periarteriolar lymphoid sheath

PBS phosphate buffered saline

PP Peyer's Patches

pLN pancreatic lymph node

SD subdominant determinant

smDC semi-mature

T1D Type 1 diabetes

Treg regulatory T cell









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

DENDRITIC CELL THERAPY FOR TYPE 1 DIABETES:
ROLE OF ANTIGEN PRESENTATION IN IMMUNE MODULATION

By

Jeannette Lo-Dauer

August 2009

Chair:Michael J. Clare-Salzler
Major:Medical Sciences Immunology and Microbiology

Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of

insulin-producing pancreatic beta cells by autoreactive T cells. Antigen-based dendritic cell

(DC) therapy has been shown to be effective in preventing T1D in NOD mice, the animal model

for autoimmune diabetes, when administered during early insulitis but fails to protect when

applied after established autoimmunity. However, the development of antigen-based DC therapy

for late intervention has been challenging due to the dynamic nature of the immune repertoire

which alters with time. As most human cases are not diagnosed until established hyperglycemia,

an intervention for late-onset prevention is highly desirable. We have developed a DC treatment

paradigm for the induction of tolerance that is not limited to early intervention. We find that by

bypassing natural antigen processing, DC presenting synthetic subdominant and ignored antigen

determinants can more effectively prime naive T cells into regulatory function and protect NOD

mice from T1D. Development of diabetes was significantly delayed in mice receiving a short-

term treatment of immature DC presenting a subdominant determinant (p=0.05) as compared to

recipients of PBS, unpulsed or dominant determinant-pulsed DC. Mice receiving sustained

treatment of DC presenting an ignored determinant were completely protected (p=0.04). We

also reveal that DC therapy with/without preloading with T D-associated Ag results in









immediate and sustained immune modulation characterized by robust proliferation of T and B

lymphocytes. Of interest, DC-treated mice have higher frequencies of CD4+Foxp3+ and IL-10

producing T cells compared to PBS controls. Additionally, CD4+CD25+ regulatory cells from

DC-treated mice have superior suppressor function, with the effect enhanced in antigen-pulsed

DC recipients. Overall, these results suggest that DC therapy provides a previously unknown

long-lasting effect on immune processes in an antigen-dependent and antigen-independent

manner. We describe the reprogrammed cell populations and show alterations in their cytokine

production and function. Importantly, we identify peptide determinants most effective in late

antigen-based therapy for T1D based on a fundamental principle of determinant immunogenicity

that extends to any autoantigen-based disease. These findings will aid the understanding of how

DC therapy modulates the immune response and guide the engineering of DC therapies for

prevention of T1D and other dynamic antigen-based disorders.









CHAPTER 1
LITERATURE REVIEW

Type 1 Diabetes

Type 1 diabetes (T1D) is an autoimmune metabolic disease characterized by insulin

deficiency as a result of the loss of pancreatic islet beta cells(l). Insulin, an endocrine hormone,

is produced by beta cells to mediate the uptake of glucose from blood into cells for carbohydrate

and lipid metabolism. Chronic destruction of beta cells by autoreactive T cells disrupts insulin

production leading to hyperglycemia and secondary complications including neuropathy,

nephropathy, blindness, and cardiovascular disease(2).

Epidemiology: Incidence and Prevalance

Type 1 diabetes accounts for nearly 10% of all cases of diabetes and afflicts as many as 1

in 300 persons in the United States(2). It is estimated that 15,000 persons under the age of 20 are

newly diagnosed annually at a rate of 19 per 100,000 each year(3) with prevalence rising to

1,087,800 cases by the year 2010 in the U.S.(4). There is significant variation in the incidence of

T1D worldwide, with more than a 400-fold variation among reporting countries. T1D is rare in

China, India, and Venezuela where the incidence is only 0.1 per 100,000 while Sardinia and

Finland report a much higher incidence at nearly 37 cases per 100,000 individuals each year(5).

T1D afflicts more non-Hispanic whites than non-whites(6) but occurs in males and females

equally(7). The incidence of T1D is on the rise in almost all populations worldwide with the

greatest increases in countries with a low incidence(8).

Etiology: Genetic and Environmental Factors

Genetic and environmental factors are implicated in the development of T1D. The human

leukocyte antigen (HLA) genes, particularly class II, on chromosome 6 are considered to provide

a considerable risk, accounting for up to 50% of familial aggregation in T1D(9). The HLA









genes, DR4-DR8 and DR3-DQ2, contribute the greatest risk for T1D since these haplotypes have

been found in 90% of children diagnosed with T1D(10). The second most important genetic

susceptibility factor is attributed to the VNTR region of the insulin gene located on chromosome

11. Shorter forms of a variable number tandem repeat on the insulin gene promoter are

associated with disease, while longer forms confer protection (11,12). The cytotoxic T

lymphocyte antigen 4 (CTLA-4) and lymphoid tyrosine phosphatase (LYP) genes, which control

the negative regulation of T-cell activation, are also implicated in susceptibility to T1D (13).

However less than 10% of those who are genetically susceptible develop the disease and

concordance in identical twins is only 30-40%, suggesting that genetics may not be the only

factor (14,15). Environmental triggers, such as a viral infection, particularly with enteroviruses,

rotaviruses, and rubella, has been linked to increased risk of T1D in genetically susceptible

individuals. Beta cell-specific infection by the Coxsackie B4 virus has been described and the

virus has been isolated from the pancreas of fatal childhood diabetes cases(16). Additionally, the

incidence of T1D was shown to increase after epidemics of enterovirus infections (17). Still, the

viral mechanism leading to increased risk for T1D remains largely undefined. Viral infection

followed by local damage to the pancreas, or stimulation of immunological cross-reactivity to

islet antigens by viral proteins are mechanisms proposed to explain the role of viruses in T1D

(15). The stimulation of protective inflammatory cytokines during viral infection may contribute

to beta cell death by promoting the persistence of effector cells after the virus has been

cleared(18). Thus, it appears that a multifactorial interaction between autoimmune, genetic and

environmental influences collectively contribute to the development of Type 1 diabetes.

Immunopathology

T1D is caused by an autoimmune attack of insulin-producing beta cells by autoreactive T-

cells. This organ-specific autoimmune disease results from a Thl-biased response targeted to









self-antigens such as insulin and glutamic acid decarboxylase (GAD) in the presence of an

ineffective regulatory response(19). The development of T1D occurs over a period of years as

chronic progression of leukocyte infiltration of the islets leads to eventual beta cell destruction

and loss of insulin production(20). In the initial stages of beta cell death, CD8+ cytotoxic T cells

account for the bulk of leukocyte infiltration with perforin and granzyme secretion(21,22). IL-10

producing CD68+ macrophages also comprise a significant percentage of the islet infiltrate(21).

As insulitis proceeds, an increase in CD20+ B cells, but not CD138 plasma cells, is

observed(21). CD4+ helper T cells, dendritic cells, NK cells are also detectable as a minor

percentage of the leukocyte infiltrate(21). Throughout the entire inflammatory process,

cytotoxic T cells and macrophages remain the predominant immune cell subset in the islet.

However, once beta cell destruction is complete, the frequency of all subsets of immune cells

decline dramatically suggesting that immune cells enter the islet only when viable beta cells are

present(21).

Existing Therapies for Type 1 Diabetes

Although advances in supportive therapy for the management of Type 1 diabetes has

improved the quality of life for those with T ID, patients have an estimated 10-15 year reduction

in life expectancy resulting from diabetes-related complications demonstrating the need for

prevention and reversal strategies(23). Serum autoantibodies to pancreatic islet proteins are

detectable before onset of Type 1 diabetes, allowing for screening of subjects at risk thereby

providing an opportunity for disease prevention. However upon T1D onset, treatment becomes

more challenging. The strategies developed for the prevention or reversal of T1D in humans,

ranging from nutritional intervention, gene therapy, islet/beta cell expansion/transplantation, and

immunotherapy have met with variable success.









Nutritional Intervention

Animal studies using the Type 1 diabetes Biobreeding (BB) rat and nonobese diabetic

(NOD) mouse models have suggested that diet can impact the development of T1D. Dietary soy,

milk, and gluten have been found to be highly diabetogenic in T1D susceptible rodents (24,25),

potentially by reducing the number of regulatory T cells(26). However others studies have found

that hydrolyzed casein-based diets protect against T1D(25). In humans, early exposure to gluten

or cereals has been linked to T1D, suggesting a role for wheat in the pathogenesis of

T1D(27,28). Conversely, dietary supplementation with vitamin D and omega-3 fatty acids has

proven to be effective in the prevention of T1D in both rodents and humans(28,29). A clinical

trial called the Nutritional Intervention to Prevent Type 1 Diabetes Pilot Study (NIP) is currently

underway to investigate the effects of increased dietary docosahexaenoic acid (DHA), an omega-

3 fatty acid naturally found in mother's breast milk and in various foods, in newborns and infants

with genetic susceptibility to T1D.

Gene Therapy

Gene therapy for Type 1 diabetes aims to genetically modify host cells using viral or

plasmid vectors to deliver novel gene constructs. The insertion of insulin/proinsulin genes into a

variety of surrogate non-beta cell types, including fibroblasts, hepatocytes, muscle cells, and

embryonic stem cells have induced insulin production(30-33) in non-endocrine cells. However,

the challenge remains in the engineering of an insulin-producing cell that can also maintain

precise control of glucose through the expression of glucose sensing or glucose transport

genes(33,34). Another approach using gene therapy for T1D aims to curb the autoimmune

response by down regulating IMHC gene expression in beta cells(33,35). Studies have shown

that transgenic NOD mice with different MHC class I and II alleles do not develop diabetes,

suggesting that IMHC recognition and antigen presentation play an integral role in the









autoimmune process(36). Thus exclusively down-regulating the expression of MHC expression

in the beta cells may be an effective means of preventing autoimmune attack of the beta cells.

Others have found that inducing IL-4 gene expression and secretion in islet cells may also stymie

the autoimmune response(35).

Beta/Islet Cell Transplantion/Regeneration

When the loss of beta cells from autoimmune attack becomes significant, the production of

insulin is compromised and insulin therapy must be initiated to maintain physiological control of

blood glucose levels. The expansion/regeneration of beta cell mass using pharmaceuticals or

transplantation can re-introduce insulin production. Exenatide, a peptide incretin mimetic that

has glucoregulatory actions, was approved by the FDA in 2005 for the treatment of Type 2

diabetes(37), but when used in combination with immunosuppressants(38) or anti-

inflammatories(39) has been shown to allow for repopulation of pancreatic islet structures with

functional beta cells. The use of various growth factors and small molecules were able to direct

the differentiation of stem cells into functional insulin-producing cells(40-42). Whole pancreas

and islet transplantation have shown limited success due to organ rejection and limitations

procuring functional islets in adequate numbers(2,43,44). However, these approaches to

increasing 3 cell mass or function only confer transient relief as survival of the beta cells is

limited due to the ensuing autoimmune attack. The engineering of encapsulated islets/beta cells

that escape fatal infiltration from autoimmune cells has been demonstrated in the NOD mouse

though its application in human clinical remains to be determined(45)

Immune Modulation

While beta cell transplantation/regeneration restores some insulin production, this strategy

only provides short-term relief from T1D as the autoimmune process is left unaddressed, leaving

the novel beta cell tissue susceptible to destruction from the ongoing autoimmune process.









Monoclonal antibody therapy has been used alone or in conjunction with beta cell replacement

techniques to suppress autoimmunity. Anti-CD3 monoclonal antibody (mAb) therapy was

initially used for the prevention of organ allograft rejection in transplant recipients, but its use

has been extended to autoimmune diseases as well. Anti-CD3 mAb binds to the CD3 receptor

on T cells, modulating the CD3-TCR complex such that it results in energy, apoptosis, or

antigenic modulation, a process in which the T cell becomes unresponsive to antigen. Murine

CD3 mAb therapy has treated autoimmunity in models of TD(46) and experimental

autoimmune encephalomyelitis (EAE)(47). Another mAb, anti-CD20 known as Rituximab, was

used to reverse T1D in transgenic NOD mice expressing human CD20 by inducing regulatory B

cells or B cell depletion(48,49). However, success using mAb in human trials has been limited.

In a clinical trial using anti-CD3 therapy, progression of T1D was delayed up to 3 years(50,51)

or preserved some insulin production(52). Clinical trials for antiCD20 mAb therapy are

currently underway. Unfortunately, unresolved issues with cytokine release syndrome, a life

threatening severe inflammatory process, and global immunosuppression have set back progress

for mAb therapy(50,52-55).

Antigen-Based Therapies

The development of Type 1 diabetes is based on the loss of tolerance to self-antigens

expressed by beta cells such GAD, insulin, and heat shock protein (HSP) 60 (56-63). Efforts to

redirect the immune response from immunity to tolerance via oral or parenteral administration of

whole or peptides of these antigens have been successful in mouse studies, but have met with

considerable setbacks in human. Clare-Salzler et al. found that early administration ofGAD65

peptides to NOD mice induced active tolerance characterized by the development of Tregs that

conferred protection when transferred to SCID NOD that had been given diabetogenic

splenocytes (64). Similar effects were found in studies using peptides of insulin and HSP60 via









alternative routes of administration (60,65). However, in a clinical trial involving genetic high-

risk non-diabetic subjects, the administration of oral or injected insulin therapy had minimal

effects on prevention of T1D(66,67). Moderate success was found in the Diamyd and

Diapep277 trials investigating GAD and HSP antigen therapy in new onset subjects, as some

reduction in loss of C-peptide appeared to correlate with treatment though no there was no effect

on reversal of diabetes (56,68,69). The failure in protection using antigen-based therapies may

be due to difficulties in translating the appropriate antigen dose and timing of treatment.

Additionally, patients susceptible to T1D often have defects in their immune cells so it is

uncertain whether the delivery of antigen to an impaired immune system may be able to correct

the autoimmunity.

Cellular Basis for Type 1 Diabetes Immunotherapy

Immunotherapy, which targets the underlying aberrancy in T1D, appears to be the most

effective strategy for T1D as this will block the autoimmune process causing beta cell

destruction as well as protect new islet grafts. Antigen-nonspecific approaches to subdue the

autoimmune process using monoclonal antibodies suppress both pathological and essential

immune processes, resulting in generalized immunosuppression/modulation that leaves an

individual susceptible to bacterial and viral threat. Antigen-specific immunotherapy that targets

only the aberrant autoimmune processes but otherwise leaves the immune system intact is ideal.

Dendritic cells, which present antigen and direct T cell responses, have been shown to induce

Ag-specific T cell tolerance possibly through the generation of regulatory T cells. Because DC

uniquely orchestrate the delicate balance between T cell immunity and regulation to self

antigens, DC-based immunotherapy is an attractive approach to immunotherapy for T1D.









Dendritic Cells

Dendritic cells (DC) are professional antigen presenting cells (APC) that direct the T cell

response toward immunity or tolerance through receptor-mediated signals and cytokine

secretion. DC were first discovered in 1972 by the group of Ralph Steinman when researchers

identified a white cell in the mouse spleen that was morphologically and functionally different

than classical white cells with the unique ability to extend and retract dendrites(70). The

immunogenic role of DC was later elucidated in a transplantation model of graft versus host

reaction. Using mixed leukocyte reaction (MLR), it was found that donor DC were more potent

than whole spleen cells in their ability to elicit rejection by recipient cells, emphasizing the role

of the major histocompatibility complex (MHC) in DC biology. In a two stage process, DC act

to stimulate T cells by first internalizing extracellular antigens through endocytosis, processing

Ag into peptide fragments within lysosomes, then transporting peptide to the cell surface for

presentation to T cells via the major histocompatibility complex (MHC) molecule. The second

stage involves the cell-to-cell interaction between DC and T cells where the Ag-MHC complex

on DC binds to the T cell receptor (TCR) providing signal 1, and T cell costimulatory molecules

such as CD80(B7.1) and CD86(B7.2) may be up regulated to bind to CD28 or CTLA-4 on the T

cell providing signal 2(71). DC act to stimulate CD4+ helper T cells through MHC class II or

CD8+ cytotoxic T cells through MHC class I. The two-signal hypothesis of lymphocyte

activation suggests that the decision between T cell activation and T cell anergy/tolerance is

mediated by whether DC provides the second costimulatory signal during antigen

recognition(71). However, the specific mechanisms as to how DC assume an immunogenic or

tolerogenic state has not been fully elucidated, and evidence suggests that their varying

immunomodulatory activity can be attributed to a variety of factors including cell lineage,

maturation state, and antigen presentation.









The DC maturation process is associated with many events such as loss of

endocytic/phagocytic receptors, up regulation of costimulatory molecules and major

histocompatibility complex class II (MHCII), change in physical attributes such as acquisition of

motility and loss of adhesive structures, and secretion of cytokines (72). Classical models

characterize immature DC (iDC) as expressing low levels of MHCII and co-stimulatory

molecules. They are considered poor stimulators of T-cells but can engulf and process antigen

efficiently. Immature steady-state DC are believed to be important mediators of peripheral

tolerance as they continuously sample and present tissue-Ag without costimulation leading to

tolerance induction through T cell energy or deletion (73). Additionally, studies using ex vivo

generated iDC have demonstrated their immunomodulatory bias toward tolerance. Machen et al.

found that the down-regulation of maturation surface markers on DC produced an immature and

tolerogenic subset of cells that significantly delayed onset of diabetes when administered to

NOD recipients (74). Others have also been able to use iDC to direct Th2 or Trl cell

polarization in culture (75,76) or human subjects (75,76) through the release of IL-10.

Stimulation of DC with TNF-a or IL-10 produces a semi-mature DC phenotype that is

characterized by high-levels of co-stimulatory molecules but low levels of pro-inflammatory

cytokines and an acquired capacity for migration. Intravenous (IV) administration of semi-

mature DC to mice have been shown to induce Ag-specific T-regulatory cells that suppressed

experimental autoimmune thyroiditis(EAT) (77). This protection was attributed to, but not

dependent on DC-induced IL-10 secretion by tolerized T-cells. Semi-mature DC have also been

found to induce anergic CD4+ and CD8+ T cells that suppressed proliferation of syngeneic T-

cells in human cell cocultures (78). Mature DC (mDC), which readily migrate to draining lymph

nodes (DLN) and express high levels of costimulatory molecules that are required for potent T









cell stimulation, have been identified as immunogenic cells. This observation is supported by

early studies in which freshly isolated immature, low-costimulatory molecule expressing DC

failed to stimulate responder T-cells in mouse MLR cultures, while cultured mature, high

costimulatory molecule expressing DC were potent activators of T-cells (79,80). Dhodapkar et

al. found that subcutaneous injection of peptide-loaded mDC stimulated a rapid induction of

IFN-y secreting CD8+ effector cells in human subjects (81). Human cell cultures also revealed

that mDC can overcome regulatory cell suppression of CD8+ cells and promote expansion of

antigen-specific CD8+ naive and memory cells (82). Furthermore, the production of pro-

inflammatory cytokines by mDC, including IL-10, IL-6, TNF-a, and IL-12, contributes to T-cell

immunity (83,84). LPS-matured DC administered with anti-CD40 have been shown to result in

the elevated production of IL-12 and a Thl phenotype in mice (84).

This classical model of DC maturation subsets is defined predominantly in terms of

phenotypic criteria, with an emphasis on the requirement of costimulatory molecule expression

for T-cell stimulation. Though the majority of the existing literature supports this general

paradigm, emerging reports of tolerance induction by mature DC subsets have prompted reviews

of our current knowledge. For instance, Feili-Hariri et al. found that mature DC were better able

to prevent diabetes development (85), while Emmer et al. found that LPS-matured DC prolonged

allograft survival when pretreated with dexamethasone (86). Another study found that DC-

mediated expansion of CD4+CD25+ regulatory cells was partially dependent on CD80/CD86

expression by DC and the production of IL-2 (87). Given these findings, it is evident the nature

of DC modulation on T-cell responses is a highly complex process that is not limited to only the

maturation phenotype. Proposed factors that may contribute to DC modulation of T-cell

responses include the type of maturation signal used to stimulate DC, class of cytokines secreted,









and activation requirements of T-cells. Pulendran found that E. coli-derived LPS stimulated DC

induced a Thl response through the secretion of IL-12, while stimulation of DC with P.

gingivalis-derived LPS resulted in a Th2 response, suggesting that minor differences in TLR

ligands may influence immune response (88). Another group found that IL-2 secretion by both

low and high-costimulatory molecule expressing-DC was sufficient to stimulate T-cell

responses, demonstrating that cytokine secretion alone was adequate for T-cell activation (89).

This observation is supported by the fact that CMV-mediated inhibition of IL-2 production by

DC results in an ineffective T-cell response and thus a persistent viral infection (90).

Additionally, the simultaneous induction of CD4 T-cell tolerance and CD8 T cell immunity in

mice by MHC class II-restricted ova-pulsed semi-mature DC and MHC class I + II-peptides-

pulsed DC, respectively, suggests that the decision between tolerance and immunity not only

depends on the DC subset, but also on the type and activation requirements of the responding T

cell (91). The tolerogenic and immunogenic nature of DC, while amply demonstrated across DC

subsets, still remains to be better understood as no definitive criteria for immune activation or

suppression as been identified.

Modification to enhance function

Dendritic cells are a heterogeneous group of cells that can exhibit many phenotypes and

functions in response to their microenvironment or in vitro stimuli. The opportunity for

clinicians to manipulate dendritic cells ex vivo allows for specific engineering of DC using

techniques that improve or enhance the desired function. Several strategies for DC modification

include the use ofretinoic acid and targeted antigen-loading.

Retinoic acid. Retinoic acid (RA) has been shown to have important immunomodulatory

effects, particularly in the gut. RA, a dietary metabolite of Vitamin A, is also locally produced

by dendritic cells found in the mesenteric lymph node (mLN) and Peyer's Patches (PP-DC)(92).









Exposure to RA through either in vitro stimulation or in vivo interaction with mLN DC or PP-DC

can induce or up regulate the expression of the gut-homing receptors a407 (CD 103) and CCR9

on T cells, B cells, and DC(92,93). RA has also been found to reduce DC-mediated production

of the inflammatory cytokine IL-12(94). Additionally, RA promotes the development of Foxp3+

regulatory T cells and inhibits their differentiation into Thl7 cells(94-97). Recently, Maynard et

al demonstrated that RA has a bidirectional role in Treg development by promoting Foxp3 Tregs

while suppressing I1-10 producing Tregs(98). These findings suggest that RA can be used to

augment the immunomodulatory effects of DC therapy.

DEC205. DEC205 is an endocytic receptor on dendritic cells that mediates the uptake and

engulfment of extracellular antigens. By fusing antigen or DNA to an antibody against the

DEC205 receptor, Ag is targeted specifically to DC and results in improved Ag uptake,

processing and presentation on MHC class I and class II molecules. Studies have found that this

strategy promotes strong T cell immunity and improves vaccination results(99,100). While this

technology has only been investigated for purposes of generating T cell immunity, the targeting

of Ag to DC may also have applications to DC therapy for the induction of antigen-specific

tolerance.

Dexamethasone. Dexamethasone (Dex) is a synthetic glucocorticoid steroid with anti-

inflammatory and immunosuppressive effects(101). Treatment of monocyte-derived DC with

Dex maintains DC in a steady state of immaturity characterized by low expression of CD80,

CD86, MHC class II, and low production of IL-12(102). These DC were resistant to maturation

following stimulation with LPS, produced high amounts ofIL-10 and maintained this phenotype

long after removal of Dex stimulation, suggesting that Dex generates DC with a durable

tolerogenic phenotype(103). Additionally, another study demonstrated that rat Dex-DC were









able to induce selective expansion of CD4+CD25+ Tregs(104). Dex-DC were also shown to

suppress colitis in SCID-mice, demonstrating immunosuppressive function in an animal model

of disease(105). Together, these findings highlight the potential of using dexamethasone to

generate tolerogenic DC for immunotherapy.

Current progress in clinical trials

The first published report of a clinical trial using dendritic cell therapy was 14 years ago

describing the use of peptide-pulsed DC for treatment of melanoma(106). Since then, over 100

trials in 15+ countries have been made, with the majority of studies focused on using DC therapy

to induce T cell immunity for the treatment of cancer(107). Fewer clinical trials using DC

therapy for tolerance induction exist, including studies for organ transplant recipients and

autoimmune disease. In 2001, a study by Dhodapkar et al investigated the use of immature

peptide-pulsed DC to induce Ag-specific tolerance. They found that a single injection of

influenza matrix peptide-pulsed DC was able to induce IL-10 producing CD8+ Tregs that

inhibited MP-specific CD8+ effector T cell function(76). Phase I clinical trials are currently

underway in Queensland and England for using tolerogenic DC in the treatment of rheumatoid

arthritis(107). Another study using tolerogenic DC is in progress to establish the safety of using

anti-sense oligonucleotides targeted to DC for prevention of Type 1 diabetes(107). These trials

have demonstrated that while DC therapy has overall proven to be safe and well-tolerated with

minimal side effects, much remains to be learned regarding how tolerogenic DC behave in

human subjects.

Regulatory T Cells

In a healthy individual, autoreactive cells are present but do not cause autoimmunity due to

effective immunoregulatory mechanisms. However in T1D the lack of an effective regulatory

response allows autoreactive T cells to become pathogenic, thereby invading and destroying the









pancreatic islet cells. In the early 1980's, a new class of "suppressor cells" that appeared to curb

autoreactivity in the body was discovered(108). Since then, the work of Sakaguchi and others

have identified these cells as regulatory T cells (Tregs), which function to suppress immune

responses against self-antigens by secreting immunosuppressive cytokines or by interacting

directly with effector T cells (108-113).

Subclasses of regulatory T cells

Naturally occurring Tregs are classically identified as CD4+CD25+Foxp3+ cells that

originate from the thymus to maintain peripheral tolerance. They constitute 5-10% of the

peripheral CD4+ T cell population and have been shown to be essential to self tolerance as

removal of this subset of cells from the mouse results in inflammation and autoreactive responses

that can only be rescued by their reconstitution(1 14). This subset of Tregs exerts their

suppressor function in a contact dependent manner. Naturally occurring Tregs become activated

by Ag following engagement of their TCR, but then suppress in an antigen nonspecific manner

by inhibiting IL-2 transcription in the responding population(115). Induced Tregs, which

develop de novo from CD4+CD25- T cells, have a CD4+CD25+Foxp3- phenotype and exert

their suppression in a contact-independent manner via secretion of soluble factors. Regulatory

Trl cells can also be induced to develop from naive or resting T cells in the presence of IL-10,

Vitamin D3, or dexamethasone( 16). Trl secrete large amounts of IL-10 to suppress effector T

cells but proliferate poorly following antigenic stimulation(115). Th3 cells, another subset of

inducible Tregs, develop with TGF-beta conditioning or Ag stimulation and secrete TGF-beta to

suppress in an antigen-nonspecific manner( 15). CD8+ Tregs that secrete IL-10 have also been

identified, and are thought to be induced through signaling from immature DC(76,117).









Role of dendritic cells in regulatory T cell development and maintenance

In addition to signaling T cells for effector function during periods of environmental

challenge, DC also have a role in maintaining tolerance through the induction of regulatory T

cells. Naive T cells can be directed to develop into Tregs following chronic exposure to

immature DC presenting self-Ag in the absence of a danger signal(75,118-120). These Tregs

then home to tissue where, under conditions of inflammation, are reactivated by resident DC

presenting self-Ag(120). IL-10 producing DC, liver DC, and lymphoid DC have all been subsets

of immature DC that were shown to induce the development of Tregs(121-123). With a role in

Ag presentation within a tolerogenic environment, it is evident that immature DC play a central

role in the induction and maintenance of Tregs in the periphery.

Antigen-Based Dendritic Cell Therapy

One of the challenges facing immunotherapy for tolerance induction is maintaining control

of the immune modulation such that the therapy targets only the aberrant autoimmune processes

while essential immune responses remain intact. The induction of global non-specific

immunosuppression or immunomodulation using nontargeted immunotherapy puts individuals at

risk for uncontrolled infections or tumors unless therapy can be effectively directed toward the

immunogenic antigen. Antigen peptide therapy has demonstrated success in animal studies.

Tian et al. prevented T1D in the NOD mouse following intranasal administration of a GAD65

peptide(64). Ramiya and others have also found that oral or IV administration of either insulin

or GAD65 antigens prevents T1D in mice(61,124-126). Although peptide immunotherapy for

tolerance induction has been widely proven in animal models(65,127-130), success in clinical

studies has been less forthcoming(59,131) possibly due to uncertainties in the translation of the

treatment regimen from animals to human. Difficulties in determining the appropriate antigen

dose, route of administration, and timing of treatment contributes to the issue of safety, with









concerns that a suboptimal treatment plan can result in the exacerbation of immunity rather than

the induction of tolerance(131-133). Antigen-based DC therapy circumvents this issue by

controlling the direction of the immune response toward the Ag using tolerogenic DC. In

autoimmune diseases such as T1D where the target antigen of the autoimmune process has been

identified, the presentation of that antigen in the context of a tolerizing signal such as those given

by immature DC can allow Ag-specific re-education of the immune system toward tolerance.

Antigen-based DC therapy provides a directed approach toward a defined target by using

tolerogenic dendritic cells pre-loaded with the target autoantigen of interest.

Type 1 Diabetes Autoantigens

In T1D, the organ-specific T-cell attack is targeted at autoantigens such as GAD65, insulin,

and IA-2 (insulinoma-associated antigen 2). Antigen-based therapy for T1D has focused on

using whole or peptide derivatives of these islet cell antigens for tolerance induction. Krueger et

al. prevented cyclophosphamide-accelerated diabetes with a single injection of insulin-pulsed,

but not ovalbumin-pulsed dendritic cells in the NOD mouse(134). Our previous work using

dendritic cells isolated from pancreatic lymph nodes presenting islet Ag prevented 100% of T1D

in NOD mice, whereas DC from inguinal lymph nodes of unpulsed DC were ineffective(135).

This strategy has also been employed in other disease paradigms to promote organ-specific

tolerance. Verginis et al. induced IL-10 secreting CD4+25+ T-cells that inhibited the

development of experimental autoimmune thyroiditis (EAT) in mice after intravenous (IV)

injection with thyroglobulin-pulsed, but not ovalbumin-pulsed dendritic cells(77). Similar

results were obtained using myelin oligodendrocyte glycoprotein (MOG) peptide-pulsed DC for

protection from experimental autoimmune encephalomyelitis (EAE)(84). Clinical symptoms of

multiple sclerosis (MS) in marmosets were ameliorated following immunization with MP4, a

protein chimera of myelin basic protein, the antigen target of MS(136). Autoimmunity was also









halted in an experimental myasthenia gravis rat model that was treated with acetylcholine

receptor-pulsed dendritic cells (137). Collectively, these studies suggest that antigen-based

therapies can provide specific and effective tolerance induction.

Native Immunogenicity of Peptide Determinants

The traditional approach to antigen-based therapies has been to identify target determinants

within the autoantigen molecule that the autoimmune response is directed at, then administer

those target peptides or whole Ag in modalities that induce tolerance. Unfortunately, these

therapies are ineffective when administered at advanced stages of disease, suggesting that there

may be an altered immune repertoire with time. In T1D, the T cell reactivity is initially limited

to a few autoantigen determinants. But with time, T cell reactivity gradually expands intra &

inter-molecularly to additional determinants and antigens, chronically recruiting naive cells into

the autoreactive pool(62,138-142). This epitope spreading gives rise to an array of determinants

that have distinct immunogenic properties and possibly unique roles in autoimmune

pathogenicity. The nature of these determinants' immunogenicity can vary when presented

either as naturally processed peptides from whole antigen or as synthetic peptide fragments

(140,143,144). Determinant classes have been characterized by assessing natural and recall T

cell reactivity to overlapping 20-mers peptides of T1D autoantigens in the NOD mouse.

Stimulation with dominant determinants, defined as epitopes that are preferentially seen by

autoreactive T cells due to favored processing and presentation, elicited spontaneous T cell

responses from NOD spleen cells(144). Subdominant determinants (SD), which are secondarily

processed and presented as they do not compete as effectively with DD for presentation, also

generated a spontaneous response. Ignored determinants (ID), which are not processed and

presented, failed to induce significant spontaneous spleen cell responses. However, if mice were









immunized with ID, they were able to develop a recall response to the immunizing peptide,

though the ability to elicit recall responses vary with age of animal for unknown reasons(144).

Historically, dominant determinants (DD) are Ag determinants of choice for Ag-based

therapies as they lead to induction of T cell response. SD or ID are not believed to be involved

in the pathogenic T cell pool and thus are not used in Ag therapies. However, it has been

speculated that DD continuously recruit naive T cells into spontaneous autoimmune attack while

SD and ID, which minimally or do not activate naive T cells, have no effect on the naive T-cell

pool (144,145). Thus, DD-reactive cells are progressively exhausted from the naive pool,

leaving only non-reactive naive T cells that may be experimentally induced to respond to SD or

ID.

Olcott et al first examined this theory by testing peptide therapy in NOD mice with early

and advanced insulitis(144). To determine which classes of determinants could prime Th2

responses and prevent diabetes when administered within an extended range of the disease

process, an extensive panel ofpeptides from various beta cell antigens were used to immunize

the NOD mouse at 6 and 12 weeks of age (144). Consistent with the idea of DD-biased

exhaustion of the naive T cell repertoire, it was found that while both DD and ID were

comparable in their ability to inhibit T1D when administered early in the disease process, only

ID were effective when used during later phases of disease(144).

Unknowns of Antigen-Based Dendritic Cell Therapy

Much of the knowledge on how DC therapy affects immune responses has been gained

from clinical studies that are directed toward the induction of Ag-specific immunity for the

treatment of cancer. In the scope of translation of DC therapy from animal to human for

tolerance induction, little is actually known. Clinicians who strive to employ DC therapy for

autoimmune disease treatment have a unique hurdle in that they must be careful that they don't









exacerbate autoimmunity while attempting to treat the patient. We must learn how to maintain

DC in a steady state of immaturity to prevent unintentional activation of the immune system.

Another aspect to DC therapy that remains to be understood is how DC affect the overall

immune response. Because DC are potent APC, most studies use DC to augment Ag-specific

immune responses and thereby assess only Ag-specific modifications following DC therapy.

Additionally, studies using unpulsed DC have demonstrated protection from autoimmunity as

well(85). It remains to be determined what role Ag has in DC therapy and how DC therapy

augments Ag-independent immune responses.

NOD Mouse Model

The nonobese diabetic (NOD) mouse is a model for the spontaneous development of

autoimmune Type 1 diabetes. NOD mice were developed through selective inbreeding of the

progeny of a female mouse of the Cataract Shionogi (CTS) strain that had spontaneously

developed autoimmune diabetes. Successive inbreeding of multiple generations yield the NOD

strain that is used today(146).

NOD mice develop peri-insulitis characterized by the appearance ofleukocytes at the

periphery of pancreatic islets as early as 3-4 weeks of age, followed by marked CD4+ and CD8+

infiltration into the pancreatic islets shortly after. Islet protein specific autoantibodies are also

detectible. Onset of diabetes is seen around 12 weeks of age in females and several weeks later

in males as beta cell death progresses and insulin production is reduced, resulting in glucosuria

and non-fasting plasma glucose higher than 250 mg/dl. The incidence of diabetes is markedly

different by gender, with 90-100% of females compared to 40-60% of males developing diabetes

by 30-40 weeks of age(146). The development of diabetes is also strongly influenced by

environment. Mice housed and handled in an SPF environment are more likely to develop T1D

compared to mice in conventional housing. Multiple abnormalities in immune phenotype have









been identified in the NOD mouse including defective maturation of dendritic cells(147), age-

related decline in the function of regulatory T cells(148), deficient hemolytic complement

C5(149), defective natural killer T (NKT) cell function(150), and defective cytokine production

from macrophages(151).

Summary and Rationale

Type 1 diabetes is a debilitating disease that results in lifelong complications in spite of

continued insulin therapy. With a general lack of clinical success in interventions such as islet

cell transplantation, primary prevention or disease reversal holds the greatest hope for T1D.

There is evidence for the prevention and reversal of autoimmune diseases through tolerance

induction using both immature and mature DC as immunotherapy, but this strategy has been

limited to early intervention(85,152-154). As most human cases are not diagnosed until

established hyperglycemia, an intervention for late-onset prevention is highly desirable. Our

current work is focused on developing DC therapy for translation into the clinic setting, with a

focus on the role of peptide presentation in immune modulation. We also seek to better

understand the antigen-dependent and antigen-independent effects of DC therapy so that

clinicians can efficiently engineer DC for a variety of immune disorders ranging from

autoimmunity to cancer immunotherapy.









CHAPTER 2
ENGINEERING OF TOLEROGENIC DC

Introduction

Dendritic cells are a heterogenous population of cells with the capacity to induce divergent

T cell responses. The factors involved in the decision in whether DC direct T cell immunity or

tolerance remain to be fully elucidated as no definitive criterion have been irrevocably validated.

Anticipated DC function has been attributed largely to phenotypic characteristics on the basis of

maturation state. Generally, it is believed that immature DC (iDC) produce low levels of

inflammatory cytokines, readily uptake and process Ag, and induce T cell tolerance in the

absence of a costimulation signal. Upon maturation with stimulatory agents such as LPS or anti-

CD40, mature dendritic cells migrate to the draining LN, release pro-inflammatory cytokines,

up-regulate expression of costimulatory molecules, and present Ag to activate T cell immunity.

In clinical studies, iDC are preferentially chosen for therapies to induce tolerance, while mDC

are applied toward therapies for immune activation (155-157). However, some critics speculate

that iDC are in a physical state of dormancy, failing to migrate or interact efficiently with T cells

for any modulation of immune response, and thus may not translate to success in the clinical

setting(119,158). Others assert that iDC are an effective means to inducing Ag-specific T cell

tolerance(118,155).

As our understanding in DC biology continues to expand, researchers have developed

novel methods in the generation of DC that enhance their ability to induce T cell tolerance.

Several studies examined the use of TNF-a for in vitro stimulation of DC to generate a third

subclass of DC termed semi-mature DC (smDC). These cells produce low levels of pro-

inflammatory cytokines but express high levels ofIMHC and costimulatory molecules and have

been shown to be able to potently induce T cell tolerance to protect mouse models from









autoimmune disease (91,155,159). Others have sought to modulate DC using retinoic acid,

which resulted in the expression of gut-homing receptors that can direct tolerogenic DC to the

area of pancreatic inflammation where their action can be most beneficial(97,160). Additionally,

RA-stimulated DC have been found to induce Foxp3 expression on T cells(97,160,161).

Overall, these findings suggest that there are multiple strategies to develop tolerogenic DC.

As our goal is to engineer DC for translation into the clinical setting for the induction of

tolerance, we must first identify potential DC subsets that are safe (low risk of immune

activation) but effective (functionally potent) for tolerogenic therapy. To address these

questions, we examine the immature and semi-mature subsets of DC and characterize their

expression ofT cell costimulation markers and cytokine production. We also examine iDC

migration following subcutaneous injection as critics argued against their capacity to travel to

LN for effective T cell interaction. We then assess diabetes incidence in NOD following

treatment with iDC and smDC. Finally, we investigate how retinoic acid enhances DC

tolerogenic function for application in T1D. These studies reveal the target DC subset for use in

tolerogenic Ag-based studies, which is examined in subsequent chapters.

Materials and Methods

Animals

Female NOD/ShiLtj (NOD) and C57BL/6J (B6) mice were purchased from The Jackson

Laboratory or Animal Care Services at the University of Florida. Bone marrow donor mice were

5-8 weeks of age. Up to five mice were housed together in micro isolator cages in a specific

pathogen free (SPF) facility with access to food and water ad libitum. Mice were allowed to

acclimate to the facility for one week prior to the initiation of any studies. Three weekly footpad

injections of PBS or unpulsed DC (105 cells/mouse) were given to female NOD mice beginning

at 9 weeks of age. Development of diabetes was monitored through twice weekly urine glucose









testing using urine glucose test strips (Clinistix, Bayer). Upon detection ofglucosuria, a small of

amount of blood was collected by pricking the tail vein and testing blood glucose using the

Accuchek OneTouch glucose meter. A mouse giving 2 consecutive daily readings of blood

glucose greater than 250 mg/dl was considered to be diabetic. Retinoic acid studies were

performed in C57BL/6 mice. Mice were euthanized by CO2 asphyxiation. All mice were cared

for in accordance with the University of Florida Institutional Animal Care and Use Committee.

Bone Marrow-Derived Dendritic Cells: Isolation and Culture

The femur and tibia were removed from mice and cleaned of muscle and connective tissue.

The ends of the bones were cut and bone marrow (BM) cells were flushed out with media using a

25-5/8 gauge needle attached to a syringe. Red blood cells were removed from bone marrow

cells using ammonium chloride potassium (ACK) lysis buffer for 2 minutes at room temperature

then washed free of lysis buffer using PBS. BM-derived DC were cultured in RPMI 1640

(Cellgro) supplemented with 10% FCS (Invitrogen Life Sciences), lx penicillin/

streptomycin/neomycin (Gibco), and 10mM HEPES buffer (Gibco) at a concentration of 106

cells/ml in flat-bottom 6-well culture plates (Corning) inside a 370C humidified incubator with

5% CO2. 500 U/mL GM-CSF (R&D Systems) and 1000 U/mL IL-4 (BD Pharmingen) were

added to BM cultures to promote differentiation into DC. On day 2 or 3, half of the media was

replaced with fresh media and cytokines. TNF-a (R&D Systems) or LPS (Sigma) was used to

stimulate some cells at lOng/ml, or 0.5ug/ml, respectively, during the last 24h of culture. On day

5 or 6, cells were removed from the bottoms of wells with gentle pipeting and a cell scraper. DC

were purified using CD1 lc+ magnetic beads (MiltenyiBiotec). DC purity was determined by

flow cytometry on the basis of CD11 c+ expression.









Flow Cytometry

Cells were prepared into single-cell suspensions in FACS buffer (lx PBS / 1% FCS).

Antibody used to identify dendritic cells was CD1 c (HL3). Antibodies used to characterize DC

maturation were I-Ab (25-9-17), I-Ad(39-10-8, cross reacts with NOD I-Ag7), CD80 (16-10A1),

CD86 (GL1), and CD103 (M290) for the gut-homing receptor. Live cells were gated from dead

cells on the basis of forward/side scatter or with 7AAD (amino-antimycin D) labeling. Isotype

controls include mouse IgG3K, rat IgG2a, hamster IgG1K, and hamster IgGlI. All antibodies

were purchased from BD Pharmingen. FACS Calibur equipment (BD Pharmingen) was used to

collect flow cytometry data and results were analyzed using FCS Express (BD Pharmingen).

Cytokine Analysis

Supernatants from DC cultures were collected immediately prior to DC isolation for

cytokine secretion analysis. The quantity ofIL-10, IL-2, TNF-a and IFN-y was measured using

the Beadlyte Cytokine Detection System 1 Kit (Millipore). All measurements were run in

duplicate. Cytokine levels were analyzed using the Luminex instrumentation (Austin, TX) and

Upstate Signaling Beadlyte software (Charlottesville, VA).

Retinoic Acid

In vitro

BM-derived DC were cultured as described previously. Retinoic acid was used to

stimulate some BM cells at 10 ng/ml during the last 24h of culture.

In vivo

We considered a simple alternative to in vitro stimulation of DC by using a topical

application of an FDA-approved RA gel to condition the injection site microenvironment.

Tretinoin (Clay-Park), an all-trans retinoic acid gel prescribed for the topical treatment of acne

vulgaris, was applied to a 1" x 1" shaved region of skin at the nape of the neck of B6 mice. Mice









were placed under isoflurane anesthesia at 4%-5% for induction, 1%-2% for maintenance as

controlled by an anesthesia machine throughout the procedure. Mice were monitored for their

state of consciousness using toe/tail pinch or palpebral reflex. RA gel was applied to cover

exposed skin several hours before DC injection, once daily for up to 5 days including a "pre-

conditioning day" in which no DC injection occurred. At various time points post injection, we

collected cells from gut LN, draining axillary and cervical LN, and non draining inguinal LN to

examine for the presence CFSE+ cells.

Migration Studies

DC were labeled with 5'-6-carboxyfluorecein diacetate succinimyl ester (CFSE)

(Molecular Probes) at 10uM for 10-15 minutes in an incubator, then washed 3x with ice-cold

PBS to quench reaction. Cells were resuspended in PBS and injected into the footpads of 5-8wk

old NOD females (70,000 DC/mouse), or 8 wk old B6 mice (1 x 106 DC/mouse). Mice were

sacrificed at 24, 48, and 72h to track migration. Cells from the draining and non-draining lymph

nodes were harvested and analyzed by flow cytometry to detect for CFSE fluorescence.

Results

Immature Dendritic Cells Express Lower Levels of T-cell Costimulatory Molecules
Relative to TNF-a semi-mature and LPS-Stimulated Mature DC

To identify phenotypic characteristics of functionally tolerogenic DC, we first aimed to

produce and characterize three subsets of DC that were generated using various stimulatory

reagents. Evidence in the literature suggests that the addition of activating agents such as TNF-a

or LPS can induce maturation of DC subsets that are functionally and phenotypically distinct

(77,86,118,121,155). We cultured bone marrow precursors of NOD mice in the presence ofIL-4

and GM-CSF for 5-6 days, then purified the cells using CD1 Ic magnetic beads. We defined this

group of cells as immature DC. To some cells, we added TNF-a (10 ng/ml) or LPS (0.5 ug/ml)









during the last 24 h of culture for the development of semi-mature or mature DC, respectively.

The DC subsets were then characterized by expression of cell surface markers using flow

cytometry. We assessed DC for expression of maturation markers CD80 (B7.1) and CD86

(B7.2), which are known to be involved in T-cell costimulation through interactions with CD28

and CTLA-4 respectively. We also assessed expression ofI-A 7, the MHC class II molecule

expressed on NOD DC (using I-Ad cross reactive Ab) which interacts with TCR of CD4+ cells.

Up regulation of T cell costimulatory molecule expression on DC will provide the secondary

signal needed by T cells in conjunction with TCR engagement to promote T cell activation. As

shown in Fig. 2-1, we find that the unstimulated iDC subset was represented by a heterogenous

group of cells with some spontaneous maturation ranging from CD801 -medCD8610-hiI-Ad-lo. TNF-

a stimulated semi-mature DC were CD80medCD86medI-Ad-hi, while LPS-stimulated mDC were

uniformly found to be CD80hiCD86hil-Ad-hi. This confirms that immature DC express lower

levels ofT cell costimulatory molecules than semi-mature and mature DC, and suggests that iDC

may be less likely to induce immunogenic T cell responses.

Immature and Semi-Mature Dendritic Cells Produce Lower Levels of Pro-Inflammatory
Cytokine IFN-y Relative to Mature Dendritic Cells

Next we examined cytokine production in supernatants collected from DC subsets cultures

following 4-5 days of culture. As seen in Fig. 2-2, we show that mDC were robust producers of

pro-inflammatory cytokines IFN-y and TNF-a. On the contrary, immature DC produced

relatively marginal levels, with 10-fold lower IFN- y and 8-fold lower TNF-a relative to mDC.

Semi-mature DC also produced low levels of IFN-y (TNF-a was not included in this subset's

analysis since there was an external source confounder), with 5-fold lower secretion of IFN-y

relative to mDC. All DC subsets produced low levels of IL-10. These findings signify that iDC

and smDC are unlikely to promote an inflammatory response as they produce low levels of TNF-









a and IFN-y, while mDC may be biased toward a more inflammatory condition through its

substantial release of TNF-a and IFN-y.

Immature Dendritic Cells Migrate from Injection Site to Draining Lymph Node

Proponents of mDC for immunotherapy argue that iDC fail to efficiently migrate to

draining lymph node (dLN) to prime T-cells into a tolerogenic response (155,156). To verify

that iDC can migrate from injection site to draining lymph nodes, we injected 35,000 CFSE-

labeled iDC into each footpad of mice and harvested cells from draining and non-draining LN to

locate CFSE+ cells. Within just 24 hours, we were able to detect CFSE+ cells in the popliteal

dLN while no fluorescent cells were detected in non draining LN. We found 1.91% of a sample

of dLN cells were CFSE+ (Fig. 2-3). With 5 x 105 1 x 106 cells in a LN, we can estimate that

9550 19100 cells were CFSE+ in the total LN population suggesting that 27-55% of injected

iDC migrated to the popliteal LN, a proportion more than sufficient for peptide presentation and

T-cell priming.

Immature Dendritic Cells are Superior to Semi-Mature Dendritic Cells for Induction of
Tolerance

Given our findings from phenotypic analysis of the DC subsets, we chose to investigate the

functional potential of the iDC and smDC subsets for immunotherapy. To determine whether

iDC or smDC can prevent T1D in NOD mice with advanced insulitis, we administered 3 weekly

footpad injections of either iDC or smDC beginning at 9 weeks of age, then observed mice for

development of diabetes. As shown in the Kaplan-Meier survival curves in Fig. 2-4, neither iDC

nor smDC treated mice were significantly protected from T1D. However, we do note that there

was an initial delay in incidence of T1D in both groups of treated mice, followed by a drop in

protection in the smDC-treated mice while iDC-treated mice appear to be protected for a longer









period. We anticipate that while significant protection was not achieved using unpulsed iDC, the

addition of antigen peptide loading or other treatments may refine the tolerogenicity of iDC.

In vitro Retinoic Acid Stimulation Increases Expression of Maturation and Gut-Homing
Receptors on Dendritic Cells

Retinoic acid has been shown to have important immunomodulatory effects. RA can

induce or up regulate the expression of the gut homing receptor a407 (CD 103) as well as inhibit

T cells from differentiation into Thl7 cells, suggesting that RA can be used to augment the

immunomodulatory effects of DC therapy(92,94-97). To characterize the effects ofretinoic acid

stimulation on iDC, we added RA (10 ng/ml) during the last 24h of C57BL/6J BM cultures to

iDC and examined the cells for expression of maturation markers and the gut-homing receptor

CD103. In vitro RA stimulation increased the expression of gut-homing receptor CD103, with

0.36% and 1.02% of iDC and mDC expressing CD103+ compared to 2.49% of RA stimulated

cells (Fig. 2-5 panels A-C). Using iDC and LPS-stimulated DC as controls for comparison, we

also found that RA stimulation resulted in maturation of iDC as indicated by increased I-Ab and

CD80 expression (Fig. 2-5 panels D-F).

To facilitate the translation of RA stimulation into the clinical setting, we devised a simple

method of pre-conditioning the injection site microenvironment with a topical application of a

FDA-approved RA gel, thereby bypassing an in vitro GMP modification step of DC. We

investigated the efficacy of this procedure to exert similar immunomodulatory effects on the

injected DC by pre-conditioning the injection site skin for 1 day prior to DC injection, again

several hours before injection of CFSE-labeled DC, and again at 24 and 48 hours post-injection

to maintain the RA environment for residual DC, if any, remaining in the injection site. At 24

and 48 hours post-injection, we harvested cells from gut LN and spleen to examine for CFSE+









cells. Unfortunately, we were unable to detect CFSE+ cells in the spleen or gut LN (data not

shown).

Discussion

Dendritic cell therapy is an attractive approach to immunotherapy for T D as DC are

uniquely able to orchestrate the delicate balance between T cell immunity and regulation to self

antigens. However, the specific mechanisms as to how DC assume an immunogenic or

tolerogenic state has not been fully elucidated, and evidence suggests that their varying

immunomodulatory activity can be attributed to a variety of factors including expression of T

cell costimulatory molecules and production of Thl or Th2 pivoting cytokines. Ex vivo

engineering of DC provides clinicians with the opportunity to manipulate DC with a variety of

stimulatory agents that can modify or enhance constitutive DC function. We examined several

methods of generating bone marrow-derived DC for T1D therapy to identify the best tolerogenic

DC product. We found that iDC released low levels of TNF-a and IFN-y (Fig. 2-2). Semi-

mature DC also produced similarly low levels of IFN-y (TNF-a was not measured since an

external source was added for culture). Conversely, mDC were potent producers of the pro-

inflammatory cytokines TNF-a and IFN-y, releasing 5-10 fold higher levels compared to iDC

and smDC (Fig. 2-2). Only mDC exhibited consistent high expression of T cell costimulatory

molecules CD80, CD86 and MHC class II, while iDC and smDC expressed low to moderate

levels of these same maturation markers, respectively (Fig. 2-1). We also sought to provide

evidence for efficient iDC migration to draining LN following subcutaneous injection. Critics of

iDC speculate that in the absence of maturation, iDC have suboptimal migratory capacity that

may render insufficient DC-T cell interactions in LN. However, we found that 27-55% of

injected DC traveled to the draining LN within 24h, demonstrating that iDC were capable of









migrating to LN enabling interaction with T cells for potential priming into tolerance (Fig. 2-3).

Collectively this data highlighted iDC and smDC as DC subsets with therapeutic potential.

Next we examined the capacity for iDC and smDC to induce tolerance in vivo. We found

that iDC and smDC treatments were well tolerated in NOD mice and treatment did not

accelerated T1D despite the moderately elevated expression of costimulatory molecules in the

smDC subset. Unfortunately neither DC subset was able to protect NOD mice with advanced

insulitis from development of T1D, though it appeared iDC recipients benefitted from a

noteworthy delay in development of T1D (Fig 2-4). This data suggested that further refinement

in the engineering of iDC could prolong the durability of protection.

Lastly, we investigated the effect of retinoic acid stimulation on DC, which has been

shown to induce the expression of gut homing receptors in DC and inhibit Thl7 T cell

development. We first characterized DC phenotype following in vitro stimulation with RA and

found that RA enhanced maturation with up-regulation of I-Ab-hi and CD80med (Fig.2-5). RA

stimulation allowed a nearly 7-fold increase in the expression of the gut-homing receptor CD 103

relative to unstimulated DC, and a nearly 2-fold increase relative to LPS-stimulated DC.

However, the overall proportion of CD103 expressing cells within the total stimulated population

remains relatively small, thus the concentration of RA in culture may require adjustment in

future studies to improve this effect. We also examined whether RA microenvironment

conditioning could induce iDC homing to the gut LN, particularly the pLN where DC-T-cell

interactions may be optimally located to facilitate modulation of the pancreatic inflammation.

Unfortunately, we were not able to detect any CFSE-labeled DC in gut or draining nodes

following RA conditioning of the injection site microenvironment. This may be due to

inadequate cell harvesting techniques or a suboptimal concentration of RA in the injection site









microenvironment. Further dosing studies may be conducted to investigate whether higher RA

treatment can improve cell homing, though careful consideration must be given to balance its

advantages over the possibility of severe skin irritation.

Overall, we find that with low production of pro-inflammatory IFN-y and TNF-a, low

expression of costimulatory molecules and efficient migratory capacity, iDC have the greatest

potential for safe and effective application into DC therapies for tolerance induction. Of

importance, the observed delay in T1D development in NOD mice treated with iDC highlights

the therapeutic potential of this DC subset. We also find that the modest improvement in DC

expression of CD103 with RA stimulation (including additional effect on induction of Foxp3+

Tregs with RA-DC is described in chapter 5) suggests that RA stimulated-DC may be worthy of

further characterization in tolerance studies. In subsequent chapters, we examine how to develop

better engineer iDC therapy for translation into late intervention using subclasses of Ag peptides

for DC pulsing that may confer significant T1D protection.



















CDM CIM


U 10 '


Ia'rl
wH lcu~


NJ fl


Figure 2-1. Characterization of DC by flow cytometry. Bone marrow-derived DC from 6 week-
old female NOD mice were cultured without activation agents (immature iDC), or in
the presence of TNF-a (semi-mature smDC), or LPS (mature mDC) and examined for
expression of maturation markers. Black line = isotype control, red line = iDC, green
line = smDC, and blue line = mDC. Data shown is representative of>5 similar
assessments by flow cytometry.


29001

28001
12501T
11001


M


U


=I iDC
S smDC
- mDC


IFN-g TNF-a IL-10


Figure 2-2. Luminex analysis of cytokine production by DC subsets. Supernatants collected
from NOD bone marrow-derived DC cultures were analyzed for the presence of
cytokines following 5-6 days of culture. iDC = immature DC, smDC = semi-mature
DC, mDC = mature DC *TNF-a data was not reported for smDC since an external
source was added for its culture.


z S

I,.
*'/ ", -.
.. ..
\: A i \
> fAV SM^ 1 '
i. \ w *' A


E m B












popliteal LN


U&4 -i r-----i -----
0.1% 0.01 % 0,06
768

512 7 ____B_____________

256 0.09/o '
1.91i

1id id 1 ? id CS 1E i
> CFSE


Figure 2-3. Migration of iDC. CFSE-labeled iDC were injected into footpads of NOD mice
(35,000 cells per footpad). At 24 hours following injection, cells were harvested from
non-draining cervical LN and draining popliteal LN and analyzed by flow cytometry
to detect for CFSE+ cells. Data shown is representative of results from 2
experiments.


100
80


- PBS(N=10)
--iDC (N=10)
-- TNF (N=14)


8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
age (weeks)

Figure 2-4. Diabetes incidence in NOD mice following DC therapy. Female NOD mice received
three weekly footpad injections of PBS, immature DC, or TNF-stimulated semi-
mature DC beginning at 9 weeks of age. A mouse was considered to be diabetic
following two consecutive daily measurements of blood glucose > 250 mg/dl. Red
arrows depict time of DC injection.


cervical LN














0.36









Forward Scatter



6.85% 9.A6%








80;, ;2 3.45%'

-- CD80


249

g:


1L45 36.~ 4%.


17.45% .3.6.4 %,








41.62% 4.70%


1.02




,-' -~.-:
-.~ ~
* *..


Figure 2-5. Effect ofretinoic acid on dendritic cell maturation. Expression of the gut-homing
receptor CD103 was examined in CD1 lc+ gated C57BL/6J bone marrow cultures of
A) unstimulated cells, B) RA-stimulated cells, and C) LPS-stimulated cells. CD103
positivity was determined by isotype control. Maturation markers were examined in
D) unstimulated cells E) RA-stimulated cells and F) LPS-stimulated cells by flow
cytometry. Cells analyzed are collected from Day 6..









CHAPTER 3
ROLE OF ANTIGEN PEPTIDE IN DC THERAPY

Introduction

Though antigen-based DC therapies for T1D have been shown to be effective in the NOD

mouse, many depend on a restricted regimen in which the timing for the initiation of therapy is

critical to the disease outcome (162). Since the immunopathology of T1D develops early and

ensues for a significant period before clinical symptoms of diabetes manifest, early intervention

may not always be possible. Additionally, individuals without family history of T1D are

unlikely to undergo screening for early prevention. Thus, a reliable immunotherapeutic approach

that can be initiated within a wider range of time for late-onset prevention or established-disease

reversal is highly desirable.

Traditional antigen-based DC therapies have focused on identifying target autoantigens or

peptide epitopes within self-Ag that are dominantly-expressed and are primary targets of the

autoimmune response. These whole antigens or dominant determinants (DD) are then

administered in modalities that induce tolerance, and this treatment has been shown to prevent

T1D as well as other autoimmune diseases in mice when applied early in the autoimmune

process. Unfortunately, this strategy becomes ineffective when administered at advanced stages

of T1D, suggesting that there may be an altered immune repertoire with time (103,144).

Dominant determinants within an autoantigen are preferentially recognized by autoreactive

T-cells in the early disease stage and primed into effector function, contributing to the disease

process. Subdominant determinants (SD), which do not compete as effectively as DD, and

ignored determinants (ID), which are not processed and presented at all, are not believed to be

involved in the pathogenic T cell pool and thus are not used in Ag therapies. However, it may be

possible that DD continuously recruit naive T cells into spontaneous autoimmune attack while









SD and ID, which minimally or do not activate naive T cells, have a minimal effect on the naive

T cell pool (144,145). Thus, DD-reactive cells are progressively exhausted from the naive pool,

while uncommitted naive T-cells remain available to be primed into regulatory function by SD

and ID even at later stages of autoimmunity.

We aimed to examine how using different classes of determinants in Ag-based DC therapy

can affect disease outcome when the treatment is initiated after the autoimmune process is well

established. We begin treatment using DC pulsed with synthetic peptides of DD, SD, or ID in 9

week-old NOD mice with advanced insulitis. By using DC pre-loaded with synthetic peptides,

we can reduce peptide competition in vivo and bypass natural antigen processing to selectively

present the desired Ag epitopes.

Materials and Methods

Animals

Mice were cared for as described in the Materials and Methods section of Chapter 2. Mice

in short-term treatment studies were treated with one DC injection per week for three weeks,

then observed for development of diabetes. Mice in long-term treatment study received once

weekly DC injections up until euthanized for organ harvest. All mice received 100,000 DC per

injection, divided into 50,000 DC per footpad. Cells were suspended in PBS for injection.

Peptides

Peptides were purchased from Peptides International (Louisville, KY) and Bio-Synthesis,

Inc. (Lewisville, TX) and determined to be of> 90% purity by HPLC analysis. All peptides are

tested to be endotoxin-free. Lyophilized peptides were dissolved in RPMI media at 1mg/mL,

then sterile filtered using a syringe apparatus (Gibco). Once resuspended in media, peptides

were stored at 40C as a working solution for up to 2 months. Lyophilized peptides were stored at

-200C indefinitely. Dominant determinants used were insulin 39-23 (SHLVEALYLVCGERG),









GAD65217-236 (EYVTLKKMREIIGWPGGSGD), and proinsulin C19-A3

(GSLQPLALEGSLQKRGIV). Subdominant determinant used was GAD6578-97

(KPCNCPKGDVNYAFLHATDL). Ignored determinant used was GAD65260-279

(PEVKEKGMAALPRLIAFT SE).

Dendritic Cell Peptide Pulsing

DC were pulsed with 3uM of peptide in cRPMI for 1-2h in a humidified incubator 370 C

with 5% CO2. Peptide-pulsing concentration and duration were determined in previous

experiments (unpublished data). Cells were washed 3x and resuspended in lx PBS at 106

cells/mL for injection.

Histology

NOD pancreata were immediately removed from the mouse following CO2 asphyxiation.

Each pancreas was dissected along its longitudinal axis to preserve its anatomical asymmetry and

distributed for fixed and frozen processing. For fresh samples, pancreas were embedded in

Tissue-Tek OCT (Bayer) and stored at -800 C until immunostaining. For fixed samples,

pancreas were fixed in 4% paraformaldehyde at room temperature overnight, then transferred to

PBS. Further tissue processing, including sectioning and staining, were performed by the

University of Florida's Molecular Pathology and Immunology Core. Briefly, sections through

the fixed-pancreas were paraffin-embedded, sectioned and collected 100 micron apart, then

stained with H&E. Six non-adjacent tissue sections from each animal were used for insulitis

grading and islet number enumeration. Islets were visualized by light microscopy at 40x, and

were counted and graded blindly by a single observer. Grading scores were assigned as follows:

0 = no infiltration, 1 = peri-insulitis, 2 = <50% intra-insulitis, 3 => 50% intra-insulitis.









ELISA for Global Suppression Analysis

Eight-week old female NOD mice received PBS, unpulsed, or peptide-pulsed DC

injections as described previously, once weekly for three consecutive weeks. One week

following the last injection, mice were immunized in the footpad with 100ug/mouse of KLH

(Calbiochem) in Alum (Pierce) weekly for two weeks. Ten to fourteen days following the final

KLH immunization, serum was collected from mice for the detection of antibodies to KLH by

ELISA (Life Diagnostics).

Statistical Analysis

Data was analyzed using the Kaplan-Meier survival curve with log-rank test to determine

if treatment provided protection. Student's t-test was also used to identify statistical differences.

The Grubbs test identifies outliers in triplicate wells of proliferation assays. A criterion of

p<0.05 was used to define significance.

Results

Subdominant Determinant-Pulsed DC Therapy Protects Against T1D

Work by Kaufman's group demonstrated that DD are ineffective for tolerance induction

when applied as peptide therapy during advanced insulitis. We examined the use of DD from the

insulin, pro-insulin, and GAD molecules as peptide-based DC therapy and confirmed that DD

were not protective in the context of iDC when given to older NOD mice (data not shown). Thus

we next sought to assess the potential of using other classes of determinants. Subdominant

determinants, which do not compete as competitively with DD for processing and presentation,

may have a larger repertoire of naive T cells available for priming. By pre-loading DC with SD

before infusion into mice, we can bypass peptide competition in vivo and allow these epitopes to

be presented more efficiently to evoke tolerogenic responses from naive T cells. We gave three

weekly treatments of either PBS, unpulsed DC, or SD-pulsed DC to 9 week-old NOD mice and









observed for development of T1D. As shown in Fig. 3-1, we found that recipients of SD-DC,

but not PBS or unpulsed DC, were protected from T1D (p=0.05). Interestingly, we noted that

SD-DC were able to significantly delay T1D in 100% of SD-DC recipients through the 17th

week of age while 40% of PBS controls were diabetic. This suggests that complete protection

was conferred for over 5 weeks following administration of the last DC treatment, however the

protection was not durable for the life of the animal.

Pancreatic Islet Survival is Enhanced in Subdominant Determinant-Pulsed DC Recipients

Though SD-DC were not able to completely protect mice from T1D, we were encouraged

to learn that SD-DC delayed development of diabetes and that protection was significant. To

better understand how DC therapy affected SD-DC treated mice, we harvested pancreas

following treatment and compared islet survival to mice that had received unpulsed or DD-

pulsed DC. We found that while the grade of T cell infiltration into islets was similar in all

treatment groups of mice (data not shown), there was a consistently larger number of islets

remaining in mice receiving Ag-pulsed DC therapy (Fig3-2). Specifically, non-diabetic mice in

both Ag-DC treated groups had a greater number of islets compared to non-diabetic unpulsed DC

mice. Diabetic mice receiving SD-DC had a greater number of islets compared to the unpulsed

DC group. Of interest, diabetic SD-DC treated mice had an average islet number similar to that

of non-diabetic unpulsed controls.

It is known that beta cell regeneration, while slow, does occur in the presence of the

autoreactive T cell response that continuously destroys islet tissue. However, a balance in favor

of regulatory T cells versus immunogenic T cells may be able to push the overall response

toward tolerance. It is possible that the islet preservation observed was achieved through the

induction of regulatory T cells that was enhanced with Ag-pulsed DC treatment, and to a greater

extent with SD-pulsed DC treatment, but was not enough to completely quench the inflammation









generated from the pathogenic T cells. The issue of regulatory T cell induction by DC therapy

will be further discussed in Chapter 5.

Ignored Determinant-Pulsed DC Therapy Confers Complete Protection Against T1D But
May Require Continuous Treatment

Subdominant determinant-pulsed DC treated mice were protected from T1D as

demonstrated by a significant delay in disease onset. However, complete protection would be

ideal in the clinical setting. As ignored determinants do not naturally elicit T cell responses, we

hypothesized that a larger pool of naive T cells remain available for priming into tolerance

compared to SD, which generates a moderate T cell response. This advantage in available naive

T cell pool size may translate into better protection. Therefore, we performed another study

using ID-pulsed DC in 9 week old NOD mice with advanced autoimmunity. We administered

three weekly injections of PBS, unpulsed, or ID-pulsed DC to mice and observed them for the

development of T1D. Surprisingly, we found that ID-DC treatment was not able to significantly

protect mice from T1D though we did observe an initial delay in T1D development all (Fig 3-3).

We speculated whether this was due to a lack of constitutive presentation of the ID that is needed

to maintain a regulatory cell response, so we refined this study to include repetitive injections

that allowed for consistent presentation of the normally unpresented determinant. As shown in

Fig 3-4, we found that continued treatment of mice with DC presenting the ID confers complete

protection up to the 17th week of age while other groups became diabetic (p=0.04).

T1D Specific Peptide-Pulsed DC Therapy Does Not Alter Natural Immunity to
Environmental Challenge: KLH Study

Because we observed an initial delay in development of T1D in all mice receiving DC

therapy, it is uncertain whether the apparent DC-induced protection against T1D is actually due

to an overall dampening of the immune response. We sought to evaluate whether DC therapy

confers specific protection against T1D, or whether the observed protection was an artifact of









global immunosuppression that renders mice tolerant to all immune challenges. We aimed to

test this by evaluating the ability of DC-treated mice to respond to a non-T1D specific challenge.

We administered either PBS, unpulsed, or ID-pulsed DC therapy as described previously, then

immunized the mice with keyhole limpet hemocyanin (KLH), a protein commonly used to

examine and elicit immune responses. Two weeks following KLH immunization, we collected

serum from the treated mice to detect if an antibody response was mounted against KLH. As

shown in Fig 3-5, there were no differences in the ability ofDC-treated mice to generate an

antibody response to KLH challenge as compared to PBS treated mice, demonstrating that

normal immune processes were intact and the protection previously observed can be attributed to

T1D specific protection.

Discussion

Type 1 diabetes is a dynamic autoimmune disorder characterized by T cell-mediated

destruction of pancreatic islets driven by an expanding T cell autoreactivity toward beta cell

autoantigens. Published studies using Ag-based DC therapy commonly aimed to identify the

target antigen of the immune response and re-administered peptide determinants of those

antigens that were found to elicit the pathogenic response using tolerogenic DC. However, the

efficacy of this strategy deteriorated if initiation of treatment was delayed until advanced stages

of the autoimmune process, suggesting an altered immune repertoire with time. We speculated

that dominant determinants identified to be the initiators of the autoimmune response chronically

recruits naive T cells into the pathogenic pool, thus the re-administration of these determinants

only reactivated cells that were programmed to respond pathogenically. However, subdominant

or ignored determinants, which have a minimal impact on naive T cell activation, should have

large pools of naive T cells available for priming into tolerance when we bypass natural antigen

processing to experimentally present these peptides. To test this idea, we treated 9 week old









NOD mice with advanced autoimmunity using various classes of antigen determinant-pulsed DC

and evaluated protection from T1D. We found that DD DC were not able to protect mice when

treatment was initiated during advanced autoimmunity. However, recipients of SD-DC and ID-

DC were significantly protected from T1D with a delay in initial onset of diabetes, though ID-

DC required continued treatment to achieve complete protection. This requirement may be due

to a lack of constitutive ID-presentation necessary to maintain the pool of regulatory cells. This

theory is further reinforced by the histological observation that SD-DC treated mice had greater

islet survival compared to other groups that were not protected. When we examined the islets of

mice treated with unpulsed, DD, and SD-pulsed DC (this analysis was performed before

initiation of ID-DC study), we found that non-diabetic mice receiving Ag-pulsed DC therapy had

a greater number of islets compared to unpulsed DC recipients. Diabetic SD-DC treated mice

had a significantly greater number of islets compared to the diabetic unpulsed DC group

(p=0.02). Of interest, diabetic SD-treated mice had a similar average islet count to non-diabetic

unpulsed mice. The grade of islet infiltration was similar in all treatment groups, though it is

unclear whether the observed infiltrate were regulatory or inflammatory T cells. Cell subset

specific antibody-staining of pancreas in future studies will enable identification of these

lymphocytes.

To exclude the possibility that the observed protection from T1D was due to global

immunosuppression, we examined whether NOD mice could generate a normal immune

response to a non-T1D related antigen challenge following treatment with DC therapy. We

immunized PBS, unpulsed DC, and ID-DC treated mice with KLH and examined their serum

antibody responses. All DC treated mice were able to mount antibody responses to KLH in a









manner comparable to PBS controls, demonstrating that normal immune responses were intact

and the previously observed protection could be attributed to diabetes-specific protection.

Overall, these results provide evidence for the existence of a relationship between native

determinant immunogenicity and developing immune repertoires in dynamic Ag-based disorders

that was previously unknown. These findings suggest that the selection of autoantigen peptide

for use in therapies aimed to prime naive T cells has a critical impact on the efficacy of

protection against dynamic autoimmune diseases. Thus, special measures should be taken to

consider the factors of the timing of treatment initiation, native peptide immunogenicity, and the

state of naive T cell repertoire in the design of Ag-based therapies. As our results indicated, SD

and ID may be best for induction of tolerance when treatment is initiated after established

autoimmunity. Taken together, these findings can guide the engineering of Ag-based DC

therapies for the prevention of TD and other dynamic autoimmune diseases.














100


So


u00


40


20


0


t t.T*
Z LZ

tft~
rC.
|o
- *
"S X
^ ^


p = 0.05
p= 0.36


-- PBS (N=1o)
Sun Ipulsed I)C (N=1o)
SD-GADI D (N=lo)


I ) i ....t.......t........i J J


8 9 10 11 12 13 14 15 16 17 8 19 20 21 22 23 24 25 26 27
age (weeks)


Figure 3-1. Kaplan-Meier survival curves of female NOD mice following PBS, unpulsed, and
SD-pulsed DC therapy. Red arrows denote time and frequency of DC treatment. P-
values represent difference compared between PBS and DC treatment groups.


60-




. 40-


20
e 20-
-


- diabetic
1= non-diabetic


unpulsed DC DD DC SD-DC
N=7 N=9 N=8


Figure 3-2. Histological assessment of islet number in NOD mice following DC treatment.
Pancreas were fixed in 4% paraformaldehyde and paraffin embedded. 4um sections
were collected at 100um intervals, then stained with H&E. All pancreas analyzed
were collected from mice ages 19wks+. Islets were counted blindly by a single
observer. (p-value represents comparison to diabetic unpulsed DC group)

















100


8o


-- PBS (N=12)
--u--pulsed DC (N-l2)
-- ID-GAD DC (N-12)


40


20 -


0 r r I I I I t I I I 1 I I I I I 1 I I 1 1
7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41
agI" (weeks)


Figure 3-3. Kaplan-Meier survival curves of female NOD mice following short-term PBS,
unpulsed, and ID-pulsed DC therapy. Red arrows denote time and frequency of DC
treatment. DC treated mice were not significantly protected from T1D.


So W
o a
<,o _-_-


40


20 PBS (N 4)
--ui pulsed DC (N-4)
-- ID-GAD DC (N=4)

8 9 10


t\\

-,
t


11 12 13


C f


14 15 16 17


age (weeks)



Figure 3-4. Kaplan-Meier survival curves of NOD mice following continuous PBS, unpulsed,
and ID-pulsed DC therapy. Red arrows denote time and frequency of DC treatment.
P-values represent difference compared between PBS and DC treatment groups.










70
60

= 50
40

30
S20
10
0
PBS unpulsed DC ID-pulsed DC

Figure 3-5. Antibody response following KLH immunization in control and DC treated mice.
NOD mice were treated with 3 weekly injections of either PBS or DC (N=3/group).
Two weeks following DC therapy, mice were immunized with 2 weekly injections of
KLH. Serum antibody levels were assessed 14 days following final KLH
immunization.









CHAPTER 4
ANTIGEN-INDEPENDENT EFFECTS OF DENDRITIC CELL THERAPY

Introduction

Because dendritic cells are potent antigen presenting cells, most studies use DC to augment

Ag-specific immune responses and thereby assess only Ag-specific modifications following DC

therapy. Animal studies using DC therapy for tolerance induction have primarily examined

deviation of immune responses toward the antigen of interest. Much of the knowledge on how

DC therapy affects immune responses in humans has been gained from clinical studies that are

directed toward the induction of antigen-specific immunity for the treatment of cancer. In the

scope of translation of DC therapy from animal to human for the treatment of autoimmune

disease, little is actually known as to how the overall immune response is affected.

Consequentially, there is a gap in knowledge in how DC therapy contributes to antigen

nonspecific responses. Additionally, studies using unpulsed DC have demonstrated protection

from autoimmunity as well, raising the question of what role antigen loading plays in DC

mediated immune responses (85). We sought to address these questions by evaluating antigen

independent immune responses following DC therapy. We examined spleen cell responses in the

presence and absence of TD-specific peptide stimulation in proliferation assays, characterized

early and late changes in immune cell subset frequencies and function using flow cytometry, and

assessed whether these DC-related changes occur in non autoimmune prone mouse strains.

Following identification of the immune modulation in vitro, we sought to confirm the findings in

vivo using BrdU treatment of mice. Finally, we collected RNA from spleen cells of mice to

perform gene array analysis of differential expression of cytokines and receptors genes. These

investigations will provide insight into what immunological changes occur with DC therapy, and

provide evidence for the mechanisms that may be driving these changes.









Material and Methods


Mice

Mice were treated as previously described in the Materials and Methods sections of

Chapters 2 and 3 unless otherwise noted.

Proliferation Assay

Suspensions of spleen cells were in serum-free HL-1 media (Biowhittaker Cambrex) with

the addition of penicillin/streptomycin/neomycin (Gibco), and L-glutamine (Gibco) in triplicate

with a selected peptide (25uM). Cells were cultured at 1x106 cells/well in round-bottom 96-well

plates at 370C. At 72h of culture, 1 [iCu 3H-thymidine (Amersham Biosciences) in 50 [l of

media was added per well and allowed to incorporate for 12-16h. Cells were harvested and

washed using an automated cell harvester (Perkin Elmer), and radioactivity was analyzed using a

liquid scintillation counter. Stimulation index was determined as proliferation (measured in

cpm) in response to peptide stimulation relative to proliferation without stimulation. Cpm

outliers identified by Grubb's test were removed from analysis.

Flow Cytometry

Cells were prepared into single-cell suspensions in FACS buffer (lx PBS / 1% FCS) and

blocked in Fc Block CD16/32 (2.4G2). Antibody used to identify dendritic cells was CD1 c

(HL3). Antibodies used to characterize T cells were CD3(145-2C11), CD4 (RM4-5), and CD8a

(53-6.7). Antibodies to characterize B cells were B220 (RA3-6B2) and CD19 (1D3). We also

used CD25 (PC61) and Foxp3 (FJK-16s) to assess regulatory T cell population, CD1 lb (M1/70)

to assess macrophages, and CD44 (IM7) and CD62 (MEL-14) to assess memory T cells, CD138

(281-2) for plasma cells, and CD80 (1610-A1) and CD35 (8C12) for memory B cells. Live cells

were gated from dead cells on the basis of forward/side scatter or with 7AAD (amino-antimycin

D) labeling. Isotype controls include mouse IgG3K, rat IgG2a, hamster IgG1K, and hamster









IgGlk. All antibodies were purchased from BD Pharmingen or eBiosciences. FACS Calibur

equipment (BD Pharmingen) was used to collect flow cytometry data and results were analyzed

using FCS Express (BD Pharmingen).

BrdU Study

BrdU is a synthetic nucleoside that is an analogue ofthymidine that will replace thymidine

during DNA replication and is commonly used in the detection of proliferating cells in living

tissues. To assess in vivo proliferation, we administered daily intraperitoneal injections of BrdU

(bromodeoyxyuridine) in sterile PBS (2mg/100ul/mouse) to mice (treated with PBS or DC as

previously described) for 4 days, then sacrificed the mice 1-2 days following final injection to

harvest organs, LN and spleen cells for analysis of BrdU incorporation. Mice with long-term

exposure to BrdU at this dose have a reported reduction in weight gain and exhibit general

malaise. Mice with short-term exposure (<1 week) to BrdU do not suffer from significant

distress. We monitored mice daily to observe for signs of distress such as lethargy, dehydration,

decreased grooming and mobility.

Histology

Spleens, livers, and pancreata were fixed in 10% formalin at room temperature for 24-48

hours. Tissues were embedded in paraffin and sectioned at 4 um for staining using anti-BrdU-

HRP Ab and DAB detection, and counterstained with hematoxylin. Two sections per sample

were collected 100 micron apart for analysis using Aperio's Spectrum ScanScope imaging

software. The frequency of BrdU positive cells was determined using ScanScope's image

analysis algorithm that detects positively stained cells on the basis of programmed color and

saturation sensitizers within a measured tissue area. Percent BrdU positive is calculated as area

positive/area total.









RT-PCR and Gene Array

RNA was isolated from the spleen cells of treated mice using the Ambion RNAcqueous-

4PCR isolation kit. Cells were disrupted in lysis/binding solution followed by an equal volume

addition of 64% ethanol, then purified through a filter column and washed 3x. Purified RNA

was eluted into nuclease-free water with DNAse buffer and RNAse inhibitor (Ambion). RNA

samples were submitted to the University of Florida ICBR to determine RNA purity and

concentration on the basis of RNA integrity number using the Agilent Bioanalyzer. RNA

concentrations were then standardized and prepared into cDNA using the Applied Biosystems

Taqman RT Reagents kit. cDNA was amplified using primers designed by the Lonza Gene

Array Mouse Common Cytokines and Receptors kit with SYBR Green Fastmix (Quanta) for

quantitative PCR analysis. Data was sent to Bar Harbor for analysis using their proprietary

software for Global Pattern Recognition, which uniquely determines fold change with respect to

data consistency, and normalizes experimental data on the basis of relative gene expression

rather than relying on a predetermined housekeeping gene.

Results

Homeostatic Proliferation is Observed Following DC Therapy: Immediate and Sustained
Effects

The spleen is a major site of immune cell interactions and antigen processing, with active

processes that contribute to the overall immune status(163,164). Thus, we sought to examine

cellular responses in this immune cell rich environment. To evaluate the spleen cell response

following DC therapy, we cultured spleen cells with and without peptide stimulation for 72 hours

then observed for proliferation using 3H-thymidine incorporation. We found that in the absence

of peptide stimulation, spleen cells isolated from DC treated mice had 3-14 fold increase in

proliferation compared to PBS treated mice (Fig. 4-1). This effect of spontaneous proliferation









was enhanced in mice receiving Ag-pulsed DC but did not increase with recall peptide challenge.

The proliferation was seen as soon as just 2 weeks following the last DC treatment at 14 weeks

age (Fig. 4-1A) and continued into 40 weeks of age, 29 weeks after the cessation of treatment

(Fig4-1B).

Homeostatic Proliferation Occurs in Healthy and Autoimmune Mouse Strains Following
DC Therapy

Because NOD mice have been shown to have abnormalities in the immune function of

many cell types including differences in DC phenotype and function (147,165-168), we

evaluated whether this homeostatic proliferation was a true effect of DC therapy or only an effect

associated with immunotherapy in an animal with aberrant immune cell subsets. We

administered DC therapy to the autoimmune NOD mouse model as well as the C57BL/6J and

Balb/c healthy mouse models and evaluated spleen cell proliferation. As depicted in Figure 4-2,

homeostatic proliferation following DC treatment occurs in both NOD and non-autoimmune

prone mouse models, demonstrating that DC therapy results in a reprogramming of immune cell

homeostasis. Additionally, this pattern is independent of route of administration, as B6 mice

were treated with intravenous tail vein DC injections while NOD and B6 mice were given

subcutaneous injections.

Identification of Proliferating Cell Populations

Next, we sought to distinguish proliferating cell subsets by labeling spleen cells with CFSE

in the proliferation assays. Following 72-84 hours of culture, we harvested the cells and labeled

them using fluorescent antibodies for flow cytometry. We used surface markers to identify

dendritic cells, macrophages, plasma cells, memory T and B cells, NK cells, and T and B

lymphocytes. Of the cell populations examined, we found that the majority of the proliferating

cells belong to the CD4+ and CD8+ T and B cell subsets (Fig. 4-3), collectively accounting for









72-98% of the proliferation (percentages shown in red). Other subsets comprised a small

percentage of the proliferation population (data not shown). While DC-treated mice exhibit

greater proliferation of spleen cells relative to PBS controls, the proportion of proliferating cell

subsets remain similar. Importantly, these findings remained consistent over the course of the

study ranging from assessments between 13-41 weeks of age.

DC Therapy Reprograms Cytokine Production of Proliferating T Lymphocytes

We have found that homeostatic proliferation occurs immediately after DC therapy,

remains durable long after cessation of treatment, and affects both autoimmune and healthy

mouse strains. Interestingly, the spontaneous proliferation is also independent of disease

outcome as both diabetic and protected mice demonstrate this effect. Thus, it is unclear what

impact this immune modulation may have in disease. To investigate whether these proliferating

cells have altered function, we evaluated the cytokine production of the proliferating

lymphocytes. We gated proliferating cells on the basis of CD4 and CD8 positivity and examined

their IL-10 and IFN-g production. We find that T cells from all DC recipients exhibit a shift

from IFN-g to IL-10 production. As shown in Fig. 4-4, upper left quadrant statistics show a 2-

fold increase in IL-10 and 2-fold decrease in IFN-g in CD4 cells, and a similar change in CD8

cells. No changes in cytokine production between control and DC-treated mice were seen within

the B cell population.

DC Therapy Results in Differential Gene Expression: Analysis of Cytokine and Chemokine
Genes

To understand what may be driving the proliferation, we performed a gene array analysis

examining the expression of common cytokines and receptor genes that may have a role in

directing immune cell development and function. We treated groups of mice (N=4/group) with

PBS, unpulsed DC, SD DC, and ID-DC and performed proliferation assays as described before,









then isolated RNA from the cultured cells. Following RT-PCR transcription of RNA into cDNA,

we performed qPCR to determine gene expression. We assessed differences by comparing PBS

and DC-treated mice as this is where we had observed differences in proliferation. We also

sought to detect gene expression changes between PBS and ID-DC treated mice as our earlier

studies found significant protection in this group. Lastly, we also examined for differences

between unpulsed DC recipients and antigen-pulsed DC recipients since the loading of antigen

appeared to enhance the immunomodulatory effects. We identified notable, but not statistically

significant changes in the expression ofIL-17f, CXCL12, and CCL12 within these comparison

groups (Fig. 4-5). Changes for only these genes that received both the highest ranking for each

analysis and a low p-value are described in detailed. As seen in Fig. 4-5, we found that

expression of IL-17f was reduced 2 to 11-fold in DC-treated mice. IL-17fis a pro-inflammatory

cytokine that is mainly produced in the spleen and has been found to have a role in autoimmunity

and Type 1 diabetes (169-171). Specifically, mice receiving DC therapy exhibited a 2.3-fold

reduction in expression of IL-17f compared to PBS controls (p=0.081) (Fig4-5A), while mice

receiving ID-pulsed DC exhibited an over 11-fold reduction (p=0.056) (Fig4-5D). These results

suggest that the reduction of pro-inflammatory IL-17 is not only correlated with homeostatic

proliferation of spleen cells, but also contributes to protection as ID-pulsed DC treated mice were

protected from T1D.

We also discovered differences between unpulsed and antigen-pulsed DC treated mice.

Mice receiving an antigen pulsed-DC had a 3.8-fold increase in expression of CXCL12

compared to unpulsed DC recipients (P=0.071) (Fig. 4-5F). CXCL12, also known as SDF-1, is a

chemokine that has a role in the generation and proliferation of early B cell progenitors, as well

as a role in T cell and hematopoietic stem cell (HSC) trafficking(172,173). Additionally, we









found that mice receiving SD-DC exhibited an 18.9-fold increase in expression of CCL12, also

known as MCP-5, compared to unpulsed DC recipients (p=0.056) (Fig. 4-5F). CCL12, the

ligand for CCR2, is a chemoattractant for eosinophils, monocytes, fibrocytes and macrophages

(174-176). CCL12 is constitutively expressed in thymus and lymph nodes, but under

inflammatory conditions its expression is induced in macrophages and mast cells and has a role

in allergic inflammation. Additional changes in expression of various genes were also noted

between other group comparisons, though not statistically significant (Fig. 4-5A-F).

In Situ Observation of Homeostatic Proliferation

The homeostatic proliferation observed may be driven by conditions that are only favored

in culture. To determine whether there is spontaneous cell proliferation in vivo, we treated mice

with BrdU, a synthetic thymidine analogue that is incorporated into the DNA of replicating cells,

and collected spleens to detect for BrdU incorporation. Spleens were fixed, sections, and stained

for the presence of BrdU. Using an image analysis software program that detects BrdU+ cells

within defined tissue areas, we were able to calculate percentages of proliferating cells. We

found that there was no difference in percentage of cells proliferating in mice treated PBS

compared to DC-treated mice (Fig. 4-6A), suggesting that the homeostatic proliferation only

occurs in vitro. Additionally, the percentages of BrdU+ proliferation in the spleen determined

using an image analyzer fell within expected ranges for normal mice.

To better understand potential differences between in vitro and in vivo proliferation, we

examined spleens sections to identify sites of proliferation within the splenic architecture. The

spleen has distinct morphological zones with discrete functions. The two main compartments are

the red pulp and the white pulp. The red pulp is an area for the filtering and cleaning of damaged

erythrocytes and foreign debris from blood and acts as a storage site for erythrocytes, platelets,

and iron(177). Hematopoietic stem cells, lymphocytes, and macrophages can also be found in









the red pulp. The white pulp is a major site of immune interactions where a quarter of the body's

lymphocytes resides along with DC, macrophages, and plasma cells, and consists of the

periarteriolar lymphoid sheath (PALS), the follicles, and the marginal zones. We find that the

majority of the BrdU+ cells are located within the red pulp, with some positive cells within the

germinal centers of PALS, where CD4+ cells are predominant. This distribution of cellular

proliferation was consistent between mice in all groups. A representative section is shown in

Fig. 4-7.

Because our earlier findings had shown that SD-DC treated mice had a greater number of

islets compared to PBS-treated mice, we sought to determine whether there was evidence of

increased beta cell regeneration. We examined pancreata and found no differences in BrdU

incorporation (Fig. 4-6B). As expected, BrdU incorporation in control liver tissue was also

similar in all treatment groups (Fig. 4-6C).

Discussion

Classical dendritic cell therapy has been based on antigen presentation to direct the

immune response in an antigen specific manner. Few studies have investigated the potential of

using unpulsed DC in immune modulation as DC are generally hailed for their ability to potently

present antigen, and even fewer have examined the deviation of non-antigen specific immune

responses following therapy. We present evidence demonstrating that dendritic cell therapy

modulates the immune response in an antigen-independent manner, as both unpulsed and

antigen-pulsed DC results in immune modulation. We also report that the resulting changes are

non-antigen specific as we find reprogrammed spleen cell responses in the absence of antigen

stimulation in the form of homeostatic proliferation. As shown in Fig. 4-1, this occurs

immediately just 2 weeks after treatment and remains durable up through 40 weeks of age. With

this finding, we attempted to identify the earliest this phenomenon occurred and discovered that









the effect was reproducible with only 2 weekly injections of DC and seen 2 weeks following the

last injection (data not shown). Thus we conclude that DC therapy potently reprograms the

spleen cell response immediately after treatment, as its effects require few treatments and the

response appears early, and is durable long after the cessation of treatment as the effect can be

seen in mice that are over 40 weeks, more than 29 weeks after the final DC treatment. The

homeostatic proliferation can be seen in both healthy and autoimmune mouse strains with normal

and aberrant DC phenotypes, confirming that the effect is a true immune response to DC therapy.

To characterize this immune modulation, we used flow cytometry to identify the

proliferating cell subsets and found that 72-98% of the proliferating cells were within the

lymphocytes subset, with B cells accounting for the majority of the proliferation. CD4+ and

CD8+ T cells accounted for 16-29% of the proliferation. However, these proportions were

similar to what is observed in PBS treated mice, indicating that while the proliferation is

significantly greater in DC treated mice, the relative fraction of replicating cell subsets remain

the same.

We then examined whether these proliferating cells were functionally altered following

DC therapy by assessing their cytokine profiles. Compared to proliferating cells of PBS treated

mice, we found that CD4+ and CD8+ lymphocytes from DC-treated mice exhibited a shift in

cytokine production characterized by a 2-fold increase in the release ofIL-10 and a 2-fold

decrease in IFN-y production. No such changes were observed from the B cell subset.

To understand what was driving this reprogramming of the immune response, we collected

RNA from the cultured spleen cells and performed a gene array analysis of common cytokines,

chemokines, and receptors to identify potential differential gene expression levels between the

treatment groups. We describe notable changes in gene expression between various group









comparisons. IL-17f, a gene associated with pro-inflammatory processes, was found to be

reduced over 2-fold in DC treated mice compared to PBS controls. This change was more

evident when comparing between PBS -treated mice and ID-DC treated mice as there was an 11-

fold decrease in expression of IL-17f in ID-DC treated mice relative to PBS controls. This

suggests that a reduction in IL-17f may allow for the proliferation of cell subsets that were once

inhibited by IL-17f expression. This data agrees with our previous results in which the

proliferating cells of DC treated mice had a cytokine shift biased toward the production of anti-

inflammatory IL10.

We also observed differences between recipients of unpulsed and antigen pulsed DC.

Mice receiving antigen-pulsed DC had a 3.8-fold higher expression of CXCL12 as compared to

unpulsed DC recipients. CXCL12, also known as SDF-1, has a role in the generation and

proliferation of early B cell progenitors and is also associated with T cell and HSC trafficking

(172,173,178). Leng et al reported that NOD mice have elevated expression of CXCL12 in the

bone marrow which promotes retention ofHSC and naive and regulatory T cells within the

compartment, thereby resulting in T1D due to a deficit of these cells within peripheral lymphoid

tissues where their regulatory function is needed(173). The elevated expression of CXCL12 in

the spleen cells of Ag-pulsed DC treated mice may compete with this aberrant signaling found in

NOD bone marrow. By having an expanding subset of CXCL12 expressive cells in the spleen, it

is possible that Ag-pulsed DC treated mice can benefit from a continued recruitment of HSC and

T cells into the spleen that may facilitate their interaction with other immune cells for the

generation of tolerogenic responses. Another study found that cells undergoing homeostatic

proliferation have an altered enhanced sensitivity to CXCL12 (179), suggesting there may be a

self-driven cycle that promotes retention of proliferating lymphocytes to the spleen.









Interestingly, CXCL12 and its ligand CXCR4 are also expressed on beta cells and the

proliferating ductal epithelium of pancreas that gives rise to regenerating beta cells (180).

Studies have shown that CXCL12 is required for the survival of beta cells and beta cell

progenitors and its over-expression results in protection from streptozotocin-induced T1D

(180,181). While our studies only examined gene expression in spleen cells, it is possible that

this increased expression may occur in other cell types including beta cells. Our earlier findings

of increased islet survival in SD-treated mice would support this notion.

We also found that SD-DC treated mice had an 18.9-fold increase in the expression of

CCL12, also known as MCP-5, compared to unpulsed DC recipients. CCL12, the ligand for

CCR2, is a chemoattractant for eosinophils, monocytes, fibrocytes and macrophages and has

been associated with allergic inflammation and Thl but not Th2 cells (174-176). CCL12 is

constitutively expressed in thymus and lymph nodes, but under inflammatory conditions its

expression is induced in macrophages and mast cells. Its expression has also been found to be

rapidly and significantly up-regulated following priming with antigen that may lead to CCR2-

mediated recruitment of inflammatory Thl cells (182). It is possible that the addition of antigen-

pulsing with SD-DC treatment increased expression of CCL12 although the expected

inflammation that is associated with CCL12 expression was not evident as SD-DC mice were

better protected from T1D compared to unpulsed DC recipients. It is important to note that

while both SD and ID-DC treated mice demonstrated homeostatic proliferation and protection

from T1D, the increased expression of CCL12 was only observed in the SD-DC group. This

finding, in conjunction with the observation that CCL12 expression increases with antigen

priming may support our earlier hypothesis that the constitutive presentation of SD, but not ID-









determinants, following short-term DC therapy provides the necessary signal to maintain the

pool of Ag-specific Tregs for durable Ag-pulsed DC-mediated protection.

Lastly, we examined whether the observed in vitro homeostatic proliferation occurs in

vivo. We treated mice with DC therapy, then administered a series of BrdU injections to allow

its incorporation into proliferating cells. We examined the spleen for BrdU+ cells to determine

whether there would be a greater percentage of BrdU+ cells in the spleens of mice treated with

DC compared to PBS controls, thereby proving homeostatic proliferation in vivo. However, we

saw no differences between treatment groups in BrdU positivity, suggesting the homeostatic

proliferating is only a phenomenon occurring in culture

Collectively, these results suggest that DC therapy results in Ag independent immune

modulation characterized by robust homeostatic proliferation of B and T lymphocytes in vitro.

Proliferating T cells exhibit a functional change characterized by a cytokine shift toward the

production of anti-inflammatory IL-10. Gene array analysis for potential cytokines/chemokines

that may be driving this proliferation requires further investigation, though there is some

evidence to suggest that the down-regulation of IL-17f expression may contribute to the

proliferation of a previously inhibited anti-inflammatory cell subset. These findings demonstrate

the durable potency of DC therapy in the modulation of antigen non-specific immune responses.










A 13 weeks old
270001
1500 I


6000,
E


40 weeks old


13000
C PBS
Sunpulsed DC 10500
E ID-GAD DC


E PBS
M unpulsed DC
EMl ID-GAD DC


media DD-insulin ID-GAD meala uu-insuln vlu-um
in vitro stimulation (25uM) in vitro stimulation [25uM]


Figure 4-1. Homeostatic proliferation following DC therapy is immediate and sustained. Spleen
cells from NOD mice were cultured in serum-free HL- media and 3H-thymidine was
added for incorporation during the final 16h of culture. Proliferation was assessed by
beta scintillation quantification of counts per minute (CPM). Spleen cell proliferation
is shown at A) 13 weeks of age and B) 40 weeks of age. Data shown is representative
of 10+ experiments.


600001 PBS
1 PBS
50000 unpulsed DC
20000 T


E 15000-
Q.
--

10000-

5000-


0-,-


NOD C57BL/6J
(N=3) (N=3)


a


Balb/c
(N=2)


Figure 4-2. Homeostatic proliferation is observed in healthy and autoimmune mouse models.
NOD and B6 mice were treated with 3 weekly subcutaneous injections of DC
(105/injection) beginning at 9 weeks of age. Balb/c mice were treated with 2 weekly
IV injections of DC (105/injection) beginning at 6 weeks of age. Spleen cells were
collected 2 weeks following final injection to assess 3H-thymidine proliferation in the
absence of stimulation.











5.8%
D.18%


!2:94%
'=,i'


10.5%
0.33%







.80%

11.2%
1126% .







996% "

20.2%
.246%







172% '
i .;;


5632%




4,.

















43.62%
li.






I.



L2


CFSE


Figure 4-3. Identification of proliferating cell populations. CFSE-labeled cells from female
NOD mice were analyzed for expression of CD4, CD8, and B220. Percentages in red
denote proportion of indicated cell subset within the proliferating cells. Data shown
is representative of 10+ experiments collected through a range of post-treatment
timepoints (13-41 weeks of age).


2.95%

4.5%
0.51% 744%









9.4%


76.6%
3.80%







1-16%

57.1%
5.86%







4:40%

68.8%
8.22%






.I: .
.372% :+
.***a


23.00%










23.51$











18159A


t.5


.












Gated on CD4+ proliferating cells Gated on CD8+ proliferating cells


25.009. 30.71%








32.85 11.43%

52.37% 16.32%








268A9i. 4.47%

49.149 23.56%


6.03%


16.4296 29.85%








46.27"1 7.46%

I35.95* 30.79%


27.661








4;24


**,. .* -: ,.




5.58%

31.97%








6.12%


Gated on proliferating cells

8.66% 63.90%



.. "."":



5.52% 21.92%

15.509 57.65%








7.41 19.44%

15.25* 59.17%


.: 18.64%


IFN-y
Cytokine profile of proliferating cell populations. Cells were permeablized and
stained for the intracellular cytokines IL-4 and IFN-y, gated on CD4, CD8, and B220
and evaluated for CFSE dilution.


Figure 4-4.












PBS vs all DC


2103001 P
30oo- p = 0.081 B

S H B B H n



0 -5

-10-









490|
190-1 J --



.c 1
N X L N=UJ -J 0X=N :u;-CU o00 8 O NtU











0 -- --2U

-5-

-16 B
injections ofPBS, unpulsed DC, SD-DC or ID-DC (N=3/group) beginning at 9 weeks of age. Two weeks following
490



















treatment, RNA was isolated from cultured spleen cells and transcribed into CDNA for gene expression assessment via
qPCR. A-G) Fold changes in gene expression between treatment groups are shown by statistical rank (highest rank starting
at y-axis).
-- n

















at y-axi s).









PBS vs SD-DC


1183,
600- -
17 --


14 111 I nl.
-1 g lllIB


I I n. .


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r-- -- a -m z o i
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S -- -- -- --- xPBSy vs I D-DC
PBS vs ID-DC


115-
105-
11-
8-


L


p= 0.056


[I .. / I
fAl Ii on I no


Ugo-a-- oe-z --0 h- doO-0iz jz so -> 00guga 'U V -ma&eNoo- o-0uo oo-a&CaI0--0 r-U
] xx-u- u u go dd do_ d -d >- U aU-0 S--dUuuuu -UXdu X-rjX c uC
S z U0 --
w
CD


Figure 4-5 continued.












Unpulsed vs Pulsed DC


160020j I







80020-|
2o0 p = 0.071 _


8-
C 5-





-4

-3 o 50000
850000 I I I I I I I I I I I I I I I Id I I I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I

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XU Xx U -U --U--U -U j -U- X- UUXu-- N 0
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Figure 4-5 continued.









p = 0.056
287
147


Unpulsed DC vs SD-DC


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-7
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HB BH B U I HE Bl I II IIm a Ia H ai 1H Uin HU1HBI


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Figure 4-5 continued.


"9x r -N Ju .UJ- j N 3-u -j IOXN -0 00U XUoc Uu Q
dI]No _i0 -r0 -
00 -- - --- X
0


o.,,,,,,,,,,,,,,,, ,,,,,,,,, ,,,,,,,,,,,, ,,,,,,,,,,,,,,


u -i N i N.


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T


rT


unpulsed DC


Pancreas


I


-Brdu Trmt -Brdu Ab


PBS unpulsed DC


Liver


-BrdU Trmt -Brd Ab
-BrdU Trmt -BrdU Ab


+7


PBS unpulsed DC


Figure 4-6. Frequency of BrdU proliferation in organs. Nine week-old female NOD mice were
given 3 weekly injections of PBS or DC therapy (N=3/group), then treated with BrdU
to assess its incorporation by proliferating cells. Organs were formalin fixed and
paraffin embedded, then sectioned for staining with anti-BrdU antibody. %Brdu
indicates proportion ofBrdU+ cells within a defined tissue area as determined with
image analysis software.


Spleen


-r


ID-DC


SD-DC


T


-I


SD-DC


ID-DC


0.25-

0.20-

n 0.15-

0.10-

0.05-

0.00


































Figure 4-7. In situ proliferation marked by BrdU incorporation. Spleens were fixed with 4%
paraformaldehyde, paraffin embedded, and stained for BrdU detection. Arrows
denote representative BrdU+ cells. C = capsule. GC = germinal center. MZ =
marginal zone. PALS = periarteriolar lymphoid sheath. RP = red pulp. WP = white
pulp









CHAPTER 5
MODULATION OF REGULATORY T CELL POPULATION

Introduction

In a healthy individual, autoreactive cells are present but do not cause autoimmunity due to

an effective immunoregulatory mechanism. However in T1D the lack of an adequate regulatory

response allows autoreactive T cells to become pathogenic, thereby invading and destroying the

pancreatic islet cells. Regulatory T cells (Tregs) inhibit immune responses against self-antigens

by secreting immunosuppressive cytokines or by interacting directly with effector T cells, and it

has been shown that animal models with absent or defective populations of Treg develop

autoimmune disease (108-114). Multiple studies have demonstrated that DC therapy can confer

protection against autoimmunity through the induction of regulatory T cells that inhibit the

pathogenic T cell inflammation (85,87,183-189). Our work has shown that Ag-pulsed iDC

therapy protects NOD mice from T1D, and that DC therapy induces homeostatic proliferation of

CD4+ T cells (among other subsets). However, it is unclear if Tregs are being induced and

whether they are part of the proliferating cell population. We sought to identify whether there

were changes in regulatory T cell frequency and function following DC therapy by evaluating

the proportion of CD4+Foxp3+ cells in DC treated and control mice and examining their ability

to suppress proliferation of effector cells.

Materials and Methods

Flow Cytometry

Cells were prepared into single-cell suspensions in FACS buffer (lx PBS / 1% FCS), then

stained for surface markers using CD4 and CD25 in FACS buffer in the presence of Fc block

(CD16/32) (BD Pharmingen). Cells were then fixed using Cytofix/CytoPerm reagent

(eBioscience) for 15 minutes at room temperature, then washed in PermWash (eBioscience). All









subsequent steps were performed in PermWash to maintain membrane permeability. Non-

specific staining of cells was blocked again using Fc block for 10 minutes, then cells were

labeled with Foxp3 antibody. Cells were analyzed by flow cytometer (FACS Calibur, BD

Pharmingen). Live cells were gated from dead cells on the basis of forward/side scatter or with

7AAD (amino-antimycin D) labeling. Isotype controls include mouse IgG3K, rat IgG2a, hamster

IgG1K, and hamster IgG1X. Results were analyzed using FCS Express (BD Pharmingen).

Suppressor Assay

Spleen cells were isolated as described previously. Cells were then suspended in MACS

buffer and CD4+ cells were enriched through depletion of unwanted cells using the CD4+CD25+

Regulatory Cell Isolation Kit (Miltenyi Biotec). Next, CD25+ cells were positively selected

from the pre-enriched fraction. Responder CD4+CD25+ depleted cells (105) were cultured with

suppressor CD4+CD25+ cells at 1:2 and 1:4 titrations in a round bottom 96 well plate. Cells

were cultured in serum-free media with anti-CD3e (0.05 ug/200 ul well) and ID-peptide

stimulation (25uM), then proliferation was determined using 3H-thymidine incorporation.

Retinoic Acid Treatment

Tretinoin, an all-trans retinoic acid gel prescribed for the topical treatment of acne vulgaris,

was topically applied to a 1" x 1" shaved region of skin at the nape of the neck of C57BL/6J

mice. Mice were placed under isoflurane anesthesia at 4%-5% for induction, 1%-2% for

maintenance as controlled by an anesthesia machine. Mice were monitored for their state of

consciousness using toe/tail pinch or palpebral reflex. RA gel was applied to cover exposed skin

several hours before DC injection, once daily for up to 5 days including a "pre-conditioning day"

in which no DC injection occurred.









Results

DC Therapy Results in Sustained Expansion of Regulatory T Cells

Evidence from the literature suggests that a possible mechanism for protection from DC

therapy is the induction of regulatory T cells. We sought to determine whether the DC treatment

was able to increase the frequency of CD4+Foxp3+ regulatory T cells. We treated mice with

either PBS, unpulsed DC, or ID-pulsed DC at 9 weeks of age, then collected spleens from mice

at various ages to assess spleen cell response. Spleen cells were labeled with CFSE and allowed

to proliferate in the presence and absence of peptides for 72-84 hours. Cells were then labeled

with regulatory T cell markers and analyzed by flow cytometry. As shown in Fig. 5-1, we found

that there was an over 2-fold increase in the frequency of CD4+Foxp3+ T cells in mice receiving

unpulsed DC, and an over 4-fold increase in frequency of CD4+Foxp3+ T cells in mice receiving

ID DC, demonstrating that DC therapy results in sustained expansion of regulatory T cells, and

that the effect is particularly enhanced in mice receiving ID-pulsed DC. This homeostatic

expansion of Tregs was independent of in vitro peptide stimulation, as the pattern was observed

in both stimulated (data not shown) and unstimulated cell cultures.

Regulatory T Cell Function is Enhanced Following DC Therapy

We also examined whether there were functional differences in regulatory T cells

following DC therapy. We performed a suppressor cell assay by co-culturing CD4+CD25+

depleted cells with CD4+CD25+ purified cells at various titrations of 1:2 and 1:4 in the presence

ofanti-CD3 and ID peptide stimulation. As seen in Fig. 5-2, regulatory cells from both unpulsed

and peptide pulsed treated mice demonstrated greater suppressive function in a dose dependent

manner, with the effect enhanced in the peptide-pulsed DC group. The enhanced suppression

was found to be nearly 2-3 fold greater in DC treated mice at 1:2 titration. This effect was

magnified when the titration was decreased to 1:4, where up to a 10-fold enhancement in









suppression was observed. These results demonstrate that on a cell to cell level, Tregs isolated

from DC treated mice are more potent in suppressor function than Tregs isolated from PBS

treated mice.

Retinoic Acid Increases Frequency of Regulatory T Cell Population

Another aim for our study was to identify how to better engineer tolerogenic DC. Studies

using retinoic acid (RA), a metabolite of Vitamin A, discovered that its treatment could induce

Foxp3 expression in CD4+ cells. We treated the injection site with RA using a topical gel for 3

days to condition the DC microenvironment prior to a single subcutaneous DC injection, then

isolated spleen cells from mice 24 and 48 hours after the injection. We found within 24 hours of

the DC injection, mice that were pretreated with RA had an increase in the frequency of

CD4+CD25+Foxp3+ cells from 5.14% (no RA + DC) to 8.23% (RA + PBS) and 8.23% (RA +

DC) (Fig. 5-3), and this was observed regardless of whether DC were injected. This effect

magnified at 48 hours following DC injection, as mice without RA treatment had a

CD4+CD25+Foxp3+ cell frequency of 7.07% compared to RA treated mice at 12.71 (RA +

PBS) and 13.88% (RA+ DC). As RA has been shown to induce the expression of CD103, the

gut homing receptor, we speculate that the increase in Tregs in the absence of DC injection is

due to the possibility that RA treatment conditioned existing Langerhans cells to migrate to gut

LN and interact in the T cell environment to induce Foxp3+ expression.

Discussion

Overall, our results suggest that DC treatment increases the frequency and function of

regulatory cells. We found that mice receiving DC had a 2-3 fold increase in the frequency of

CD4+Foxp3+ cells, and that the effect was further enhanced when mice received a peptide-

pulsed DC. We also assessed the ability of CD4+CD25+ cells to suppress anti-CD3 and peptide-

stimulated proliferation of CD4+CD25+ depleted effector cells and found that regulatory cells









isolated from DC treated mice has superior function in suppression exhibited by an 2-10 fold

enhancement in suppression compared to cells isolated from PBS treated mice. These findings

were consistent in all mice that received DC therapy, and were enhanced when the DC was

loaded with an antigen. Of interest, the results of the proliferation studies were verified in both

diabetic and non-diabetic mice. As Tregs are expected to promote tolerance, it is unclear why

the observed expansion in Tregs was not consistently associated with protection from T1D. It is

possible that the increased frequency and function of Tregs were not sufficient in scale to

override the inflammatory activity of effector cells. A balance in favor of Tregs over pathogenic

effector cells must be achieved to yield immune deviation to tolerance. Another potential

explanation was proposed by work from Diane Mathis's group, which demonstrated that while

defects in NOD Treg contribute to T ID, it may be an effect of over-responsive effector T cells to

self-antigen (or monoclonal Ab stimulation) that truly drive the immunopathology (190). Thus

the loss of tolerance may be related not to impaired function or decreased frequency of NOD

Tregs, but rather a decline in the ability of NOD T cell effectors to respond to fully competent

Tregs. However, our studies of Treg function examine PBS- vs. DC-treated Tregs against NOD

effectors, which in concept should be similarly impaired, so our observation of functional

differences between the treatment groups can be attributed to a true variation between PBS- and

DC-treated Tregs.

To investigate other methods to improve Treg frequency, we examined whether topical

retinoic acid treatment, which has been shown to induce Foxp3 expression, can induce Foxp3+

Tregs. We also found that RA conditioning of the injection microenvironment alone, with or

without additional administration of DC, was able to increase the frequency of splenic

CD4+CD25+Foxp3+ regulatory T cells by over 2 fold. This effect was immediately observed









with just 3 daily RA treatments. An increase in the frequency of RA-induced Foxp3+ Tregs in

conjunction with DC-therapy-induced Tregs may together be able to dampen the potent

inflammatory response from increased or over-responsive effector T cells.

Taken together, these findings suggest that DC therapy effectively improves both the

frequency and function of regulatory T cells that are imperative to the suppression of

autoreactive T cells. Importantly, the homeostatic expansion of Tregs continues after the

cessation of DC treatment. We also demonstrate that the protective effects of DC therapy can be

improved using topical RA conditioning of the injection site microenvironment located away

from the autoinflammatory site where steady-state resident DC in the skin can be recruited to

participate in immune modulation. Collectively, these results provide evidence that DC therapy

induces regulatory T cells that are more potent suppressors compared to Tregs isolated from a

PBS treated mouse, and that these cells exist in greater proportions as they continue to expand

following DC therapy.





























10 4

103,


00
0L
0
L-


CD25


16.13%
3.20%






'v: .


10.47% 70.2


2.070
6.99%









26.85% *54.


115.89
12.87',









S55. 46


CFSE+


Figure 5-1. Assessment of regulatory T cell population following DC therapy. CFSE labeled
spleen cells from female NOD mice were cultured in serum-free media without
stimulation and allowed to proliferate for 72-84h. Cells in right panels are gated on
CD4+. Data shown is representative of 3 experiments from mice aging from 13-41
weeks old.



















88













S1:2
1:4


PBS(N=3) unpulsedDC(N=3) ID-DC(N=3)
PBS (N=3) unpulsed DC (N=3) ID-DC (N=3)


Figure 5-2. Suppressor cell assay. Female 9 wk-old NOD mice were treated with 3 weekly
injections of DC, then Treg function was assessed at 13 weeks of age. CD4+CD25+
depleted cells from spleen were co-cultured with titrations of CD4+CD25+ purified
cells and stimulated with anti-CD3 and ID-peptide. Proliferation was assessed by
3H-thymidine incorporation.


RA+ PBS


(.4


Gatad an CD ,
RA+ DC


noRA+DC


65.16'. .9.41 62.43% .I-20.74 75.28 15.92

| Foxp3

Figure 5-3. Retinoic acid increases frequency of regulatory T cell population. Female C57BL/6J
mice were treated with a topical application of RA at injection site for 3 days before
injection with PBS or DC (N=3/group). Expression of CD4+CD25+Foxp3+ was
assessed in spleen cells.


i









CHAPTER 6
CONCLUSIONS

Type 1 diabetes is a metabolic disorder characterized by autoimmune destruction of the

insulin-producing pancreatic beta cells. The autoimmune process is driven by T cells reactive to

autoantigens expressed in beta cells, including GAD, ICA69, IA-2, insulin, and proinsulin in the

absence of an effective regulatory mechanism(191-195). Dendritic cells, which present antigen

and direct T cell responses, are an ideal platform for use in T1D treatment as DC therapy works

to correct the specific underlying autoimmune aberrancy in T1D. DC therapy can uniquely

control (1) the direction of the immune response through the selection of either immunogenic or

tolerogenic classes of DC, as well as (2) dictate the target antigen that the response is directed

toward through the presentation of a chosen antigen, reinforcing DC therapy to be an effective

and powerful strategy for immune modulation. However, a consensus on what defines a

tolerogenic DC and which antigens to select for DC loading remains to be established, as

multiple studies in the NOD mouse have reported protection from T1D using a range of DC

subsets and beta cell antigens or antigen determinants. Additionally, another confounding factor

lies in the requisite of early intervention. Reports of DC therapy for tolerance induction has been

successfully demonstrated when applied before or in the early stages of autoreactivity in animal

models of various autoimmune diseases, as well as in studies of transplant/graft

acceptance(85,135,196-200). However, if treatment is initiated after the autoimmune process is

advanced, efficacy in DC-mediated protection declines. While NOD mice have a predictable

timeline for T1D onset allowing for intervention to be planned accordingly, the dynamics of

autoreactivity processes in human has not been difficult to define due to multiple variations in

subtypes that compound assessment. Additionally, the majority of subjects susceptible to Type 1

diabetes lack familial history that would otherwise prompt early autoantibody screening, thus the









opportunity for early intervention in humans is low, emphasizing the need for therapy that can

treat both established and new onset disease.

We sought to understand how to better develop DC therapy for translation into the clinical

setting. We first addressed the issue of what defines a tolerogenic DC. Classical models of DC

subsets classify DC function on the basis of phenotypic characteristics associated with the

expression of maturation markers such as MHC class II and T cell costimulatory molecules and

production of cytokines. Immature DC, which have low expression of maturation markers, and

anti-inflammatory biased cytokine production, are believed to be tolerogenic while mature DC,

which have high expression of maturation markers and a pro-inflammatory bias in cytokine

production are defined as immunogenic. However, critics ofiDC speculate whether they can

migrate efficiently to dLN for critical interaction with T cells, and whether a new subset of DC

termed semi-mature DC with low production of inflammatory cytokines and high expression of

T cell costimulatory molecules may be better suited to stimulate naive T cells into regulation. We

first examined iDC migration and found that a substantial proportion of injected DC traveled to

the dLN. Then we compared the efficacy of iDC and smDC therapy for the prevention ofT1D

in 9 week old NOD mice with advanced insulitis and found that iDC were superior to smDC in

delaying of TD onset, though neither treatment conferred significant protection.

To create DC for therapy with more durable protection, we considered another aspect of

DC therapy: selection of antigen for loading prior to infusion. We and others have demonstrated

that the administration of beta cell autoantigens in a tolerogenic modality is highly effective in

preventing T1D in the NOD mouse (61,64,65,125,129,141,201). However, uncertainties in

extrapolating appropriate Ag doses and correlating treatment timeline, as well as limitations in

identifying epitopes in human has hindered its translation into the clinical setting, particularly









since studies have shown that the immune response can pivot toward immunity or tolerance

depending on antigen dose. Fortunately, antigen presentation in the context of a tolerogenic DC

may circumvent the issue of ambiguous immune deviation associated with antigen treatment

alone. Traditionally, antigens were selected for DC therapy by focusing on autoantigens or

peptide epitopes within self-Ag that are dominantly-expressed and are primary targets of the

autoimmune response. These whole antigens or dominant determinants (DD) were then

administered in modalities that induce tolerance, and has been shown to prevent T1D as well as

other autoimmune diseases in mice when applied early in the autoimmune

process(61,64,124,144). However, this strategy becomes ineffective when administered at

advanced stages of T1D (138,144). We speculated that the decline in protection is associated

with an altered immune repertoire with time as dominant determinants (DD) continuously recruit

naive T cells into spontaneous autoimmune attack while subdominant determinants (SD), which

do not compete as effectively as DD, and ignored determinants (ID), which are not processed

and presented at all, have a minimal effect on the naive T cell pool (144,145). Thus, the re-

administration of DD may simply reactivate cells that are preprogrammed to respond

pathogenically, while SD or ID can recruit the available pool of naive T cells for priming into

tolerance when natural antigen processing is bypassed experimentally to present these previously

unseen epitopes.

We compared the efficacy ofDD, SD, and ID peptide classes in DC therapy to protect 9-

week old NOD mice and found that only SD- and ID-pulsed DC were able to protect mice when

the treatment was applied in the presence of advanced autoimmune processes. Specifically, just

three weekly injections of SD-DC protected NOD from T1D with a significant delay in the onset

ofT1D, though complete protection was not achieved. Consistent with this finding, we saw that









SD-DC treated mice had a greater number of pancreatic islets compared to unpulsed and DD-DC

treated mice though the level of islet infiltration remained similar between all groups. Next we

examined whether ID-DC, which should have a comparatively larger pool of naive T cells to

prime into tolerance, would be more effective in conferring protection. However, we found that

three injections of ID-DC were not sufficient to achieve protection, as a sudden drop in survival

within 6 weeks of the last treatment dampened the treatment success. We speculated that since

ID are not constitutively presented, treatment may need to be continued to maintain the

regulatory T cell pool. We treated another cohort of mice with repetitive injections of ID-DC

and found that mice were completely protected from T1D. This total protection was not seen in

mice treated with repetitive injections of PBS or unpulsed DC, suggesting that the protection is

attributed to the ID.

To exclude the possibility that the observed protection from T1D was due to global

immunosuppression, we examined whether NOD mice could generate a normal immune

response to a non-T1D related antigen challenge following treatment with DC therapy. We

immunized PBS, unpulsed DC, and ID-DC treated mice with KLH and examined their serum

antibody responses. All DC treated mice were able to mount antibody responses to KLH in a

manner comparable to PBS controls, demonstrating that normal immune responses were intact

and the previously observed protection could be attributed to diabetes-specific protection.

Much of the current knowledge on how DC therapy affects immune responses has been

delineated from studies with a focus on antigen specific immune modulation, as we have shown

that Ag-based DC therapy-mediated protection is limited to the suppression of autoreactive

processes specific to T1D. However, whether DC therapy results in antigen non-specific

immune changes has yet to be studied in detail. The spleen is a major site of immune cell









interactions and antigen processing, with active processes that contribute to the overall immune

status(163,164). Thus, we sought to examine the spleen cell response in the absence and

presence ofT1D peptide stimulation. To our surprise, we found robust homeostatic proliferation

of spleen cells isolated from DC-treated, but not PBS-treated mice. This effect was immediate

and sustained, as the proliferation could be observed as early just two weeks following 2 DC

injections, and was durable even 29 weeks after treatment had ended. Characterization of these

cells revealed that the proliferation could be predominantly attributed to B and T lymphocytes,

though there was no evidence for the expansion of memory B or T cells within these subsets.

We found that this DC-mediated reprogramming of the immune response extended into changes

of cytokine production as well, as we observed a 2-fold shift from the production of IFN-y to the

production of anti-inflammatory IL-10. To determine what may be driving this immune

modulation, we performed a gene array analysis to identify potential changes in expression of

cytokine/chemokines genes and did not find any statistically significant difference between

treatment groups, though there was some evidence to suggest that the down-regulation of IL-17f

expression seen in DC-treated mice may have contributed to the proliferation of a previously

inhibited anti-inflammatory cell subset. This data agrees with our previous results in which the

proliferating cells of DC treated mice had a cytokine shift biased toward the production of anti-

inflammatory IL-10.

We also observed gene expression that may not delineate the cause of the spleen cell

proliferation but still contributes to our understanding of the effect of DC therapy on disease

outcome. We identified an increase in CXCL12 expression in the spleen cells of Ag-pulsed DC

treated mice compared to unpulsed DC-treated mice. CXCL12 has a role in the generation and

proliferation of early B cell progenitors and has also been found to be over-expressed in NOD









BM thereby promoting retention of HSC and naive and regulatory T cells within the BM

compartment resulting in a deficit of these cells in the peripheral lymphoid tissues where their

regulatory function is needed(173). The elevated expression of CXCL12 in the spleen cells of

Ag-pulsed DC treated mice may compete with this aberrant signaling found in NOD bone

marrow and recruit cells into the spleen for interactions leading to tolerance induction.

Additionally, CXCL12 and its ligand CXCR4 are also expressed on beta cells and the

proliferating ductal epithelium of pancreas that gives rise to regenerating beta cells and its over-

expression results in protection from streptozotocin-induced T1D (180,181). While our studies

only examined gene expression in spleen cells, it is possible that this increased expression may

occur in other cell types including beta cells. Our earlier findings of increased islet survival in

SD-treated mice would support this notion. We also found that spleen cells of SD-DC treated

mice had an increase in the expression of CCL12, which has been shown to be up-regulated

following priming with antigen (182). It is possible that the SD-pulsed DC treatment increased

trafficking of both CCL12 responsive Ag-specific Tregs and DC presenting cognate SD into the

spleen promoting their interaction to induce proliferation of these Treg. This may elucidate why

we observe proliferation of Tregs in the absence of in culture peptide stimulation, and why this

increased expression is only observed in SD but not ID groups as there should not be continued

constitutive presentation of ID following cessation of treatment to induce the up regulation of

CCL12. Proliferation observed in splenic cultures derived from ID-treated mice may also benefit

in a similar manner from infectious tolerance (202,203). However, in vivo BrdU studies

provided no evidence of homeostatic proliferation, suggesting that this phenomenon is only

occurring in vitro. Whether this is due to an accumulation of cytokines that may drive

proliferation in culture remains to be determined.









Because NOD mice have a defect in DC phenotype and function, we evaluated whether

this homeostatic proliferation was a true effect of DC therapy or an only an effect associated with

therapy using DC with an aberrant phenotype. We treated the non-autoimmune prone mouse

strains Balb/c and C57BL/6J mice with PBS or DC and observed a similar enhancement in

homeostatic proliferation in mice receiving DC therapy, confirming that the effect is a true

immune response to DC therapy.

Dendritic cells play an important role in the induction and maintenance of regulatory T

cells that may confer tolerance(75,118-121,123,184). Thus, we evaluated the effect of DC

therapy on the regulatory cell subset. We found that DC therapy resulted in the durable

expansion of CD4+Foxp3+ regulatory T cells, and this effect was further enhanced using Ag-

pulsed DC. DC therapy also improved the function of Tregs, as CD4+CD25+ regulatory cells

from DC-treated mice were able to more potently suppress CD3 and peptide-stimulated

proliferation of CD4+CD25-depleted responder cells compared to regulatory cells from PBS

controls. This Treg expansion can be further enhanced with retinoic acid, as we found that RA

pre-conditioning of the DC microenvironment was able to increase the frequency of Foxp3+

Tregs in the spleen with or without co-injection with DC. This may be due to the RA-induced up

regulation of CD103 expression on resident Langerhans cells that home to the gut and induce

Foxp3+ expression. While we observed an increase in both frequency and function of Tregs

with DC treatment, we did not observe a correlation in protection from T1D. It is possible that

the improvement was not sufficient to pivot the balance in favor of regulation in the presence of

a potent inflammatory effector T cells response that has been shown to grow with age (190).

This may be supported by our observation of increased islet survival in Ag-pulsed-DC treated

mice, but not unpulsed-DC treated mice that had similar levels of lymphocyte infiltrate, as the









undefined lymphocyte population may potentially be an influx of both pathogenic and regulatory

T cells. A balance in favor of regulatory T cells versus immunogenic T cells may be able to push

the overall response toward tolerance, but perhaps the DC therapy-mediated increase in

regulatory T cells was not enough to completely quench the inflammation generated from the

pathogenic T cells. However, RA-treatment induced Foxp3+ Tregs in conjunction with DC-

therapy-induced Tregs may together be able to dampen the potent inflammatory response from

increased or over-responsive effector T cells. Nonetheless, the latter stages of advanced

autoimmunity immediately prior to T1D onset may simply not be amenable to a one-armed

intervention; Tregs alone may not be sufficient to rescue beta cell death and the requirement for

combinatorial strategies to treat both autoimmunity and regenerate beta cell mass may become

necessary.

Collectively, these results suggest that DC therapy results in antigen-dependent and

antigen independent effects on immune modulation. We find that the choice of peptide

determinants for DC pulsing has a profound effect on the efficacy of DC therapy. SD or ID-

pulsed DC, but not DD-pulsed DC, were able to protect NOD mice with advanced autoimmunity

from development of T1D, demonstrating a role for antigen in T1D prevention. This

fundamental principle of altering native determinant presentation to accommodate a changing T

cell repertoire can be extended to the treatment of any dynamic autoantigen-based disease. We

also demonstrate that DC therapy augments the immune response in an antigen-dependent and

independent manner. Spleen cells from both unpulsed and Ag-pulsed DC treated mice, but not

PBS controls, exhibited robust homeostatic proliferation of B and T cell lymphocytes in vitro,

with the effect further enhanced in Ag-DC recipients. However, the immune modulation was

non-antigen specific, as the addition of peptide stimulation in culture did not alter the response.









Importantly, the proliferation appears to be a protective and not pathogenic response as the

expanding cells exhibit a deviation in cytokine production from pro-inflammatory to anti-

inflammatory. Furthermore, we find that DC therapy increases the frequency and enhances the

function of Tregs and that the chronic expansion of these cell populations did not require recall

Ag peptide stimulation. This reprogrammed Treg response can potentially re-establish the

balance between effector and regulatory cells back in favor of tolerance. Together, these

findings demonstrate the durable potency of DC therapy in the modulation of antigen specific

and non-specific immune responses and provide an important step toward translation into the

clinic as other peptide-based therapies for T1D have been limited to early intervention. These

discoveries will guide the engineering of DC therapies for T1D and other dynamic Ag-based

disorders.









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BIOGRAPHICAL SKETCH

Jeannette Lo-Dauer was born in Hong Kong where she lived with her parents and older

sister Paulette before moving to Florida at the age of 5. She lived in Fort Lauderdale, FL where

she attended and graduated Nova High School in 1997. Jeannette began her undergraduate

studies at the University of Florida with double majors in microbiology and cell science, and

psychology. She developed an interested for biomedical research while performing studies

examining the action of anorectic drugs in rats with select serotonin receptor depletion in the

laboratory of Dr. Neil E. Rowland. Following graduation from UF in 2001 with a Bachelors of

Science in microbiology and cell science and psychology, she continued neuroscience research

for one year in the laboratory of Dr. John M. Petitto where she investigated the role of IL-2

signaling in the immune trafficking of lymphocytes to injured neuronal environments. Jeannette

then continued on to obtain a Master of Science in public health in 2004 from the University of

South Florida, where her graduate research focused on the development of novel nitroplatinum

complexes for the treatment of cancer. Jeannette entered the Interdisciplinary Program in

Biomedical Sciences Ph.D. at the University of Florida in 2004, and joined the laboratory of Dr.

Michael Clare-Salzler in 2005 where she studied dendritic cell biology (DC) in the NOD mouse,

the autoimmune model of Type 1 diabetes (T1D). Jeannette studied factors associated with the

antigen-driven process of autoimmune activation and immune tolerance and accordingly

engineered DC therapy for the induction of tolerance. Following the completion of her doctoral

work, Jeannette plans to continue research in DC biology with Dr. Clare-Salzler, and will

subsequently relocate to Birmingham, AL with her husband Daniel Dauer as he begins his

residency training at the University of Alabama, Birmingham. Jeannette plans to remain in the

field ofimmunotherapy with a career in academic research.





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1 DENDRITIC CELL THERAPY FOR TYPE 1 DIABETES: ROLE OF ANTIGEN PRESENTATION IN IMMUNE MODULATION By JEANNETTE LO DAUER A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009

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2 2009 Jeannette Lo Dauer

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3 To Mom and Dad

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4 ACKNOWLEDG E MENTS I have been fortunate to have an extensive team of people whom I wish to thank for their relentless support during the course of my graduate career. I would first like to extend my gratitude for the immeasurable guidance provided to me by my mentor, Dr. M ichael Clare Salzler. Mike has been instrumental in training me to think critically with a sound scientific mind while feeding my curiosity in science with his vibrant and passionate discussions in immunology. His e nthusiasm and encouragement kept me ins pired during times of challenge and allowed me to emerge with renewed momentum. Mike has also been a n admirable role model both professional ly and personal ly demonstrating that in the development of a scientists career, there can be a graceful balance between academic responsibilities and family. I also wish to thank my committee members, Dr. Mark Atkinson, Dr. Eric Sobel and Dr. Jake Streit. These professo rs have provided me with insightful unbiased advice and constructive criticisms. Without their help, I could not have learned to examine and refine my own work from a broader understanding of immunology. I especially want to thank Jake, who has been a w onderful friend and motivator throughout my years as a developing scientist. I would also like to express my gratitude to the members of my laboratory, past and present, for their support and assistance during my graduate research. Dr. Changqing Xia has b een instrumental in educating me on dendritic cell biology as well as on basic principles of immunology, and has served as a selfless and patient mentor. Dr. Ruihua Peng has been a constant friend and endless supporter of me in countless ways. Both Ruihua and CQ have played a prominent role in helping me develop the technical skills I needed to perform my work. I also wish to thank Tolga Barker, Ed Paek, Dr. Michelle Rodriguez, Luke Smith, Dr. Yushi Qiu Dr. Ron Ferguson, and Dr. Sally Yuan for their ki nd support. My lab has been a home for me for the

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5 past 4 years, not because of the time committed to research, but also because these friends have provided a warm and nurturing environment that made it feel like home. My graduate studies could not have be en completed without the help of many support staff. Fred Grant and Ronnie Middleton from the SPF Pathology Mouse Colony provided me and my mice with unique attentive assistance and kind understanding. Dr Dave Ostrov kindly taught me about basic princip les of antigen protein str ucture. Steve McClella n and Nea l Benson from the flow cytometr y core provide d ser v ice above and beyond what was expected and were instrumental in my data collection I especially would like to acknowledge the members of the Atki nson lab who provided me with support, equipment and reagents when I most desperately needed their help. Finally I wish to thank my family and friends. I could not have survived the demands of graduate school without family and friends to laugh and celebrate with. My family : mom, dad, my sister Paulette, you have been unstoppable sources of support for me. I cannot thank you enough for all your sacrifices that allowed to me attain my goals. And to my love my husband Dan, you inspired and motivate d me to achieve my professional ambitions supported me as my best friend and confidante while always entertaining me with your irresistible charm and humor. Of course, t o my best friend Campbell, you are gone but never far from the heart. You were my co nnection with all that is important in life and your spirit kept me going every day.

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6 TABLE OF CONTENTS page ACKNOWLEDGEMENTS ................................................................................................................. 4 LIST OF FIGURES .............................................................................................................................. 9 LIST OF ABBREVIATIONS ............................................................................................................ 10 ABSTRACT ........................................................................................................................................ 12 CHAPTER 1 LITERATURE REVIEW ........................................................................................................... 14 Type 1 Diabetes ........................................................................................................................... 14 Epidemiology: In cidence and Prevalance .......................................................................... 14 Etiology: Genetic and Environmental Factors ................................................................... 14 Immunopathology ................................................................................................................ 15 Existing Therapies for Type 1 Diabetes .................................................................................... 16 Nutritional Intervention ....................................................................................................... 17 Gene Therapy ....................................................................................................................... 17 Beta/Islet Cell Transplantion/Regeneration ....................................................................... 18 Immune Modulation ............................................................................................................ 18 Antigen -Based Therapies .................................................................................................... 19 Cellular Basis for Type 1 Diabetes Immunotherapy ................................................................. 20 Dendritic Cells ..................................................................................................................... 21 Modification to enhance function ............................................................................... 24 Current progress in clinical trials ................................................................................ 26 Regulatory T Cells ............................................................................................................... 26 Subclasses of regulatory T cells .................................................................................. 27 Role of dendritic cells in regulatory T cell development and maintenance ............. 28 Antigen -Based Dendritic Cell Therapy ..................................................................................... 28 Type 1 Diabetes Autoantigens ............................................................................................ 29 Native Immunogenicity of Peptide Determinants ............................................................. 30 Unknowns of Antigen-Based Dendritic Cell Therapy ...................................................... 31 NOD Mouse Model ..................................................................................................................... 32 Summary and Rati onale .............................................................................................................. 33 2 ENGINEERING OF TOLEROGENIC DC .............................................................................. 34 Introduction ................................................................................................................................. 34 Materials and Methods ................................................................................................................ 35 Animals ................................................................................................................................ 35 Bone Marrow Derived Dendritic Cells: Isolation and Culture ......................................... 36 Flow Cytometry ................................................................................................................... 37 Cytokine Ana lysis ................................................................................................................ 37

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7 Retinoic Acid ....................................................................................................................... 37 In vitro ........................................................................................................................... 37 In vivo ............................................................................................................................ 37 Migration Studies ................................................................................................................. 38 Results .......................................................................................................................................... 38 Immature Dendritic Cells Express Lower Levels of T -cell Costimulatory Molecules Relative to TNF -mature and LPS -Stimulated Mature DC ................................ 38 Immature and Semi -Mature Dendritic Cells Produce Lower Levels of ProInflammatory Cytokine IFN .............................. 39 Immature Dendritic Cells Migrate from Injection Site to Draining Lymph Node .......... 40 Immature Dendritic C ells are Superior to Semi -Mature Dendritic Cells for Induction of Tolerance ..................................................................................................... 40 In vitro Retinoic Acid Stimulation Increases Express ion of Maturation and Gut Homing Receptors on Dendritic Cells ............................................................................ 41 Discussion .................................................................................................................................... 42 3 ROLE OF ANTIGEN PEPTIDE IN DC THERAPY ............................................................... 48 Introduction ................................................................................................................................. 48 Materials and Methods ................................................................................................................ 49 Animals ................................................................................................................................ 49 Peptides ................................................................................................................................ 49 Dendritic Cell Peptide P ulsing ............................................................................................ 50 Histology .............................................................................................................................. 50 ELISA for Global Suppression Analysis ........................................................................... 51 Statistical Analysis ............................................................................................................... 51 Results .......................................................................................................................................... 51 Subdominant Determinant Pulsed DC Therapy Protects Against T1D ........................... 51 Pancreatic Islet Survival is Enhanced in Subdominant Determinant -Pulsed DC Recipients ......................................................................................................................... 52 Ignored Determinant Pulsed DC Therapy Confers Complete Protection Against T1D But May Require Continuous Treatment ............................................................... 53 T1D Specific Peptide -Pulsed DC Therapy Does Not Alter Natural Immunity to Environmental Challenge: KLH Study ........................................................................... 53 Discussion .................................................................................................................................... 54 4 ANTIGEN INDEPENDENT EFFECTS OF DENDRITIC CELL THERAPY ..................... 60 Introduction ................................................................................................................................. 60 Material and Methods ................................................................................................................. 61 Mice ...................................................................................................................................... 61 Proliferation Assay .............................................................................................................. 61 Flow Cytometry ................................................................................................................... 61 BrdU Study ........................................................................................................................... 62 Histology .............................................................................................................................. 62 RT PCR and Gene Array .................................................................................................... 63 Results .......................................................................................................................................... 63

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8 Homeostatic Proliferation is Observed Following DC Therapy: Immediate and Sustained Effects .............................................................................................................. 63 Homeostatic Proliferation Occurs in Healthy and Autoimmune Mouse Strains Following DC Therapy .................................................................................................... 64 Identification of Proliferating Cell Populations ................................................................. 64 DC Therapy Reprograms Cytokine Production of Proliferating T Lymphocytes ........... 65 DC Therapy Results in Differential Gene Expression: Analysis of Cytokine and Chemokine Genes ............................................................................................................ 65 In Situ Observation of Homeostatic Proliferation ............................................................. 67 Discussion .................................................................................................................................... 68 5 MODULATION OF REGULATORY T CELL POPULATION ............................................ 82 Introduction ................................................................................................................................. 82 Materials and Methods ................................................................................................................ 82 Flow Cytometry ................................................................................................................... 82 Suppressor Assay ................................................................................................................. 83 Retinoic Acid Treatment ..................................................................................................... 83 Results .......................................................................................................................................... 84 DC Therapy Results in Sustained Expansion of Regulatory T Cells ............................... 84 Regulatory T Cell Function is Enhanced Following DC Therapy ................................... 84 Retinoic Acid Increases Frequency of Regulatory T Cell Population ............................. 85 Discussion .................................................................................................................................... 85 CONCLUSIONS ................................................................................................................................ 90 LIST OF REFERENCES ................................................................................................................... 99 BIOGRAPHICAL SKETCH ........................................................................................................... 117

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9 LIST OF FIGURES Figure page 2 1 Characteriz ation of DC by flow cytometry. ........................................................................ 45 2 2 Luminex analysis of cytokine production by DC subsets. .................................................. 45 2 3 Migration of iDC. ................................................................................................................... 46 2 4 Diabetes incidence in NOD mice following DC therapy. .................................................. 46 2 5 Effect of retinoic acid on dendritic cell maturation. .. .......................................................... 47 3 1 Kaplan -Meier survival curves of female NOD mice following PBS, unpuls ed, and SD pulsed DC therapy. .......................................................................................................... 57 3 2 Histological assessment of islet number in NOD mice following DC treatment............... 57 3 3 Kaplan -Meier survival curves of female NOD mice following short term PBS, unpulsed, and ID pulsed DC therapy. ................................................................................... 58 3 4 Kaplan -Meier survival curves of NOD mice following continuous PBS, unpulsed, and ID pulsed DC therapy. ................................................................................................... 58 3 5 Antibody response following KLH immunization in control and DC treated mice. ........ 59 4 1 Homeostatic proliferation following DC therap y is immediate and sustained. ................. 73 4 2 Homeostatic proliferation is observed in health y and autoimmune mouse models. .......... 73 4 3 Identification of proliferating cell populations. ................................................................... 74 4 4 Cytokine profile of proliferating cell populations. ............................................................... 75 4 5 Gene array an alysis of common cytokine and receptors gene expre ssion. ....................... 76 4 6 Frequency of BrdU proliferation in organs. ......................................................................... 80 4 7 In situ proliferation marked by BrdU incorporation ........................................................... 81 5 1 Assessment of regulatory T cell population following DC therapy. ................................... 88 5 2 Suppressor cell assay. ............................................................................................................ 89 5 3 Retinoic acid increases frequency o f regulatory T cell population. .................................... 89

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10 LIST OF ABBREVIATIONS Ab antibody Ag antigen B6 C57/BL6 BM bone marrow BrdU bromodeoxyu ridine CD cluster of differentiation DC dendritic cell DD dominant determinant Dex dexamethasone Foxp3 forkhead box P3 GAD glutamate decarboxylase GMP good manufacturing practices HLA human leukocyte antigen HSC hematopoietic stem cell ID ignored determinant iDC immature dendritic cell IL interleukin IV intravenous KLH keyhole limpet hemocyanin LN lymph node mDC mature dendritic cells mLN mesenteric lymph node MHC major histocompatibility complex NOD non -obese diabetic

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11 PALS periarteriolar lymphoid sheath PBS phosphate buffered saline PP Peyers Patches pLN pancreatic lymph node SD subdominant determinant s m DC semi -mature T1D Type 1 diabetes Treg regulatory T cell

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12 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DENDRITIC CELL THERAPY FOR TYPE 1 DIABETES: ROLE OF ANTIGEN PRESENTATION IN IMMUNE MODULATION By Jeannette Lo -Dauer August 2009 Chair:Michael J. ClareSal zler Major:Medical Sciences Immunology and Microbiology Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of insulin -producing pancreatic beta cells by autoreactive T cells. Antigen -based dendritic cell (DC) therapy has been shown to be effective in preventing T1D in NOD mice, the animal model for autoimmune diabetes, when administered during early insulitis but fails to protect when applied after established autoimmunity However, the development of anti g en -based DC the rapy for late intervention has been challenging due to the dynamic nature of the immune repertoire which alters with time. As most human cases are not diagnosed until established hyperglycemia, an intervention for late -onset prevention is highly desirable We have developed a DC treatment paradigm for the induction of tolerance that is not limited to early intervention We find that by bypassing natural anti g en processing, DC presenting synthetic subdominant and ignored antigen determinants can more effe ctively prime nave T cells into regulatory function and protect NOD mice from T1D. Development of diabetes was significantly delayed in mice receiving a short term treatment of immature DC presenting a subdominant determinant (p=0.05) as compared to reci pients of PBS, unpulsed or dominant determinant -pulsed DC. Mice receiving sustained treatment of DC presenting an ignored determinant were completely protected (p=0.04). We also reveal that DC therapy with/without preloading with T1D associated Ag result s in

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13 immediate and sustained immune modulation characterized by robust proliferation of T and B lymphocytes. Of interest, DC treated mice have higher frequencies of CD4+Foxp3+ and IL 10 producing T cells compared to PBS controls. Additionally, CD4+CD25+ regulatory cells from DC -treated mice have superior suppressor function, with the effect enhanced in antigen -pulsed DC recipients Overall, these results suggest that DC therapy provides a previously unknown long -lasting effect on immune processes in an antigen -dependent and antigen independent manner. We describe the reprogrammed cell populations and show alterations in their cytokine production and function. Importantly, we identify peptide determ inants most effective in late anti g en -based therapy for T1D based on a fundamental principle of determinant immunogenicity that extends to any autoantigen based disease. These findings will aid the understanding of how DC therapy modulates the immune response and guide the engineering of DC therapies for preve n tion of T1D and other dynamic anti g en -based disorders.

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14 CHAPTER 1 LITERATURE REVIEW Type 1 Diabetes Type 1 diabetes (T1D) is an autoimmune metabol ic disease characterized by insulin deficiency as a result of the loss of pancreatic islet beta cells (1). Insulin, an endocrine hormone, is produced by beta cells to mediate the uptake of glucose from blood into cells for carbohydrate and lipid metabolism. Chronic destruction of beta cells by autoreactive T cells disrupts insulin production leading to hyperglycemia and secondary complications including neuropathy, nephropathy, blindness, and cardi ovascular disease (2). Epidemiology: Incidence a nd Prevalance Type 1 diabetes accounts for nearly 10% of all cases of diabetes and afflicts as many as 1 in 300 persons in the United States (2). It is estimated that 15,000 persons under the age of 20 are newly diagnosed annually at a rate of 19 per 100,000 each year (3) with prevalence rising to 1,087,800 cas es by the year 2010 in the U.S. (4). There is significant variation in the incidence of T1D worldwide, with more than a 400-fold variation among reporting countries. T1D is rar e in China, India, and Venezuela where the incidence is only 0.1 per 100,000 while Sardinia and Finland report a much higher incidence at nearly 37 cases per 100,000 individuals each year (5). T1D afflicts more non Hispanic whites than non -whites (6) but occurs in males and females equally (7). The incidence of T1D is on the rise in almost all populations worldwide with the greatest increases in countries with a low incidence (8). Etiology: Genetic a nd Environmental Factors Genetic and environmental factors are implicated in the development of T1D. The human leukocyte antigen (HLA) genes, particularly class II, on chromosome 6 are considered to provide a considerable risk, accounting for up to 50% of familial aggregation in T1D (9). The HLA

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15 genes, DR4 DR8 and DR3 DQ2, contribute the greatest risk for T1D since these haplotypes have been found in 90% of children diagnosed with T1D (10) The second most important genetic susceptibility factor is attributed to the VNTR region of the insulin gene located on chromosome 11. Shorter forms of a variable number tandem repe at on the insulin gene promoter are associated with disease, while longer forms confer protection (11,12) The cytotoxic T lymphocyte antigen 4 (CTLA 4) and lymphoid tyrosine phosphatase (LYP) genes, which control the negative regulation of T -cell activation, are also implicated in susceptibility to T1D (13) However less than 10% of those who are genetically susceptible develop the disease and concordance in identical twins is only 3040%, suggesting that genetics may not be the only factor (14,15) Environmental triggers, such as a viral infection, particularly with enteroviruses, rotaviruses, and rubella, has been linked to increased risk of T1D in genetically susceptible individuals. Beta cell -specific infection by the Coxsackie B4 virus has been described and the virus has been isolated from the pancreas of fatal childhood diabetes cases (16). Additionally, the incidence o f T1D was shown to increase after epidemics of enterovirus infections (17) Still, the viral mechanism leading to increased risk for T1D remains largely undefined. Viral infection followed by local damage to the pancreas, or stimulation of immunological cross -reactivity to islet antigens by viral proteins are mechanisms proposed to explain the role of viruses in T1D (15) The stimulation of protective inflammatory cytokines during viral infection may contribute to beta cell death by promoting the persistence of effector cells after the virus ha s been cleared (18) Thus, it appears that a multifactorial interaction between autoimmune, genetic and environmental influences collectively contribute to the development of Type 1 diabetes. Immunopathology T1D is caused by an autoimmune attack of insulin-producing beta cells by autoreactive T cell s. This organ -specific autoimmune disease results from a Th1 -biased response targeted to

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16 self antigens such as insulin and glutamic acid decarboxylase (GAD) in the presence of an ineffective regulatory response (19) The development of T1D occurs over a period of years as chronic progression of leukocyte infiltration of the islets leads to eventual beta cell destruction and loss of insulin production (20) In the initial stages of beta cell death, CD8+ cytotoxic T cells account for the bulk of leukocyte infiltration with perforin and granzyme secretion(21,22) IL producing CD68+ macrophages also comprise a significant percentage of the islet infiltrate (21) As insulitis proceeds, an increase in CD20+ B cells, but not CD138 plasma cells, is observed (21) CD4+ helper T cells, dendritic cells, NK cells are also detectable as a minor percentage of the leukocyte infiltrate (21) Throughout the entire inflammatory process, cytotoxic T cells and ma crophages remain the predominant immune cell subset in the islet. However, once beta cell destruction is complete, the frequency of all subsets of immune cells decline dramatically suggesting that immune cells enter the islet only when viable beta cells are present (21) Existing T herapies for Type 1 Diabetes Although advances in supportive therapy for the management of Type 1 diabetes has improved the quality of life for those with T1D, patients have an estimated 10 15 year reduction in life expectancy resulting from diabetes -related complications demonstrating the need for prevention and reversal strategies (23) Serum autoantibodies to pancreatic islet proteins are detectable before onset of Type 1 diabetes, allowing for screening of subjects at risk thereby providing a n opportunity for disease prevention. However upon T1D onset, treatment becomes more challenging. The strategies developed for the prevention or reversal of T1D in humans, ranging from nutritional intervention, gene therapy, islet/beta cell expansion/tra nsplantation, and immunotherapy have met with variable success.

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17 Nutritional I ntervention Animal studies using the Type 1 diabetes Biobreeding (BB) rat and nonobese diabetic (NOD) mouse models have suggested that diet can impact the development of T1D. Die tary soy, milk, and gluten have been found to be highly diabetogenic in T1D susceptible rodents (24,25) potentially by reducing the number of regulatory T cells (26) However others studies have found that hydrolyzed casein -based diets protect against T1D (25) In humans, early exposure to gluten or cereals has been linked to T1D, suggesting a role for wheat in the pathogenesis of T1D (27,28). Conversely, dietary supplementation with vitamin D and omega 3 fatty acids has proven to be effective in the prevention of T1D in both rodents and humans (28,29) A clinical trial called the Nutritional Intervention to Prevent Type 1 Diab etes Pilot Study (NIP) is currently underway to investigate the effects of increased dietary d ocosahexaenoic acid (DHA) an omega 3 fatty acid naturally found in mother's breast milk and in various foods, in newborns and infants with genetic susceptibility to T1D. Gene Therapy Gene therapy for Type 1 diabetes aims to genetically modify host cells using viral or plasmid vectors to deliver novel gene constructs. The insertion of insulin/proinsulin genes into a variety of surrogate non -beta cell types, includ ing fibroblasts, hepatocytes, muscle cells, and embryonic stem cells have induced insulin production (30 33) in non-endocrine cells. However, the challenge remains in the engineering of an insulin-producing cell that can also maintain precise control of glucose through the expression of glucose sensing or glucose tran sport genes (33,34) Another approach using gene therapy for T1D aims to curb the autoimmune response by down regulating MHC gene expression in beta cells (33,35) Studies have shown that transgenic NOD mice with different MHC class I and II alleles do not develop diabetes, suggesting that MHC recognition and antigen presentation play an integral role in the

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18 autoimmune pr ocess (36) Thus exclusively downregulating the expression of MHC expression in the beta cells may be an effective means of preventing autoimmune attack of the beta cells. Others hav e found that inducing IL 4 gene expression and secretion in islet cells may also stymie the autoimmune response (35) Beta /Islet Cell Transplantion/R egeneration When the loss of beta cells from autoimmune attack becomes significant, the production of insulin is compromised and insulin therapy must be initiated to maintain physiological control of blood glucose levels. The expansion/rege neration of beta cell mass using pharmaceuticals or transplantation can re introduce insulin production. Exenatide, a peptide incretin mimetic that has glucoregulatory actions, was approved by the FDA in 2005 for the treatment of Type 2 diabetes (37) but when us ed in combination with immunosuppressants (38) or anti infl ammatories (39) has been shown to allow for repopulation of pancreatic islet structures with functional beta cells. The use of various growth factors and small molecules were able to direct the differ entiation of stem cells into functional insulin -producing cells (40 42) Whole pancreas and islet transplantation have shown limited success due to organ rejection and limitations procuring functional islets in adequate numbers (2,43,44) However, these approaches to increasing cell mass or function only confer transient relief as survival of the beta cells is limited due to the ensuing autoimmune attack. The engineering of encapsulated islets/beta cells that escape fatal infiltration from autoimmune cells has been demonstrated in the NOD mouse though its application in human clinical remains to be determined(45) Immun e Modulation While be ta cell transplantation/regeneration restores some insulin production, this strategy only provides short -term relief from T1D as the autoimmune process is left unaddressed, leaving the novel beta cell tissue susceptible to destruction from the ongoing auto immune process.

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1 9 Monoclonal antibody therapy has been used alone or in conjunction with beta cell replacement techniques to suppress autoimmunity. Anti -CD3 monoclonal antibody (mAb) therapy was initially used for the prevention of organ allograft rejectio n in transplant recipients, but its use has been extended to autoimmune diseases as well. Anti CD3 mAb binds to the CD3 receptor on T cells, modulating the CD3 TCR complex such that it results in anergy, apoptosis, or antigenic modulation, a process in wh ich the T cell becomes unresponsive to antigen. Murine CD3 mAb therapy has treated autoimmunity in models of T1D (46) and experim ental autoimmune encephalomyelitis (EAE) (47) Another mAb, anti CD20 known as Rituximab, was used to reverse T1D in transgenic NOD mice expressing human CD20 by inducing regulatory B cells or B cell depletion (48,49) However, success using mAb in human trials has been limited. In a clinical trial using a nti CD3 therapy, progression of T1D was delayed up to 3 years (50,51) or preserved some insulin production (52) Clinical trials for antiCD20 mAb therapy are currently underway. Unfortunately, unresolved issues with cytokine release syndrome, a life threatening severe inflammatory process, and global immunosuppression have set back progress for mAb therapy (50,5255) Antigen-B ased Therapies T he development of Type 1 diabetes is based on the loss of tolerance to self -antigens expressed by beta cells such GAD, insulin, and heat shock protein (HSP) 60 (56 63) Efforts to redirect the immune response from immunity to tolerance via oral or parenteral administr ation of whole or peptides of these antigens have been successful in mouse studies, but have met with considerable setbacks in human. Clare -Salzler et al. found that early administration of GAD65 peptides to NOD mice induced active tolerance characterized by the development of Tregs that conferred protection when transferred to SCID NOD that had been given diabetogenic splenocytes (64) Similar effects were found in studies using peptides of insulin and HSP60 via

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20 alternative routes of administration (60,65) However, in a clinical trial involving genetic high risk non-diabetic subjects, the administration of oral or injected insulin ther apy had minimal effects on prevention of T1D (66,67) Moderate success was found in the Diamyd and Diapep277 trials investigating GAD and HSP antigen therapy in new onset subjects, as some reduction in loss of C -peptide appeared to correlate with treatment though no there was no effect on reversal of diabetes (56,68,69) The failure in protection using antigenbased therapies may be due to difficulties in translating the appropriate antigen dose and timing of treatment. Additionally, patients susceptible to T1D often have defects in their immune cells so it is uncertain whether the delivery of antigen to an impaired immune system may be able to correct the autoimmunity. Cellular Basis f or Type 1 Diabetes Immunotherapy Immunotherapy, which targets the underlyi ng aberrancy in T1D, appears to be the most effective strategy for T1D as this will block the autoimmune process causing beta cell destruction as well as protect new islet grafts. Antigen -nonspecific approaches to subdue the autoimmune process using monoc lonal antibodies suppress both pathological and essential immune processes, resulting in generalized immunosuppression /modulation that leaves an individual susceptible to bacterial and viral threat. Antigen -specific immunotherapy that targets only the abe rrant autoimmune processes but otherwise leaves the immune system intact is ideal. Dendritic cells, which present antigen and direct T cell responses, have been shown to induce Ag -specific T cell tolerance possibly through the generation of regulatory T c ells. Because DC uniquely orchestrate the delicate balance between T cell immunity and regulation to self antigens, DC -based immunotherapy is an attractive approach to immunotherapy for T1D.

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21 Dendritic Cells Dendritic cells (DC) are professional antigen pr esenting cells (APC) that direct the T cell response toward immunity or tolerance through receptor -mediated signals and cytokine secretion. DC were first discovered in 1972 by the group of Ralph Steinman when researchers identified a white cell in the mou se spleen that was morphologically and functionally different than classical white cells with the unique ability to extend and retract dendrites (70) The immunogenic role of DC was later elucidated in a transplantation model of graft versus host reaction. Using mixed leukocyte reaction (MLR), it was found that donor DC were more potent than whole spleen cells in their ability to elicit rejection by recipient cells, emphasizing the role of the major histocompatibility complex (MHC) in DC biology. In a two stage process, DC act to stimulate T cells by first internalizing extracellular antigens through endocytosis, processing Ag into peptide fragments within lysosomes, then transporting peptide to the cell surface for presentation to T cells via the major histocompatibility complex (MHC) molecule. The second stage involves the cell -to -cell interaction between DC and T cells where the Ag MHC complex on DC binds to the T cell receptor (TCR) providing signal 1, and T cell costimulatory molecules such as CD80(B7.1) and CD86(B7.2) may be up regulated to bind to CD28 or CTLA 4 on the T cell providing signal 2 (71) DC act to stimulate CD4+ helper T cells through MHC class II or CD8+ cytotoxic T cells through MHC class I The two -signal hypothesis of lymphocyte activation suggests that the decision between T cell activation and T cell anergy/tolerance is mediated by whether DC provides the second costimulatory signal during antigen recognition (71) However, the specific mechanisms as to how DC assume an immunogenic or tolerogenic state has not been fully elucidated, and evidence suggests that their varying immunomodulatory activity can be attributed to a variety of factors including cell lineage, maturation state, and antigen presentation.

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22 The DC maturation process is associated with many events such as loss of endocytic/phagocytic receptors, up regulation of costimulatory molecules and major histocompatibility complex class II (MHCII), change in physical attributes such as acquisition of motility and loss of adhesive structures, and secretion of cytokines (72) Classical models characterize immature DC (iDC) as expressing low levels of MHCII and co -stimulatory molecules. They are considered poor stimulators of T -cells but can engulf and process antigen efficiently. Immature steady -state DC are believed to be important mediators of peripheral tolerance as they continuously sample and present tissue -Ag without costimulation leading to tolerance induction through T cell anergy or deletion (73) Addition ally, studies using ex vivo generated iDC have demonstrated their immunomodulatory bias toward tolerance. Machen et al. found that the downregulation of maturation surface markers on DC produced an immature and tolerogenic subset of cells that significan tly delayed onset of diabetes when administered to NOD recipients (74) Others h ave also been able to use iDC to direct Th2 or Tr1 cell polarization in culture (75,76) or human subjects (75,76) through the release of IL 10. Stimulation of DC with TNF 10 produces a semi -mature DC phenotype that is characterized by highlevels of co -stimulatory molecules but low levels of proinflammatory cytokines and an acquired capacity for migration. Intravenous (IV) administration of semi mature DC to mice have been shown to induce Ag-specific T regulatory cells that suppre ssed experimental autoimmune thyroiditis(EAT) (77) This protection was attributed to, but not dependent on DC -induced IL 10 secretion by tolerized T -cells. Semi -mature DC have also been found to induce anergic CD4+ and CD8+ T cells that suppressed proliferation of syngeneic T cells in human cell cocultures (78) Mature DC (mDC), which readily migrate to draining lymph nodes (DLN) and expre ss high levels of costimulatory molecules that are required for potent T

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23 cell stimulation, have been identified as immunogenic cells. This observation is supported by early studies in which freshly isolated immature, low -costimulatory molecule expressing DC failed to stimulate responder T -cells in mouse MLR cultures, while cultured mature, high costimulatory molecule expressing DC were potent activators of T -cells (79,80) Dhodapkar et al. found that subcutaneous injection of peptide -loaded mDC stimulated a rapid induction of IFN subjects (81) Human cell cultures also revealed that mDC c an overcome regulatory cell suppression of CD8+ cells and promote expansion of antigen -specific CD8+ nave and memory cells (82) Furthermore, the production of proinflammatory cytokines by mDC, including IL 6, TNF 12, contributes to T -cell immunity (83,84) LPS -matured DC administered with anti CD40 have been shown to result in the elevated production of IL 12 and a Th1 phenotype in mice (84) This classical model of DC maturation subsets is defined predominantly in terms of phenotypic criteria, with an emphasis on the requirement of costimula to ry molecule express ion for T cell stimulation. Though the majority of the existing literature supports this general paradigm, emerging reports of tolerance induction by mature DC subsets have prompted reviews of our current knowledge. For instance, Feili -Hariri et al. foun d that mature DC were better able to prevent diabetes development (85) while Emmer et al. found that LPS -matured DC prolonged allograft survival when pretreated with dexamethasone (86) Another study found that DC mediated expansion of CD4+CD25+ regulatory cells was partially dependent on CD80/CD86 expression by DC and the production of IL 2 (87) Given these findings, it is evident the nature of DC modulation on T -cell responses is a highly complex process that is not li mited to only the maturation phenotype. Proposed factors that may contribute to DC modulation of T -cell responses include the type of maturation signal used to stimulate DC, class of cytokines secreted,

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24 and activation requirements of T -cells. Pulendran f ound that E. coli -derived LPS stimulated DC induced a Th1 response through the secretion of IL 12, while stimulation of DC with P. gingivalis derived LPS resulted in a Th2 response, suggesting that minor differences in TLR ligands may influence immune resp onse (88) Another group found that IL -2 secretion by both low and high-costimulatory mole cule expressing DC was sufficient to stimulate T -cell responses, demonstrating that cytokine secretion alone was adequate for T -cell activation (89) This observation is supported by the fact that CMV -mediated inhibition of IL 2 production by D C results in an ineffective T-cell response and thus a persistent viral infection (90) Additionally, the simultaneous induction of CD 4 T -cell tolerance and CD8 T cell immunity in mice by MHC class II -restricted ova -pulsed semi -mature DC a nd MHC class I + II -peptides pulsed DC, respectively, suggests that the decision between tolerance and immunity not only depends on the DC subset, but also on the type and activation requirements of the responding T cell (91) The tolerogenic and immunogenic nature of DC, while amply demonstrated across DC subsets, still remains to be better understood as no definitive criteria for immune activation or suppression as been identified. Modification to enhance function Dendritic cells are a heterogeneous group of cells that can exhibit many phenotypes and functions in response to their microenvironment or in vitro stimuli. The opportunity for clinicians to manipulate dendritic cells ex v ivo allows for specific engineering of DC using techniques that improve or enhance the desired function. Several strategies for DC modification include the use of retinoic acid and targeted antigen -loading. R etinoic acid Retinoic acid (RA) has been show n to have important immunomodulatory effects, particularly in the gut. RA a dietary metabolite of Vitamin A, is also locally produced by dendritic cells found in the mesenteric lymph node (mLN) and Peyers Patches (PP DC)(92)

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25 Exposure to RA through either in vitro stimulation or in vivo interaction with mLN DC or PP -DC can induce or up regulate the expression of the gut and CCR9 on T cells, B cells, and DC (92,93) RA has also been found to reduce DC -mediated production of the inflammatory cytokine IL 12(94) Additionally, RA promotes the development of Foxp3+ regulatory T cells and inhibits thei r differentiation into Th17 cells (94 97) Recently, Maynard et al demonstrated that RA has a bidirectional role in Treg development by promoting Foxp3 Tregs whil e suppressing Il 10 producing Tregs (98) These findings suggest that RA can be used to augment the immunomodulatory effects of DC therapy. DEC205. DEC205 is an endocytic receptor on dendritic cells that mediates the uptake and engulfment of extracellular antigens. By fusing antigen or DNA to an antibody against the DEC205 receptor, Ag is targeted specifically to DC and results in improved Ag uptak e, processing and presentation on MHC class I and class II molecules. Studies have found that this strategy promotes strong T cell immunity and improves vaccination results (99,100) While this technology has only been investigated for purposes of generating T cell immunity, the targeting of Ag to DC may also have applica tions to DC therapy for the induction of antigen-specific tolerance. Dexamethasone Dexamethasone (Dex) is a synthetic glucocorticoid steroid with anti inflammatory and immunosuppressive effects (101) Treatment of monocyte -derived DC with Dex maintains DC in a steady state of immaturity characterized by low expression of CD80, CD86, MHC cla ss II, and low production of IL 12(102) Th ese DC were resistant to maturation following stimulation with LPS, produced high amounts of IL 10 and maintained this phenotype long after removal of Dex stimulation, suggesting that Dex generates DC with a durable tolerogenic phenotype (103) Additionally, another study demonstrated that rat DexDC were

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26 able to induce selective expansion of CD4+CD25+ Tregs (104) Dex DC were also shown to suppress colitis in SCID -mice, demonstrating immunosuppressive function in an animal model of disease (105) Together, these findings highlight the potential of using dexamethasone to generate tolerogenic DC for immunotherapy. Current progress in clinical trials The first published report of a clinical trial using dendritic cell therapy was 14 years ago describing the use of peptide -pulsed DC for treatment of melanoma (106) Since then, over 100 trials in 15+ countries have been made, with the majo rity of studies focused on using DC therapy to induce T cell immunity for the treatment of cancer (107) Fewer clinical trials using DC therapy for tolerance induction exist, including studie s for organ transplant recipients and autoimmune disease. In 2001, a study by Dhodapkar et al investigated the use of immature peptide -pulsed DC to induce Ag-specific tolerance. They found that a single injection of influenza matrix peptide -pulsed DC was able to induce IL 10 producing CD8+ Tregs that inhibited MP -specific CD8+ effector T cell function (76) Phase I clinical trials are currently underway in Queensland and England for using tolerogenic DC in the treatment of rheumatoid arthritis (107) Another study using tolerogenic DC is in progress to establish the safety of using anti -sense oligonucleotides targeted to DC for prevention of Type 1 diabetes (107) These trials have demonstrated that while DC therapy has overall proven to be safe and well -tolerated with minimal side effects, much remains to be learned regarding how tolerogenic DC behave in human subjects. R egulator y T C ells In a healthy individual, autoreactive cells are present but do not cause autoimmunity due to effective immunoregulatory mechanisms. However in T1D the lack of an effective regulatory response allows autoreactive T cells to become pathogenic, thereby invading and destroying the

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27 pancreatic islet cells. In the early 1980s, a new class of suppressor cells that appeared to curb autoreactivity in the body was discovered (108) Since then, the work of Sakaguchi and others have identi fied these cells as regulatory T cells (Tregs), which function to suppress immune responses against self antigens by secreting immunosuppressive cytokines or by interacting directly with effector T cells (108113) Subclasses of regulatory T cells Naturally occurring Tregs are classically identified as CD4+CD25+Foxp3+ cells that originate f rom the thymus to maintain peripheral tolerance. They constitute 510% of the peripheral CD4+ T cell population and have been shown to be essential to self tolerance as removal of this subset of cells from the mouse results in inflammation and autoreactive responses that can only be rescued by their reconstitution(114) This subset of Tregs exerts their suppressor function in a contact dependent manner. Naturally occurring Tregs be come activated by Ag following engagement of their TCR, but then suppress in an antigen nonspecific manner by inhibiting IL 2 transcription in the responding population (115) Induced Tregs, which develop de novo from CD4+CD25T cells, have a CD4+CD25+Foxp3 phenotype and exert their suppression in a contact independent manner via secretion of soluble factors. Regulatory Tr1 cells can also be i nduced to develop from nave or resting T cells in the presence of IL 10, Vitamin D3, or dexamethasone (116) Tr1 secrete large amounts of IL 10 to suppress effector T cells but proliferate poorly following antigenic stimulation (115) Th3 cells, another subset of inducible Tregs, develop with TGF -beta conditioning or Ag stimulation and secrete TGF -beta to suppress in an antigen -nonspecific manner (115) CD8+ Tregs that secrete I L 10 have also been identified, and are thought to be induced through signaling from immature DC (76,117)

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28 Role of dendritic cells in regulatory T cell development and maintenance In addition to signaling T cells for effector function during periods of environmental challenge, DC also have a role in maintaining tolerance through the induction of regulatory T cells. Nave T cells can be directed to develop into Tregs fol lowing chroni c exposure to immature DC present ing self -Ag in the absence of a danger signal (75,118120) These Tregs then home to tissue where, under conditions of inflammation, are reactivated by resident DC presenting self -Ag (120) IL 10 producing DC, liver DC, and lym phoid DC have all been subsets of immature DC that were shown to induce the development of Tregs (121123) With a role in Ag presentation within a tolerogenic environment, it is evident that immature DC play a central role in the induction and maintenance of Tregs in the periphery. Antigen-Based Dendritic Cell Therapy One of the challenges facing immunotherapy for tolerance induction is maintaining control of the immune modulation such that the therapy targets only the aberrant autoimmune p rocesses while essential immune responses remain intact. The induction of global non -specific immunosuppression or immunomodulation using nontargeted immunotherapy puts individuals at risk for uncontrolled infections or tumors unless therapy can be effect ively directed toward the immunogenic antigen. Antigen peptide therapy has demonstrated success in animal studies. Tian et al. prevented T1D in the NOD mouse following intranasal administration of a GAD65 peptide (64) Ramiya and others have also found that oral or IV administration of either insulin or GAD65 antigens prevents T1D in mice (61,124126) Although peptide immunotherapy for tolerance induction has been widely proven in animal m odels (65,127130) success in clinical studies has been less forthcoming (59,131) possibly due to uncertainties i n the translation of the treatment regimen from animals to human. Difficulties in determining the appropriate antigen dose, route of administration, and timing of treatment contributes to the issue of safety, with

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29 concerns that a suboptimal treatment plan can result in the exacerbation of immunity rather than the induction of tolerance (131133) Antigen -based DC therapy circumvents this issue by controlling the direction of the immune response toward the Ag using tolerogenic DC. In autoimmune diseases such as T1D where the target antigen of the autoimmune pr ocess has been identified, the presentation of that antigen in the context of a tolerizing signal such as those given by immature DC can allow Ag -specific re -education of the immune system toward tolerance. Antigen -based DC therapy provides a directed approach toward a defined target by using tolerogenic dendritic cells pre loaded with the target autoantigen of interest. Type 1 D iabetes A utoantigens In T1D, the organ -specific T -cell attack is targeted at autoantigens such as GAD65, insulin, and IA 2 (insul inoma associated antigen 2). Antigen-based therapy for T1D has focused on using whole or peptide derivatives of these islet cell antigens for tolerance induction. Krueger et al. prevented cyclophosphamide accelerated diabetes with a single injection of i nsulin-pulsed, but not ovalbumin -pulsed dendritic cells in the NOD mouse (134) Our previous work using dendritic cells isolated from pancreatic lymph nodes presenting islet Ag prevented 100% of T1D in NOD mice, whereas DC from inguinal lymph nodes of unpulsed DC were ineffective (135) This strategy has also been employed in other disease paradigms to promote organ -specific tolerance. Verginis et al. induced IL 10 secreting CD4+25+ T cells that inhibited the development of experimental autoimmune thyroiditis (EAT) in mice after intravenous (IV) injection with thyroglobulin -pulsed, but not ovalbumin -pulsed dendritic cells (77) Similar results were obtained using myelin oligodendrocyte glycoprotein (MOG) peptide pulsed DC for protection from experimental autoimmune encephalomyelitis (EAE) (84) Clinical symptoms of multiple sclerosis (MS) in marmosets were ameliorated following immunization with MP4, a protein chimera of myelin basic protein, the antigen target of MS (136) Autoimmunity was also

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30 halted in an experimental myas thenia gravis rat model that was treated with acetylcholine receptor -pulsed dendritic cells (137) Collectively, these studies suggest that antigen based therapi es can provide specific and effective tolerance induction. Native Immunogenicity of Peptide Determinants The traditional approach to antigen -based therapies has been to identify target determinants within the autoantigen molecule that the autoimmune resp onse is directed at, then administer those target peptides or whole Ag in modalities that induce tolerance. Unfortunately, these therapies are ineffective when administered at advanced stages of disease, suggesting that there may be an altered immune repertoire with time. In T1D, the T cell reactivity is initially limited to a few autoantigen determinants. But with time, T cell reactivity gradually expands intra & inter -molecularly to additional determinants and antigens chronically recruiting nave cel ls into the autoreactive pool (62,138142) This epitope spreading gives rise to an array of determinants that have distinct immunogenic properties and possibly unique roles in autoimmune pathogenicity. The nature of these determinants immunogenicity can vary when presented either as naturally processed peptides from whole antigen or as synthetic peptide fragments (140,143,144) Determinant classes have been characterized by assessing natural and recall T cell reactivity to overlapping 20 -mers peptides of T1D autoantigens in the NOD mouse. Stimulation with dominant d eterminants defined as epitopes that are preferentially seen by autoreactive T cells due to favored processing and presentation, elicited spontaneous T cell responses f rom NOD spleen cells (144) Subdominant determinants (SD), which are secondarily processed and presented as they do not compete as effectively with DD for presentation, also generated a spontaneous response. Ignored determinants (ID) which are not processed and presented, failed to induce significant spontaneous spleen cell responses. However, if mice were

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31 immuni zed with ID, they were able to develop a recall response to the immunizing peptide, though the ability to elicit recall responses vary with age of animal for unknown reasons (144) Historically, dominant determinants (DD) are Ag determinants of choice for Ag -based therapies as they lead to induction of T cell response. SD or ID are not bel ieved to be involved in the pathogenic T cell pool and thus are not used in Ag therapies. However, it has been speculated that DD continuously recruit nave T cells into spontaneous autoimmune attack while SD and ID, which minimally or do not activate na ve T cells, have no effect on the naive T cell pool (144,145) Thus, DD reactive cells are progressively exhausted from the nave pool, l eaving only non reactive nave T cells that may be experimentally induced to respond to SD or ID. Olcott et al first examined this theory by testing peptide therapy in NOD mice with early and advanced insulitis (144) To determine which classes of determinants could prime Th2 resp onses and prevent diabetes when administered within an extended range of the disease process, an extensive panel of peptides from various beta cell antigens were used to immunize the NOD mouse at 6 and 12 weeks of age (144) Consistent with the idea of DD biased exhaustion of the nave T cell repertoire, i t was found that while both DD and ID were comparable in their ability to inhibit T1D when administered early in the disease process, only ID were effective when used during later phases of disease (144) Unknowns of AntigenBased Dendritic Cell Therapy M uch of the knowledge on how DC therapy affects immune responses has been gained from clinical studies that are directed toward the induction of Ag -specific immunity for the treatment of cancer. In the scope of translation of DC therapy from animal to huma n for tolerance induction, little is actually known. Clinicians who strive to employ DC therapy for autoimmune disease treatment have a unique hurdle in that they must be careful that they dont

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32 exacerbate autoimmunity while attempting to treat the patien t. We must learn how to maintain DC in a steady state of immaturity to prevent unintentional activation of the immune system. Another aspect to DC therapy that remains to be understood is how DC affect the overall immune response. Because DC are potent A PC, most studies use DC to augment Ag-specific immune responses and thereby assess only Ag-specific modifications following DC therapy. Additionally, studies using unpulsed DC have demonstrated protection from autoimmunity as well (85) It remains to be determined what role Ag has in DC therapy and how DC the rapy augments Ag independent immune responses. NOD Mouse Model The nonobese diabetic (NOD) mouse is a model for the spontaneous development of autoimmune Type 1 diabetes. NOD mice were developed through selective inbreeding of the progeny of a female mous e of the Cataract Shionogi (CTS) strain that had spontaneously developed autoimmune diabetes. Successive inbreeding of multiple generations yield the NOD strain that is used today(146) NOD mice develop peri -insulitis characterized by the appearance of leukocytes at the periphery of pancreatic islets as early as 3 4 weeks of age, followed by marked CD4+ and CD8+ infiltration into the pancreatic islets shortly after. Islet protein specific autoantibodies are also detectible. Onset of diabetes is seen around 12 weeks of a ge in females and several weeks later in males as beta cell death progresses and insulin production is reduced, resulting in glucosuria and non -fasting plasma glucose higher than 250 mg/dl. The incidence of diabetes is markedly different by gender, with 90100% of females compared to 40 60% of males developing diabetes by 3040 weeks of age (146) The development of diabetes is also strongly influenced by environment. Mice housed and handled in an SPF environment are more likely to develop T1D compared to mice in conventional housing. Multiple abnormalities in immune phenotype have

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33 been identif ied in the NOD mouse including defective maturation of dendritic cells (147) age related decline in the function of regulatory T cells (148) deficient hemolytic complement C5 (149) defective natural killer T (NKT) cell function (150) and defective cytokine production from macrophages (151) Su mmary and Rationale Type 1 diabetes is a debilitating disease that results in lifelong complications in spite of continued insulin therapy. With a general lack of clinical success in interventions such as islet cell transplantation, primary prevention or disease reversal holds the greatest hope for T1D. There is evidence for the prevention and reversal of autoimmune diseases through tolerance induction using both immature and mature DC as immunotherapy, but this strategy has been limited to early intervention(85,152154) As most human cases are not diagnosed until established hyperglycemia, an intervention for late onset prevention is highly desirable Our current work is focused on developing DC therapy for translation into the clinic setting, with a focus on the role of peptide presentation in immune modulation. We also seek to better understand the antigendependent and antigenindependent effects of DC therapy so that clinicians can efficiently engineer DC for a variety of immune disorders ranging from auto immunity to cancer immunotherapy.

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34 CHAPTER 2 ENGINEERING OF TOLER OGENIC DC Introduction Dendritic cells are a heterogenous population of cells with the capacity to induce divergent T cell responses. The fact ors involved in the decision in whether DC direct T cell immunity or tolerance remain to be fully elucidated as no definitive criter ion have been irrevocably validated Anticipated DC function has been attributed largely to phenotypic characteristics on the basis of m aturation state Generally, it is b elieved that immature DC (iDC) produce low levels of inflammatory cytokines, readily uptake and process Ag, and induce T cell tolerance in the absence of a costimulation signal Upon maturation with stimulatory agents such as LPS or anti CD40, m ature dend ritic cells migrate to the draining LN release pro inflammatory cytokines, up -regulate expression of costimulatory molecules, and present Ag to activate T cell immunity. In clinica l studies, iDC are preferentially chosen for therapies to induce tolerance while mDC are applied toward therapies for immune activation (155157) However, some critics speculate that iD C are in a physical state of dormancy, failing to migrate or interact efficiently with T cells for any modulation of immune response, and thus may not translate to success in the clinical setting (119,158) Others assert that iDC are an effective means to inducing Ag -specific T cell tolerance (118,155) As our understanding in DC biology continues to expand researchers have developed novel methods in the generation of DC that enhance their ability to induce T cell tolerance. Several studies examined the use of TNF in vitro sti mulation of DC to generate a third subclass of DC termed semi -mature DC (smDC) These cells produce low levels of pro inflammatory cytokines but express high levels of MHC and costimulatory molecules and have been shown to be able to potently induce T cel l tolerance to protect mouse models from

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35 autoimmune disease (91,155,159) Others have sought to modulate DC using retinoic acid, which resulted in the expression of gut homing receptors that can direct tolerogenic DC to the area of pancreatic inflammation where their action can be most beneficial (97,160) Additionally, RA -stimulated DC have been found to induce Foxp3 expression on T cells (97,160,161) Overall, these findings suggest that there are multiple strategies to develop tolerogenic DC. As our goal is to engineer DC for translation into the clinical setting for the induction of tolerance, we must first id entify potential DC subsets that are safe (low risk of immune activation) but effective ( functionally potent ) for tolerogenic therapy To address these questions, we examine the immature and semi -mature subsets of DC and characterize their expression of T cell costimulation markers and cytokine production. We also examine iDC migration following subcutaneous injection as critics argued against their capacity to travel to LN for effective T cell interaction. We then assess diabetes incidence in NOD following treatment with iDC and smDC. Finally, we investigate how retinoic acid enhances DC tolerogenic function for application in T1D. These studies reveal the target DC subset for use in tolerogenic Ag -based studies, which is examined in subsequent chapters. Materials and Methods Animals Female NOD/ShiLtj (NOD) and C57BL/6J (B6) mice were purchased from The Jackson Laboratory or Animal Care Services at the University of Florida. Bone marrow donor mice were 5 8 weeks of age. Up to five mice were hou sed together in micro isolator cages in a specific pathogen free (SPF) facility with access to food and water ad libitum. Mice were allowed to acclimate to the facility for one week prior to the initiation of any studies. Three weekly footpad injections of PBS or unpulsed DC (105 cells/mouse) were given to female NOD mice beginning at 9 weeks of age. Development of diabetes was monitored through twice weekly urine glucose

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36 testing using urine glucose test strips (Clinistix, Bayer). Upon detection of gluc osuria, a small of amount of blood was collected by pricking the tail vein and testing blood glucose using the Accuchek OneTouch glucose meter. A mouse giving 2 consecutive daily readings of blood glucose greater than 250 mg/dl was considered to be diabet ic. Retinoic acid studies were performed in C57BL/6 mice. Mice were euthanized by CO2 asphyxiation. All mice were cared for in accordance with the University of Florida Institutional Animal Care and Use Committee Bone Marrow -Derived Dendritic Cells: Isolation and C ulture The femur and tibia were removed from mice and cleaned of muscle and connective tissue. The ends of the bones were cut and bone marrow (BM) cells were flushed out with media using a 255/8 gauge needle attached to a syringe. Red blo od cells were removed from bone marrow cells using ammonium chloride potassium (ACK) lysis buffer for 2 minutes at room temperature then washed free of lysis buffer using PBS. BM -d erived DC were cultured in RPMI 1640 (Cellgro) supplemented with 10% FCS (I nvitrogen Life Sciences), 1x penicillin/ streptomycin/neomycin (G ibco), and 10mM HEPES buffer (Gi bco) at a concentration of 106 cells/ml in flat bottom 6 -well culture plates (Corning) inside a 37C humidified incubator with 5% CO2. 5 00 U/mL GM CSF (R&D Sy stems) and 1000 U/mL IL 4 (BD Pharmingen) w ere added to BM cultures to promote differentiation into DC. On day 2 or 3, half of the media was replaced with fresh media and cytokines. TNF (Sigma) was used to stimula te some c ells at 10ng/ml, or 0.5ug / ml, respectively, during the last 24h of culture. On day 5 or 6, cells were removed from the bottoms of wells with gentle pipeting and a cell scraper. DC were purified using CD11c + magnetic beads (MiltenyiBiotec). DC purity was determi ned by flow cytometry on the basis of CD11c+ expression.

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37 Flow Cytometry Cells were prepared into single -cell suspensions in FACS buffer (1x PBS / 1% FCS). Antibody used to identify dendritic cells was CD11c (HL3). Antibodies used to characterize DC matur ation were I -Ab (25 9 17), I -Ad ( 39108, cross reacts with NOD I -Ag7), CD80 (16 10A1), CD86 (GL1), and CD103 ( M290) for the gut -homing receptor. Live cells were gated from dead cells on the basis of forward/side scatter or with 7AAD (amino antimycin D) labeling. Isotype were purchased from BD Pharmingen. FACS Calibur equipment (BD Pharmingen) was used to collect flow cytometry data and results were analyzed using FCS Express (BD Pharmingen). Cytokine A nalysis Supernatants from DC cultures were collected immediately prior to DC isolation for cytokine secretion analysis. The quantity of IL 10, IL 2, TNF and IFN the Beadlyte Cytokine Detection System 1 Kit ( Millipore ). All measurements were run in du plicate. Cytokine levels were analyzed using the Luminex instrumentation (Austin, TX) and Upstate Signaling Beadlyte software (Charlottesville, VA). Retinoic Acid In vitro BM -derived DC were cultured as described previously. Retinoic acid was used to stimulate some BM cells at 10 ng/ml during the last 24h of culture. In vivo We considered a simple alternative to in vitro stimulation of DC by using a topical application of an FDA approved RA gel to condition the injection site microenvironment. Tretinoin (Clay -Park) an all -trans retinoic acid gel prescribed for the topical treatment of acne vulgaris, was applied to a 1 x 1 shaved region of skin at the nape of the neck of B6 mice Mice

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38 were placed under isoflurane anesthesia at 4%5% for induction, 1% 2% for maintenance as controlled by an anesthesia machine throughout the procedure Mice were monitored for their state of consciousness using toe/tail pinch or palpebral reflex. RA gel was appl ied to cover exposed skin several hours before DC injection, once daily for up to 5 days including a pre conditioning day in which no DC injection occurred At various time point s post injection, we collected cells from gut LN, draining axillary and cervical LN, and non draining inguinal LN to examine for the presence CFSE+ cells. Migration S tudies DC were labeled with 5 6 -carboxyfluorecein diacetate succinimyl ester (CFSE) (Mole cular Probes) at 10uM for 10 15 minutes in an incubator, then washed 3 x with ice -cold PBS to quench reaction. Cells were resuspended in PBS and injected into the footpads of 5 8wk old NOD females (70,000 DC/mouse) or 8 wk old B6 mice (1 x 106 DC/mouse) Mice were sacrificed at 24, 48, and 72h to track migration Cells from the draining and non-draining lymph nodes were harvested and analyzed by flow cytometry to detect for CFSE fluorescence Results Immature Dendritic Cells Express Low er Levels of T-cell Costimulatory Molecules Relative to TNF -mature and LPS -Stimulated Mature DC To identify phenotypic characteristics of fu nctionally tolerogenic DC we first aimed to produce and characterize three subsets of DC that were generated using variou s stimulatory reagents. Evidence in the literature suggests that the addition of activating agents such as TNF or LPS can induce maturation of DC subsets that are functionally and phenotypically distinct (77,86,118,121,155) We cultured b one marrow precursors of NOD mice in the presence of IL 4 and GM CSF for 5 6 days then purified the cells using CD11c magnetic beads We defined this group of cells as immature DC. To some cells, w e adde d TNF 0.5 ug/ ml)

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39 during the last 24 h of culture for the development of s emi -m ature or m ature DC, respectively The DC subsets were then characterized by expression of cell surface markers using flow cytometry. We assessed DC for expression of m aturation markers CD80 (B7.1) and CD86 (B7.2), which are known to be involved in T -cell costimulation through interactions with CD28 and CTLA 4 respectively We also assessed expression of I -Ag7, the MHC class II molecule expressed on NOD DC (using I -Ad cross reactive Ab) which interacts with TCR of CD4+ cells. Up regula tion of T cell costimulatory molecule expression on DC will provide the secondary signal needed by T cell s in conjunction with TCR engagement to promot e T cell activation. As shown in Fig. 2 1, we find that the unstimulated iDC subset was represented by a heterogenous group of cells with some spontaneous maturation ranging from CD80lo medCD86lo hiI -Adl o. TNF emi -mature DC were CD80medCD86medI -Adhi, while LPS -stimulated mDC were uniformly found to be CD80hiCD86hiI -Adhi. This confirms that immature DC express lower levels of T cell costimulatory molecules than semi -mature and mature DC, and suggests that iDC may be less likely to induce immunogenic T cell responses. Immature and Semi -Mature Dendritic Cells Produce Low er Levels of Pro -Inflammatory Cytokine IFN Relative to Mature Dendritic Cells Next we examine d cytokine production in supernatants collected from DC subsets cultures following 4 5 days of culture As seen in Fig. 2 2 w e show that mDC were robust producers of pro -inflammatory cytokines IFN DC produced relatively marginal levels, with 10 -fold lower IFN -fold lower TNF Semi -mature DC also produced low levels of IFN n this subsets analysis since there was an external source confounder), w ith 5 -fold lower secretion of IFN relative to mDC. A ll DC subsets produced low levels of IL 10. These findings signify that iDC and smDC are unlikely to promote an inflammatory r esponse as they produce low levels of TNF -

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40 while mDC may be bias ed towa rd a more inflammatory condition through its substantial release of TNF Immature D endritic C ells Migrate from Injection Site to Draining Lymph Node Proponents of mDC for immunotherapy argue that iDC fail to efficiently migrate to draining lymph node (dLN) to prime T -cells into a tolerogenic response (155,156) To verify that iDC can migrate from injection site to draining lymph nodes, we injected 35,000 CFSE labeled iDC into each footpad of mice and harvested cells from draining and non-draining LN to locate CFSE+ cells. Within just 24 hours, we were able to detect CFSE+ cells in the popliteal d LN while no fluorescent cells were detected in non draining LN. We found 1.91% of a sample of dLN cells were CFSE+ (Fig. 2 3). With 5 x 105 1 x 106 cells in a LN, we can estimate that 9550 19100 cells were CFSE+ in the total LN population suggesting that 2755% of injected iDC migrated to the popliteal LN, a proportion more than sufficient for peptide presentation and T cell priming. Immature Dendritic Cells are Superior to Semi -Mature Dendritic Cells for Induction of Tolerance Given our findings from phenotypic analysis of the DC subsets, we cho se to investigate the functional potential of the iDC and smDC subsets for im munotherapy. To determine whether iDC or smDC can prevent T1D in NOD mice with advance d insulitis, we administered 3 weekly footpad injections of either iDC or sm DC beginning at 9 weeks of age, then observed mice for development of diabetes. As shown in the Kaplan -Meier survival curves in Fig 2 4 neither iDC nor smDC treated mice were significantly protected from T1D. However, we do note that there was an initial delay in incidence of T1D in both groups of treated mice, followed by a drop in protection in the smDC treated mice while iDC treated mice appear to be protected for a longer

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41 period. We anticipate that while significant protection was not achieved using unpulsed iDC, the addition of antigen peptide loading or other treatments may refine the tolerogenicity of iDC. In vitro Retinoic Acid Stimulation Increases Expression of Maturation and Gut -Homing Receptors on D endritic C ells Retinoic acid has been shown to have important immunomodulatory effects. RA can induce or up regulate the expres sion of the gut homing receptor T cells from differentiation into Th17 cells, suggesting that RA can be used to augment the immunomodulatory effects of DC therapy (92,9497) To characterize the effects of retinoic acid stimulation on iDC, we added RA (10 ng/ml) during the last 24h of C57BL/6J BM culture s to iDC and examined the cells for expression of maturation markers and the gut -homi ng receptor CD103. In vitro RA stimulation increased the expression of gut -homing receptor CD103, with 0.36% and 1.02% of iDC and mDC expressing CD103+ compared to 2.49% of RA stimulated cells (Fig. 2 5 panels A C). Using iDC and LPS -stimulated DC as con trols for comparison, we also found that RA stimulation resulted in maturation of iDC as indicated by increased I -Ab and CD80 expression (Fig. 2 5 panels D -F). To facilitate the translation of RA stimulation into the clinical setting, we devised a simple m ethod of pre -conditioning the injection site microenvironment with a topical application of a FDA approved RA gel, thereby bypassing an in vitro GMP modification step of DC. We investigated the efficacy of this procedure to exert similar immunomodulatory effects on the injected DC by pre -conditioning the injection site skin for 1 day prior to DC injection again several hours before injection of CFSE -labeled DC and again at 24 and 48 hours post injection to maintain the RA environment for residual DC, if any, remaining in the injection site. At 24 and 48 hours post injection, w e harvested cells from gut LN and sp leen to examine for CFSE+

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42 cells. Unfortunately, we were unable to detect CFSE+ cells in the spleen or gut LN (data not shown). Discussion Dendri tic cell therapy is an attractive approach to immunotherapy for T1D as DC are uniquely able to orchestrate the delicate balance between T cell immunity and regulation to self antigens. However, the specific mechanisms as to how DC assume an immunogenic or tolerogenic state has not been fully elucidated, and evidence suggests that their varying immunomodulatory activity can be attributed to a variety of factors including expression of T cell costimulatory molecules and production of Th1 or Th2 pivoting cytokines. Ex vivo engineering of DC provides clinicians with the opportunity to manipulate DC with a variety of stimulatory agents that can modify or enhance constitutive DC function. We examined several methods of generating bone marrow -derived DC for T1D therapy to identify the best tolerogenic DC product. We found that iDC released low levels of TNF IFN (Fig. 2 2) Semi mature DC also produced similarly low levels of IFN (TNF external source was added for culture). Conversely, mDC were potent producers of the pro inflammatory cytokines TNF IFN 10 fold higher levels compared to iDC and smDC (Fig. 2 2) Only mDC exhibited consistent hi gh expression of T cell costimulatory molecules CD80, CD86 and MHC class II while i DC and smDC expressed low to moderate levels of these same maturation markers, respectively (Fig. 2 1) We also sought to provide evidence for efficient iDC mig ration to draining LN following subcutaneous injection. Critics of iDC speculate that in the absence of maturation, iDC have suboptimal migratory capacity that may render insufficient DC T cell interactions in LN. However, we found that 27 55% of injecte d DC traveled to the draining LN within 24h, demonstrating that iDC were capable of

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43 migrating to LN enabling interaction with T cells for potential priming into tolerance (Fig. 2 3). Collectively this data highlighted iDC and smDC as DC subsets with thera peutic potential. Next we examined the capacity for iDC and smDC to induce tolerance in vivo We found that iDC and smDC treatments were well tolerated in NOD mic e and treatment did not accelerated T1D despite the moderately elevated expression of costimu latory molecules in the smDC subset Unfo rtunately neither DC subset was able to protect NOD mice with advanced insulitis from development of T1D though it appeared iDC recipients benefitted from a noteworthy delay in development of T1D (Fig 2 4) This data suggested that further refinement in the engineering of iDC could prolong the durability of protection. Lastly, we investigate d the effect of retinoic acid stimulation on DC, which has been shown to induce the expression of gut homing receptors in DC and inhibit Th17 T cell development We first characterized DC phenotype following in vitro stimulation with RA and found that RA enhanced matura tion with up regulation of I -Abhi and CD80med (Fig.2 5 ). RA sti mulation allowed a nearly 7 -fold i n crease in the expression of the gut -homing receptor CD103 relative to unstimulated DC, and a nearly 2 -fold increase relative to LPS -stimulated DC. However, the overall proportion of CD103 expressing cells within the total stimulated population remains relatively small, thus the concentration of RA in culture may require adjustment in future studies to improve this effect. We also examined whether RA microenvironment conditioning could induce iDC homing to the gut LN particularly the pLN where DC T cell interaction s may be optimally located to facilitate modulation of the pancreatic inflammation Unfortunately, w e were not able to detect any CFSE labeled DC in gut or draining nodes following RA conditioning of the injection site microenvironment. This may be due t o inadequate cell harvesting techniques or a suboptimal concentration of RA in the injection site

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44 microenvironment. Further dosing studies may be conducted to investigate whether higher RA treatment can improve cell homing, though careful consideration must be given to balance its advantages over the possibility of severe skin irritation. Overall, we find that with low production of pro-inflammatory IFN and TNF low expression of costimulatory molecules and efficient migratory capacity, iDC have the gr eatest potential for safe and effective application into DC therapies for tolerance induction. Of importance, the observed delay in T1D development in NOD mice treated with iDC highlights the therapeutic potential of this DC subset We also find that the modest improvement in DC expression of CD103 with RA stimulation (including additional effect on induction of Foxp3+ Tregs with RA DC is described in chapter 5) suggests that RA stimulatedDC may be worthy of further characterization in tolerance studies. In subsequent chapters, we examine how to develop better engineer iDC therapy for translation into late intervention using subclasses of Ag peptides for DC pulsing that may confer significant T1D protection.

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45 Fig ure 2 1. Characterization of DC by flow cytometry. Bon e marrow -derived DC from 6 week old female NOD mice were cultured without activation agents (immature i DC), or in the presence of TNF ( s emi -mature sm DC ) or LPS ( m ature m DC ) and examined for expression of maturation markers. Black line = isotype control, red line = iDC, green line = smDC, and blue line = mDC. Data shown is representative of >5 similar assessments by flow cytometry. Fig ure 2 2. Luminex analysis of cytokine production by DC subsets. Supernatants collect ed from NOD bone marrow -derived DC cultures were analyzed for the presence of cytokines following 5 6 days of culture iDC = immature DC, smDC = semi -mature DC, mDC = mature DC *TNF for smDC since an external source was added for its culture. IFN-g TNF-a IL-10 0 50 100 150 200 250 300 350 1100 1250 2800 2900iDC smDC mDC pg/ml

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46 Figure 2 3. Migration of iDC. CFSE -labeled iDC were injected into footpads of NOD mice (35,000 cells per footpad). At 24 hours following injection cells were harvested from non -d raining cer vical LN and draining popliteal LN and analyzed by flow cytometry to detect for CFSE+ cells Data shown is representative of results from 2 experiments. Figure 2 4. Diabetes incidence in NOD mice following DC therapy. Female NOD mice received three weekly footpad injections of PBS, immature DC, or TNF -stimulated semi mature DC beginning at 9 weeks of age. A mouse was considered to be diabetic following two consecutive daily measurements of blood glucose > 250 mg/dl. Red arrows depict time of DC injection. 0 20 40 60 80 100 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 % Protectedage (weeks ) PBS (N=10) iDC (N=10) TNF (N=14)

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47 Figure 2 5 Effect of retinoic acid on dendritic cell maturation E xpression of the gut homing receptor CD103 was examined in CD11c+ gated C57BL/6J bone marrow cultures of A) unstimulated cells, B) RA -stimulated cells, and C) LPS -stimulated cells CD103 positivity was determined by isotype control. M aturation markers were examined in D) unstimulated cells E) RA -stimulated cells and F) LPS -stimulated cells by flow cytometry. Cells analyzed are collect ed from Day 6 Forward ScatterCD103CD80I -AbA B C D E F

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48 CHAPTER 3 ROLE OF ANTIGEN PEPT IDE IN DC THERAPY Introduction Though antigen -based DC therapies for T1D have been shown to be effective in the NOD mouse, many depend on a restricted regimen in which the timing for the initiation of therapy is critical to the disease outcome (162) Since the immunopathology of T1D develops early and ensues for a significant period before clinical symptoms of diabetes manifest early intervention may not always be possible. Additionally, individuals without family history of T1D are unlikely to undergo screening for early prevention. Thus, a reliable immunotherapeutic approach that can be initiated within a wider range of tim e for late -onset prevention or established -disease reversal is highly desirable. Traditional antigen based DC therapies have focused on identifying target autoantigens or peptide epitopes within self -Ag that are dominantly -expressed and are primary targets of the autoimmune response. These whole antigens or dominant determinants (DD) are then administer ed in modalities that induce tolerance and this treatment has been shown to prevent T1D as well as other autoimmune diseases in mice when applied early in the autoimmune process. Unfortunately, this strategy becomes ineffective when administered at advanced stages of T1D suggesting that there may be an altered immune repertoire with time (103,144) D ominant determinants within an autoant igen are preferentially recognized by autoreactive T cells in the early disease stage and primed into effector function, contributing to the disease process. Subdominant determinants (SD) which do not compete as effectively as DD, and ignored determinant s (ID), which are not processed and presented at all, are not believed to be involved in the pathogenic T cell pool and thus are not used in Ag therapies. However, it may be possible that DD continuously recruit nave T cells into spontaneous autoimmune a ttack while

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49 SD and ID, which minimally or do not activate nave T cells, have a minimal effect on the nave T cell pool (144,145) Thus, DD reactive cells are progressively exhausted from the nave pool, while uncommitted nave T cells remain available to be primed into regulatory function by SD and ID even at later stages of autoimmunity. We aimed to examine how using different classes o f determinants in Ag based DC therapy can affect disease outcome when the treatment is initiated after the autoimmune process is well established. We begin treatment using DC pulsed with synthetic peptides of DD, SD, or ID in 9 week -old NOD mice with adva nced insulitis. By using DC pre -loaded with synthetic peptides, we can reduce peptide competition in vivo and bypass natural antigen processing to selectively present the desired Ag epitopes. Materials and Methods Animals Mice were cared for as described in the Materials and Methods section of Chapter 2. Mice in short term treatment studies were treated with one DC injection per week for three weeks, then observed for development of diabetes. Mice in longterm treatment study received once weekly DC inje ctions up until euthanized for organ harvest. All mice received 100,000 DC per injection, divided into 50,000 DC per footpad. Cells were suspended in PBS for injection. Peptide s Peptides were purchased from Peptides International (Lou is ville, KY) and Bio -Synthesis, Inc. (Lewisville, TX) and determined to be of > 90% purity by HPLC analysis. All peptides are tested to be endotoxin -free. L yophilized peptides were dissolved in RPMI media at 1mg/mL, then sterile filtered using a syringe ap paratus (Gibco). Once re suspended in media, peptides were stored at 4C as a working solution for up to 2 months. Lyophilized peptides were stored at 20C indefinitely. Dominant determinant s used were 23 (SHLVEALYLVCGERG),

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50 GAD65217236 (EYVT LKKMREIIGWPGGSGD), and proinsulin C19 -A3 (GSLQPLALEGSLQKRGIV). Subdominant determinant used was GAD657897 (KPCNCPKGDVNYAFLHATDL ). Ignored determinant used was GAD65260279 (PEVKEKGMAALPRLIAFTSE). Dendritic Cell Peptide P ulsing DC were pulsed with 3uM of peptide in cRPMI for 1 2h in a humidified incubator 37 C with 5% CO2. Peptide pulsing concentration and duration were determined in previous experiments (unpublished data). Cells were washed 3x and resuspended in 1x PBS at 106 cells/mL for injection Histology NOD pancreata were immediately removed from the mouse following CO2 asphyxiation. Each pancreas was dissected along its longitudinal axis to preserve its anatomical asymmetry and distributed for fixed and frozen processing. For fresh samples, pancreas were embedded in Tissue Tek OCT (Bayer) and stored at 80 C until immunostaining. For fixed samples, pancreas were fixed in 4% paraformaldehyde at room temperature overnight, then transferred to PBS. Further tissue processing, including section ing and staining, were performed by the University of Floridas Molecular Pathology and Immunology Core. Briefly, sections through the fixedpancreas were paraffin -embedded, sectioned and collected 100 micron apart, then stained with H&E. Six non adjacen t tissue sections from each animal were used for insulitis grading and islet number enumeration Islets were visualized by light microscopy at 40x, and were counted and graded blindly by a single observer. Grading scores were assigned as follows: 0 = no infiltration, 1 = peri insulitis, 2 = <50% intra insulitis, 3 = insulitis.

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51 ELISA for Global Suppression A nalysis Eig ht -week old female NOD mice receive d PBS, unpulsed, or peptide -pulsed DC injections as described pr eviously, once weekly for three consecutive weeks. One week following the last injection, mice were immunized in the footpad with 100ug/mouse of KLH (Calbiochem ) in Alum (Pierce) weekly fo r two weeks. Ten to fourteen days following the final KLH immuniza tion, serum was collected from mice for the detection of antibodies to KLH by ELISA ( Life Diagnostics ). Statistical Analysis Data was analyzed using the Kaplan -Meier survival curve with logrank test to determine if treatment provided protection. Stude nts t -test was also used to identify statistical differences. The Grubbs test identifies outliers in triplicate wells of proliferation assays A criterion of p<0.05 w as used to define significance Results Subdominant Determinant Pulsed DC Therapy Protects A gainst T1D Work by Kaufman s group demonstrated that DD are ineffective for tolerance induction when applied as peptide therapy during advanced insulitis We examined the use of DD from the insulin, p ro insulin, and GAD molecules as peptide -base d DC therapy and confirmed that DD were not protective in the context of iDC when given to older NOD mice (data not shown). Thus we next sought to assess the potential of using other classes of determinants. Subdominant determinants, which do not compete as competitively with DD for processing and presentation, may have a larger repertoire of nave T cells available for priming. By pre loading DC with SD before infusion into mice, we can bypass peptide competition in vivo and allow these epitopes to be p resented more efficiently to evoke tolerogenic responses from nave T cells. We gave three weekly treatments of either PBS, unpulsed DC, or SD -pulsed DC to 9 week -old NOD mice and

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52 observed for development of T1D. As shown in Fig. 31, we found that recipi ents of SD DC, but not PBS or unpulsed DC, were protected from T1D (p=0.05). Interestingly, we noted that SD DC w ere able to significantly delay T1D in 100% of SD -DC recipients through the 17th week of age while 40% of PBS controls were diabetic. This suggests that complete protection was conferred for over 5 weeks following administ ration of the last DC treatment, however the protection was not durable for the life of the animal Pancreatic Islet Survival is Enhanced in S u bdominant Determinant P ulsed D C R ecipients Though SD DC were not able to completely protect mice from T1D, we were enc ouraged to learn that SD DC delay ed development of diabetes and that protection was significant To better understand how DC therapy affected SD DC treated mice, we ha rvested pancreas following treatment and compared islet survival to mice that had received unpulsed or DD puls ed DC. We found that while the grade of T cell infiltration into islets was similar in all treatment groups of mice (data not shown), there was a consistently large r number of islets remaining in mice receiving Ag -pulsed DC therapy (Fig3 2). Specif ically, non -diabetic mice in both Ag DC treated group s had a greater number of islets compared to non-diabetic unpulsed DC mice. D iabetic mice receiving SD DC had a greater number of islets compared to the unpulsed DC group. Of interest, diabetic SD DC treated mice had an average islet number similar to that of non -d iabetic unpulsed controls. It is known that beta cell re generation, while slow, does occur in the presence of the autoreactive T cell response that continuously destroys islet tissue. However, a balance in favor of regulatory T cells versus immunogenic T cells may be able to push the overall response toward tolerance. It is possible that th e islet preservation observed was achieved through the induction of regulatory T cells that was enhanced with Ag pulsed DC treatment, and to a greater extent with SD -pulsed DC treatment, but was not enough to completely quench the infl ammation

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53 generated from the pathogenic T cells. The issue of regulatory T cell induction by DC therapy will be further discussed in Chapter 5. Ignored Determinant -Pulsed DC Therapy Confers Complete Protection Against T1D But May Require Continuous Treatme nt Subdominant determinant -pulsed DC treated mice were protected from T1D as demonstrated by a significant delay in disease onset. However, complete protection would be ideal in the clinical setting. As ignored determinants do not naturally elicit T cell responses we hypothesized that a larger pool of nave T cells remain available for priming into tolerance compared to SD, which generates a moderate T cell response. This advantage in available nave T cell pool size may translate into better protection Therefore, we performed another study using ID pulsed DC in 9 week old NOD mice with a dvanced autoimmunity. We administered three weekly injections of PBS, unpulsed, or ID -pulsed DC to mice and observed them for the development of T1D. Surprisingly w e found that ID DC treatment was not able to significantly protect mice from T1D though we did observe an initial delay in T1D development all (Fig 3 3 ). We speculated whether this was due to a lack of constitutive presentation of the ID that is needed to maintain a regulatory cell response, so we refined this study to include repetitive injections that allow ed for consistent presentation of the normally unpresented determinant. As shown in Fig 3 4 w e fou nd that continued treatment of mice with DC presenting the ID confers complete protection up to the 17th week of age while other groups became diabetic (p=0.04) T1D Specific Peptide Pulsed DC Therapy Does Not Alter Natural Immunity to Environmental Challenge: KLH Study Because we observed an init ial delay in development of T1D in all mice receiving DC therapy, it is uncertain whether the apparent DC -induced protection against T1D is actually due to an overall dampening of the immune response. We sought to evaluate whether DC therapy confers speci fic protection against T1D, or whether the observed protection was a n artifact of

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54 global immunosuppression that renders mice tolerant to all immune challenges. We aimed to test this by evaluating the ability of DC treated mice to respond to a non T1D spec ific challenge. We administered either PBS, unpulsed, or ID -pulsed DC therapy as described previously, then immunized the mice with keyhole limpet hemocyanin (KLH), a protein commonly used to examine and elicit immune responses. Two weeks following KLH i mmunization, we collected serum from the treated mice to detect if a n antibody response was mounted against KLH. As shown in Fig 3 5, there were no differen ces in the ability of DC treated mice to generate an antibody response to KLH challenge as compared to PBS treated mice, demonstrating that normal immune processes were intact and the pr otection previously observed can be attributed to T1D specific protection. Discussion Type 1 diabetes is a dynamic autoimm une disorder characterized by T cell -mediated destruction of pancreatic islets driven by an e xpanding T cell autoreactivity toward beta cell autoantigens. Published s tudies using Ag -based DC therapy commonly aimed to identif y the target antigen of the immune response and re administered peptide deter minants of those antigens that were found to elicit the pathogenic response using tolerogenic DC However, the efficacy of this strategy deteriorated if initiation of treatment was delayed until advanced stages of the autoimmune process suggesting an alt ered immune repertoire wit h time. We speculated that dominant determinants identified to be the initiators of the autoimmune response chronically recruits nave T cells into the pathogenic pool, thus the re administration of these determinants only reactivated cells that were programmed to respond pathogenically. However, subdominant or ignored determinants, which have a minimal impact on nave T cell activ ation, should have large pools of nave T cells available for priming into tolerance when we bypass natural antigen processing to experimentally present these peptides. To test this idea, we treated 9 week old

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55 NOD mice with advanced autoimmunity using var ious classes of antigen determinant -pulsed DC and evaluated protection from T1D. We found that DD DC were not able to protect mice when treatment was initiated during advanced autoimmunity. However, r ecipients of SD DC and ID DC were significantly protec ted from T1D with a delay in initial onset of diabetes, though ID DC required continued treatment to achieve complete protection. This requirement may be due to a lack of constitutive ID -presentation necessary to maintain the pool of regulatory cells. Th is theory is further reinforced by the histological observation that SD DC treated mice had greater islet survival compared to other groups that were not protected. When we examined the islets of mice treated with unpulsed, DD, and SD pulsed DC (this anal ysis was performed before initiation of ID DC study), we found that non -diabetic mice receiving Ag -pulsed DC therapy had a greater number of islets compared to unpulsed DC recipients. Diabetic SD DC treated mice had a significantly greater number of islet s compared to the diabetic unpulsed DC group (p=0.02). Of interest, diabetic SD treated mice had a similar average islet count to non -diabetic unpulsed mice. The grade of islet infiltration was similar in all treatment groups, though it is unclear whether the observed infiltrate were regulatory or inflammatory T cells. Cell subset specific antibody-staining of pancreas in future studies will enable identification of these lymphocytes. To exclude the possibility that the observed protection from T1D was d ue to global immunosuppression, we examined whether NOD mice could generate a normal immune response to a non T1D related antigen challenge following treatment with DC therapy. We immunized PBS, unpulsed DC, and ID -DC treated mice with KLH and examined th eir serum antibody responses. All DC treated mice were able to mount antibody responses to KLH in a

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56 manner comparable to PBS controls, demonstrating that normal immune responses were intact and the previously observed protection could be attributed to dia betes -specific protection. Overall, these results provide evidence for the existence of a relationship between native determinant immunogenicity and developing immune repertoires in dynamic Ag -based disorders that was previously unknown. T hese fin dings suggest that the selection of autoantigen peptide for use in therapies aimed to prime nave T cells has a critical impact on the efficacy of protection against dynamic autoimmune disease s. Thus, special measures should be taken to consider the facto rs of the timing of treatment initiation, native peptide immunogenicity, and the state of nave T cell repertoire in the design of Ag -based therapies. As our results indicated, SD and ID may be best for induction of tolerance when treatment is initiated a fter established autoimmunity. Taken together, these findings can guide the engineering of Agbased DC therapies for the prevention of T1D and other dynamic autoimmune diseases.

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57 Figure 3 1. Kaplan Meier survival curves of female NOD mice following PBS, unpulsed, and SD pulsed DC therapy. Red arrows denote time and frequency of DC treatment. P values represent difference compared between PBS and DC treatment groups. Figure 3 2. Histological assessment of islet number in NOD mice following DC treatmen t. Pancreas were fixed in 4% paraformaldehyde and paraffin embedded. 4um sections were collected at 100um intervals, then stained with H&E. All pancreas analyzed were colle cted from mice ages 19wks+. Islets were counted blindly by a single observer. (p -value represents comparison to diabetic unpulsed DC group) unpulsed DC DD DC SD-DC 0 20 40 60diabetic non-diabetic Average Islet# N=7 N=9 N=8*p=0.02

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58 Figure 3 3 Kaplan -Meier survival curves of female NOD mice following short term PBS, unpulsed, and ID pulsed DC ther apy. Red arrows denote time and frequency of DC treatment. DC treated m ice were not significantly protected from T1D. Figure 3 4 Kaplan -Meier survival curves of NOD mice following continuous PBS, unpulsed, and ID pulsed DC therapy. Red arrows denote time and frequency of DC treatment. P values represent difference compa red between PBS and DC treatment groups.

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59 F igure 3 5 Antibody response following KLH immunization in control and DC treated mice. NOD mice were treated with 3 weekly injections of either PBS or DC (N=3/group) Two weeks following DC therapy, mice were immunized with 2 weekly injections of KLH. Serum antibody levels were assessed 14 days following final KLH immunization. 0 10 20 30 40 50 60 70 PBS unpulsed DC ID pulsed DC anti -KLH IgG (units/ml)

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60 CHAPTER 4 ANTIGEN INDEPENDENT EFFECTS OF D ENDRITIC C ELL THERAPY Introduction Because dendritic cells are potent antigen presenting cells most studies use DC to augment Ag -specific immune responses and thereby assess only Ag-specific modifications following DC therapy. Animal studies using DC therapy for tolerance induction have primarily examined devia tion of immune responses toward the antigen of interest. Much of the knowledge on how DC therapy affects immune responses in humans has been gained from clinical studies that are directed toward the induction of antigen -specific immunity for the treatment of cancer. In the scope of translation of DC therapy from animal to human for the treatment of autoimmune disease little is actually known as to how the overall immune response is affected. Consequentially, there is a gap in knowledge in how DC therapy contributes to antigen nonspecific responses. Additionally, studies using unpulsed DC have demonstrated protection from autoimmunity as well raising the questi on of what role antigen loading plays in DC mediated immune responses (85) We sought to address these questions by evaluating antigen independent i mmune responses following DC therapy. We examined spleen cell responses in the presence and absence of T1D -specific peptide stimulation in proliferation assays characterized early and late changes in immune cell s ubset frequencies and function using flow cytometry and assessed whether these DC related changes occur in non autoimmune prone mouse strains. Following identification of the immune modulation in vitro we sought to confirm the findings in vivo using BrdU treatment of mice. Finally, we collect ed RNA from spleen cells of mice to perform gene array analysis of differential expression of cytokines and receptors genes. These investigations will provide insight into what immunological changes occur with DC therapy, and provide evidence for the mechanisms that may be driving these changes.

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61 Material and Methods Mice Mice were treated as previously described in the Materials and Methods sections of Chapters 2 and 3 unless otherwise noted. Proliferation A ssay Suspensions of spleen cells were in serum -free HL 1 media (Biowhittaker Cambrex) with the addition of penicillin/streptomycin/neomycin (Gibco), and L -glutamine (Gibco) in triplicate with a selected peptide (25uM). Cells were cultured at 1x106 cells/well in roundbottom 96-well plates at 37C. At 72h of culture, 1 Cu 3H thymidine (Amersham Biosciences) in 50 l of media was added per well and allowed to incorporate for 12 16h. Cells were harvested and washed using an automated cell harvester (Perkin Elmer), and radioactivity was analyz ed using a liquid scintillati on counter. Stimulation index was determined as proliferation (measured in cpm) in response to peptide stimulation relative to proliferation without stimulation. Cpm outliers identified by Grubbs test were removed from analy sis. Flow C ytometry Cells were prepared into single -cell suspensions in FACS buffer (1x PBS / 1% FCS) and blocked in Fc Block CD16/32 (2.4G2) Antibody used to identify dendritic cells was CD11c (HL3). Antibodies used to characterize T cells were CD3 (1452C11) CD4 (RM4 5) and CD8 a (53 6.7) Antibodies to characterize B cells were B220 (RA3 6B2) and CD19 (1D3) We also used CD25 (PC61) and Foxp3 (FJK 16s) to assess regulatory T cell population, CD11b (M1/70) to assess macrophages, and CD44 (IM7) and C D62 (MEL 14) to assess memory T cells, CD138 (2812) for plasma cells, and CD80 (1610A1) and CD35 (8C12) for memory B cells. Live cells were gated from dead cells on the basis of forward/side scatter or with 7AAD (amino antimycin D) labeling. Isotype co

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62 or eBiosciences FACS Calibur equipment (BD Pharmingen) was used to collect flow cytometry data and results were analyzed using FCS Express (BD Pharmingen). BrdU S tudy BrdU is a synthetic nucleoside that is an analogue of thymidine that will replace thymidine during DNA replication and is commonly used in the detection of proliferating cells in living tissues. To assess in vivo proliferation, we administered daily intraperitoneal injections of BrdU (bromodeoyxyuridine) in sterile PBS (2mg/100ul/mouse) to mice (treated with PBS or DC as previously described) for 4 days, then sacrificed the mice 1 2 days following final injection t o harvest organs, LN and spleen cells for analysis of BrdU incorporation. Mice with long term exposure to BrdU at this dose have a reported reduction in weight gain and exhibit general malaise. Mice with short term exposure (<1 week) to BrdU do not suffe r from significant distress. We monitored mice daily to observe for signs of distress such as lethargy, dehydration, decreased grooming and mobility. Histology Spleen s liver s and pancrea ta were fixed in 10% formalin at room temperature for 24 48 hours Tissues were embedded in paraffin and sectioned at 4 um for staining using anti BrdU HRP Ab and DAB detection and counterstained with hematoxylin. Two sections per sample were collected 100 micron apart for analysis using Aperios Spectrum ScanS cope im aging software. The frequency of BrdU positive cells was determined using ScanScopes image analysis algorithm that detects positively stained cells on the basis of programmed color and saturation sensitizers within a measured tissue area. Percent BrdU p ositive is calculated as area positive/area total.

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63 RT -PCR and Gene A rray RNA was isolated from the spleen cell s of treated mice using the Ambion RNAcqueous 4PCR isolation kit. Cells were disrupted in lysis/binding solution followed by an equal volume addi tion of 64% ethanol, then purified through a filter column and washed 3x. Purified RNA was eluted into nuclease -free water with DNAse buffer and RNAse inhibitor (Ambion). RNA samples were submitted to the University of Florida ICBR to determine RNA purit y and concentration on the basis of RNA integrity number using the Agilent Bioanalyzer RNA concentrations were then standardized and prepared into cDNA using the Applied Biosystems Taqman RT Reagents kit. cDNA was amplified using primers designed by the Lonza Gene Array Mouse Common Cytokin es and Receptors kit with SYBR G reen Fast mix (Quanta) for quantitative PCR analysis Data was sent to Bar Harbor for analysis using their proprietary software for Global Pattern Recognition, which uniquely determines fold change with respect to data consistency, and normalizes experimental data on the basis of relative gene expression rather than relying on a predetermined housekeeping gene. Results Homeostatic P ro liferation is Observed F ollowing DC Therapy: Immediat e a nd Sustained Effects The spleen is a major site of immune cell interactions and antigen processing, with active processes that contribute to the overall immune status (163,164) Thus, we sought to examine cellular responses in this immune cell rich environment. To evaluate the spleen cell response following DC therapy, we cultured spleen cells with and without peptide stimulation for 72 hours then observed for proliferation using 3H thymidine incor poration We found that in the absence of peptide stimulation, spleen cells isolated from DC treated mice had 3 14 fold increase in proliferation compared to PBS treated mice (Fig. 4 1). This effect of spontaneous proliferation

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64 was enhanced in mice recei ving Ag pulsed DC but did not increase with recall peptide challenge. The proliferation was seen as soon as just 2 weeks following the last DC treatm ent at 14 weeks age (Fi g. 4 1A) and continued into 40 weeks of age 29 weeks after the cessation of treatm ent (Fig4 1B). Homeostatic P roliferation Occurs i n Healthy and Autoimmune Mouse Strains Following DC Therapy Because NOD mice have been shown to have abnormalities in the immune function of many cell types including differences in DC phenotype and function (147,165168) we evaluated whether this homeostatic proliferation was a true effect of DC therapy or only an effect associated with immuno therapy in an animal wi th aberrant immune cell subsets We administered DC therapy to the autoimmune NOD mouse model as well as the C57BL/6J and Balb/c healthy mouse models and evaluated spleen cell proliferation. As depicted in Figure 4 2, homeostatic proliferation following DC treatment occurs in both NOD and no n autoimmune prone mouse models, demonstrating that DC therapy results in a reprogramming of immune cell homeostasis. Additionally, this pattern is independent of route of administration, as B6 mice were treated with intravenous tail vein DC injections while NOD and B6 mice were given subcutaneous injections. Identification of Proliferating Cell Populations Next, we sought to distinguish proliferating cell subsets by labeling spleen cells with CFSE in the proliferation assays. Following 7284 hours of culture, we harvested the cells and labeled them using fluorescent antibodies for flow cytometry. We used surface markers to identify dendritic cells, macrophages, plasma cells, memory T and B cells, NK cel ls, and T and B lymphocytes Of the cell populations examined, we found that the majority of the proliferating cells belong to the CD4+ and CD8+ T and B cell subsets (Fig 4 3) collectively accounting for

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65 7298% of the proliferation (percentages shown in red) Other subsets comprised a small percentage of the proliferation population (data not shown). While DC treated mice exhibit greater proliferation of spleen cells relative to PBS controls, the proportion of proliferating cell subsets remain similar. Importantly, these findings remained consistent over the course of the study ranging from assessments between 1341 weeks of age. DC Therapy Reprograms Cytokine Production o f Proliferating T Lymphocytes We have found that homeostatic proliferation occurs immediately after DC therapy, remains durable long after cessation of treatment, and affects both autoimmune and healthy mouse strains. Interestingly, t he spontaneous proliferation is also independent of disease outcome as both diabetic and protected mic e demon strate this effect. Thus, it is unclear what impact this immune modulation may ha ve in disease. To investigate wh ether these proliferating cells have altered function, we evaluated the cytokine production of the proliferating lymphocytes. W e gate d proliferating cells on the basis of CD4 and CD8 positivity and examined their IL 10 and IFN -g production. We find that T cells f rom all DC recipients exhibit a shift from IFN -g to IL 10 production. As shown in Fig. 4 4, u pper left quadrant statistics s how a 2 fold increase in IL 10 and 2 -fold decrease in IFN -g in CD4 cells, and a similar change in CD8 cells. No changes in cytokine production between control and DC treated mice were se en within the B cell population. DC Therapy Results i n Differential G ene Expression : Analysis o f Cytokine a nd Chemokine Genes To understand what may be driving the proliferation, we performed a gene array analysis examining the expression of common cytokines and receptor genes that may have a role in directing immune cell development and function. We treated groups of mice (N=4/group) with PBS, unpulsed DC, SD DC, and ID DC and performed proliferation assays as described before,

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66 then isolated RNA from the cultured cells. Following RT -P CR transcription of RNA into cDNA, we performed qPCR to determine gene expression. We assessed differences by comparing PBS and DC treated mice as this is where we had observed differences in proliferation. We also sought to detect gene expression changes between PBS and ID DC treated mice as our earlier studies found significant protection in this group. Lastly, we also examined for differences between unpulsed DC recipients and antigen -pulsed DC recipients since the loading of antigen appeared to enhan ce the immunomodulatory effects. We identified notable but not statistically significant changes in the expression of IL 17f, CXCL12, and CCL12 within these comparison group s (Fig. 4 5) Changes for only these genes that received both the highest rankin g for each analysis and a low p -value are described in detailed. As seen in Fig. 4 5 we fou nd that expression of IL 17 f was reduced 2 to 11 -fold in DC -treated mice. IL 17f is a proinflammatory cytokine that is mainly produced in the spleen and has been found to have a role in autoimmunity and Type 1 diabetes (169171) Specifically, mice receiving DC therapy exhibited a 2.3 -fold reduction in expression of IL 17f compared to PBS cont rols (p=0.081) (Fig4 5A) while mice receiving ID -pulsed DC exhibited an over 11 -fold reduction (p=0.056) (Fig4 5D) These results suggest that the reduction of pro -inflammatory IL 17 is not only correlated with homeostatic proliferation of spleen cells, but also contributes to protection as ID pulsed DC treated mice were protected from T1D. We also discovered differences between unpulsed and antigen-pulsed DC treated mice. Mice receiving an antigen pulsed -DC had a 3.8 -fold increase in expression of CXCL12 compared to unpulsed DC recipients (P=0.071) (Fig. 4 5F). CXCL12, also known as SDF 1, is a chemokine that has a role in the generation and proliferation of early B cell progenitors, as well as a role in T cell a nd hematopoietic stem cell (HSC) trafficking (172,173) Additionally, w e

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67 found that mice receiving SD -DC exhibited an 18.9 -fold increase in expression of CCL12, also known as MCP 5, compared to unpulsed DC recipients (p=0.056) (Fig. 4 5F). CCL12, the ligand for CCR2, is a chemoattractant for eosinophils, monocytes, fibrocytes and macrophages (174176) CCL12 is constitutively expressed in thymus and lymph nodes, but under inflammatory conditions its expression is induced in macrophages and mast cells and has a role in allergic inflammation. Additional changes in expression of various genes were also noted betwee n other group comparisons though not statistically significant (Fig. 4 5A F). In Situ Observation of Homeostatic Proliferation The homeostatic proliferation observed may be driven by conditions that are only favored in culture. To determine whether there is spontaneous cell proliferation in vivo we treated mice with BrdU, a synthetic thymidine analogue that is incorporated into the DNA of replicating cells, and collected spleens to detect for BrdU incorporation. Splee ns were fixed, sections, and stained for the presence of BrdU. Using an image analysis software program that detects BrdU+ cells within defined tissue areas, we were able to calculate percentages of proliferating cells. We found that there was no differe n ce in percentage of cells proliferating in mice treated PBS compared to DC -treated mice (Fig. 4 6 A ), suggesting that the homeostatic proliferation only occurs in vitro Additionally, the percentages of BrdU+ proliferation in the spleen determined using a n image analyzer fell within expected ranges for normal mice. To better understand potential differences between in vitro and in vivo proliferation, we examined spleens sections to identify sites of proliferation within the splenic architecture. The splee n has distinct morphological zones with discrete functions. The two main compartments are the red pulp and the white pulp. The red pulp is an area for the filtering and cleaning of damaged erythrocyte s and foreign debris from blood and acts as a storage site for erythrocytes, platelets, and iron(177) Hematopoietic stem cells, lymphocytes, and macrophages can also be found in

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68 the red pulp. The white pulp is a major site of immune interactions where a quarter of the bodys lymphocytes resides along with DC, macrophages, and plasma cells and consists of the periarteriolar lymphoid sheath (PALS), the follicles, and the marginal zones. We find that the majority of the BrdU+ cells are located within the red pulp, with some positive cells within the germinal centers of PALS, where CD4+ cells are predominant. This distribution of cellular proliferation was consistent between mice in all groups. A representative section is shown in Fig. 4 7. Because our earlier findings had shown that SD DC treated mice had a greater number of islets compared to PBS treated mice, we sought to determine whether there was evidence of increased beta cell regeneration. We examined pancreata and found no differences in BrdU incorporation (Fig. 4 6B). As expected, BrdU incorporation in control liver tissue was also similar in all treatment groups (Fig. 4 6C). Discussion Classical d endritic cell therapy has been based on antigen presentation to direct the immune response in an a ntigen specific mann er Few studies have investigated the potential of using unpulsed DC in immune modulation as DC are generally hailed for their ability to potently present antigen, and even fewer have examined the deviation of non antigen specific immune responses followi ng therapy. We present evidence demonstrating that de ndritic cell therapy modulates th e immune response in an antigen independent manner, as both unpulsed and antigen -pulsed DC results in immune modulation. We also report that th e resulting changes are n onantigen specific as we find reprogrammed spleen cell responses in the absence of antigen stimulation in the form of homeostatic proliferation. As shown in Fig. 4 1 this occurs immediately just 2 weeks after treatment and remains durable up through 40 weeks of age. With this finding, w e attempted to identify the earliest this phenomenon occurred and discovered that

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69 the effect was reproducible with only 2 weekly injections of DC and seen 2 weeks following the last injection (data not shown). Thus we conclude that DC therapy potently reprograms the spleen cell response immediately after treatment as its effects require few treatments and the response appears early and is durable long after the cessation of treatment as the effect can be seen in mice th at are over 40 weeks, more than 29 weeks after the final DC treatment. The homeostatic proliferation can be seen in both healthy and autoimmune mouse strains with normal and aberrant DC phenotypes, confirming that the effect is a true immune response to D C ther apy. To characterize this immune modulation, we used flow cytometry to identify the proliferating cell subsets and found that 72 98% of the proliferating cells were within the lymphocytes subset, with B cells accounting for the majority of the prolif eration. CD4+ and CD8+ T cells accounted for 16 29% of the proliferation. However, these proportions were similar to what is observed in PBS treated mice, indicating that while the proliferation is significantly greater in DC treated mice, the relative f raction of replicating cell subsets remain the same. We then examined whether these proliferating cells were functionally altered following DC therapy by assessing their cytokine profiles. Compared to proliferating cells of PBS treated mice, we found that CD4+ and CD8+ lymphocytes from DC treated mice exhibited a shift in cytokine production characterized by a 2 -fold increase in the release of IL 10 and a 2 -fold decrease in IFN To underst and what was driving this reprogramming of the immune response, we collected RNA from the cultured spleen cells and performed a gene array analysis of common cytokines, chemokines, and receptors to identify potential differential gene expression levels bet ween the treatment groups. We describe notable changes in gene expression between various group

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70 comparisons. IL 17f, a gene associated with proinflammatory processes, was found to be reduced over 2 -fold in DC treated mice compared to PBS controls. This change was more evident when comparing between PBS treated mice and ID DC treated mice as there was an 11 fold decrease in expression of IL 17f in ID DC treated mice relative to PBS controls. This suggests that a reduction in IL 17f may allow for the pr oliferation of cell subsets that were once inhibited by IL 17f expression. This data agrees with our previous results in which the proliferating cells of DC treated mice had a cytokine shift biased toward the production of anti inflammatory IL10. We also observed difference s between recipients of unpulsed and antigen pulsed DC. Mice receiving antigen -pulsed DC had a 3.8 -fold higher expression of CXCL12 as compared to unpulsed DC recipients. CXCL12, also known as SDF 1, has a role in the generation and pr oliferation of early B cell progenitors and is also associated with T cell and HSC trafficking (172,173,178) Leng et al reported that NOD mice have elevated expressi on of CXCL12 in the bone marrow which promotes retention of HSC and nave and regulatory T cells within th e compartment thereby resulting in T1D due to a deficit of these cells with in peripheral lymphoid tissues where their regulatory function is needed (173) The elevated expression of CXCL12 in the spleen cells of Ag -pulsed DC treated mice may compete with this aberrant signaling found in NOD bo ne marrow. By having an expanding subset of CXCL12 expressive cells in the spleen, it is possible that Ag -pulsed DC treated mice can benefit from a continued recruitment of HSC and T cells into the spleen that may facilitate their interaction with other immune cells for the generation of tolerogenic responses. Another study found that cells undergoing homeostatic proliferation have an altered enhanced sensitivity to CXCL12 (179) suggesting there may be a self -driven cycle that promotes retention of proliferating lymphocytes to the spleen.

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71 Interestingly, CXCL12 and its ligand CXCR4 are also expressed on beta cells and the proliferating ductal epithelium of pancreas that gives rise to regenerating beta cells (180) Studie s have shown that CXCL12 is re quired for the survival of beta cells and beta cell progenitors and its over -expression results in protection from streptozotocin induced T1D (180,181) While our studies only examined gene expression in spleen cells, it is possible that this increased expression may occur in other cell types includ ing beta cells. Our earlier findings of increased islet survival in SD treated mice would support this notion. We also found that SD DC treated mice had an 18.9 -fold increase in the expression of CCL12, a lso known as MCP 5, compared to unpulsed DC recipie nts. CCL12 the ligand for CCR2 is a chemoattractant for eosinophils, monocytes, fibrocytes and macrophages and has been associated with allergic inflammation and Th1 but not Th2 cells (174176) CCL12 is constitutively expressed in thymus and lymph nodes, but under inflammatory conditions its expression is induced in macrophages and mast cells Its express ion has also been found to be rapidly and significantly up -regulated following priming with antigen that may lead to CCR2 mediated recruitment of inflammatory Th1 cells (182) It is possible that the addition of antigenpulsing with SD DC treatment increased expression of CCL12 although the expected infla mmation that is associated with CCL12 expression was not evident as SD DC mice were better protected from T1D compared to unpulsed DC recipients. It is important to note that while both SD and ID DC treated mice demonstrated homeostatic proliferation and protection from T1D, the increased expression of CCL12 was only observed in the SD -DC group. This finding, in conjunction with the observation that CCL12 expression increases with antigen priming may support our earlier hypothesis that the constitutive pr esentation of SD, but not ID -

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72 determinants, following short -term DC therapy provides the necessary signal to maintain the pool of Ag -specific Tregs for durable Ag -pulsed DC -mediated protection. Lastly, we examined whether the observed in vitro homeostatic p roliferation occurs in vivo We treated mice with DC therapy, then administered a series of BrdU injections to allow its incorporation into proliferating cells. We examined the spleen for BrdU+ cells to determine whether there would be a greater percenta ge of BrdU+ cells in the spleens of mice treated with DC compared to PBS controls, thereby proving homeostatic proliferation in vivo However, we saw no differences between treatment groups in BrdU positivity, suggesting the homeostatic proliferating is o nly a phenomenon occurring in culture Collectively, these results suggest that DC therapy results in Ag independent immune modulation characterized by robust homeostatic proliferation of B and T lymphocytes in vitro Proliferating T cells exhibit a functi onal change characterized by a cytokine shift toward the production of anti inflammatory IL 10. Gene array analysis for potential cytokines/chemokines that may be driving this proliferation requires further investigation though there is some evidence to suggest that the downregulation of IL 17f expression may contribute to the proliferation of a previously inhibited anti inflammatory cell subset. These findings demonstrate the durable potency of DC therapy in the modul ation of antigen non -specific immune response s.

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73 Figure 4 1. Homeostatic proliferation following DC therapy is immediate and sustained. Spleen cells from NOD mice were cultured in serum -free HL media and 3H thymidine was added for incorporation during the final 16h of culture. Proliferation was assessed by beta scintillation quantification of counts per minute (CPM). Spleen cell proliferation is shown at A) 13 weeks of age and B) 40 weeks of age. D ata show n is representative of 10+ experimen ts. Figure 4 2. Homeostatic proliferation is observed in healthy and autoimmune mouse models. NOD and B6 m ice were treated with 3 weekly subcutaneous injections of DC (105/injection) beginning at 9 weeks of age Balb/c mice were treated with 2 weekly IV injections of DC (105/injection) beginning at 6 weeks of age. Spleen cells were collected 2 weeks following final injection to assess 3H -thymidine proliferation in the absence of stimulation. 13 weeks old media DD-insulin ID-GAD 0 2000 4000 6000 15000 unpulsed DC ID-GAD DC 27000 7400PBS in vitro stimulation (25uM)cpm 40 weeks old media DD-insulin ID-GAD 0 2000 4000 6000 8000 10500 13000PBS unpulsed DC ID-GAD DC in vitro stimulation [25uM]cpm A B NOD C57BL/6J Balb/c 0 5000 10000 15000 20000 50000 60000PBS unpulsed DC cpm(N=3) (N=3) (N=2)

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74 Figure 4 3. Identification of proliferating cell populations. CFSE labeled cells from female NOD mice were analyzed for expression of CD4, CD8, and B220. Percentages in red denote proportion of indicated cell subset within the proliferating cells. Data shown is representative of 10+ experiments collec ted through a range of post -treatment timepoints (13 41 weeks of age). CD4 CD8 B220PBS UnpulsedDCID -GAD DC CFSE10.5% 5.8% 76.6% 11.2% 4.5% 57.1% 20.2% 9.4% 68.8%

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75 Figure 4 4. Cytokine profile of proliferating cell populations. Cells were permeablized and stained for the intracellular cytokines IL 4 and IFN and evaluated for CFSE dilution.

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76 Figure 4 5. Gene array analysis of common cytokine and receptors gene expression. Female NOD mice were treated with 3 weekly injections of PBS, unpulsed DC, SD DC or ID DC (N=3/group) beginning at 9 weeks of age. Two weeks foll ow ing treatment, RNA was isolated from cultured spleen cells and transcribed into CDNA for gene expression assessment via qPCR. A G) Fold changes in gene expression between treatment groups are shown by statistical rank (highest rank starting at y axis). PBS vs all DC IL17F IL23A IL13 IFNA1 IL12RB1 IL11 CXCL2 IL18 CSF2 IL21 IL24 CCR4 IL19 IL12RB2 IL3RA CCL28 IL23R CXCL1 IL10 IL2RA LIF IFNB1 IL28B IL2RB GENOMIC3 IL17A IL18R1 IL7R CXCL10 IL15RA PF4 IL12A CCR5 IL7 CX3CL1 CX3CR1 IL10RB IL4 IL4RA IL5 IL6 IL9R CCR1 CXCR4 TNF KIT CCL4 CXCR5 IL1A TGFB1 CCR7 IL2RG IL17RA IL5RA IL21R IL6RA IL27RA IL6ST CSF1 IL1B IL1RN CCL5 IL8RB CCR2 RN18S KITL IFNG IL12B CCL2 IL11RA1 CCL3 IL27 CXCR3 IL22 IL1R1 CCL1 CXCL5 CCR8 IL9 IL3 CCL7 IL25 PPBP CXCL9 IL13RA2 IL22RA1 IL15 IL1RL1 LIFR IL2 DARC CCR3 CSF3 CXCL12 CCL12 CCL11 -15 -10 -5 0 10 60 300 210300fold change p = 0.081 PBS vs Unpulsed DC IL23A CSF2 IL13 CCR4 CCR8 IL12RB1 IL19 IL21 CX3CR1 CXCL1 IL28B LIF CXCL2 GENOMIC3 IL9 IL9R CXCL10 IL7 IL10 IFNA1 IFNG IL1R1 IL17F IL2RB IL11 CCL28 IL21R IL2RA CCR5 IL6 IL5 IL12RB2 CCL12 IFNB1 IL3RA IL24 CXCR3 IL12A KIT CCR7 IL7R IL4RA CXCR5 CX3CL1 IL18 CCL1 IL1RN IL17A TNF IL15RA CSF1 IL2RG CCL5 CCR2 CXCR4 IL10RB IL6ST IL17RA RN18S CCL3 TGFB1 IL1A CCL4 IL6RA IL27RA IL5RA IL11RA1 IL23R KITL CXCL12 IL18R1 CCL2 CXCL5 CCL7 IL27 IL4 IL1RL1 IL8RB PF4 CCR1 CCR3 IL1B IL12B IL15 IL2 CSF3 CXCL9 IL13RA2 IL22RA1 IL25 IL3 PPBP DARC CCL11 LIFR IL22 -27 -16 -5 -2 1 4 7 190 490 790fold change A B

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77 Figure 4 5 continued. PBS vs ID-DC IL17F IL23A IL13 LIF KITL CSF3 CXCL1 IL12RB1 CXCL2 IL1A CCL1 IL1B IL4 IL18R1 IL23R IL3RA IL3 IFNB1 IL17A CCL28 IL10 CX3CL1 IL21 CCR8 IL19 IL10RB IL2RB IL11RA1 IL24 KIT IL28B IFNA1 IL1RN IL27RA IL12RB2 IL18 IL2RG CCR4 IL8RB TGFB1 IL2RA IL5 TNF IL7R IL6RA PF4 CXCR5 IL22 GENOMIC3 CCR7 CXCR4 RN18S IL5RA IL12B IL15RA IL6ST IL4RA CXCL10 IL17RA CCL4 IL6 PPBP IL21R CCL5 IL11 CX3CR1 CCR5 IFNG CXCR3 IL9R IL12A CCR2 CCR1 CSF2 CCL3 IL7 CCL7 CSF1 CXCL5 IL1R1 CCL2 IL15 IL9 IL1RL1 LIFR IL27 CXCL9 IL13RA2 IL22RA1 IL25 IL2 CCL12 CCR3 CXCL12 DARC CCL11 -15 -10 -5 0 5 8 11 105 115fold change p = 0.056 PBS vs SD-DC IFNA1 IL11 IL13 LIFR IL24 CCL12 CXCL2 IL12RB1 IL23A IL23R IL12RB2 IL17F IL18 IL15RA IL27RA IL21 IFNB1 CCL4 IL9 CSF2 IL2 CCR1 CSF1 PF4 CCL28 IL25 PPBP CCL2 CCR5 IL27 IL1RN CCL5 IL6RA IL6 IL2RB IL2RA IL5RA IL18R1 IL3RA CCR7 IL10 KIT TGFB1 IL4RA IL6ST IL7R IL9R CCL3 IL2RG TNF IL17RA CXCR4 DARC CXCR5 IL5 IL22 CCR8 IL10RB IL8RB CCR4 CXCL9 IL12B IL13RA2 IL22RA1 IL12A RN18S CXCL10 IL1R1 IL4 CCL1 IL11RA1 CXCR3 IL1A CX3CR1 CX3CL1 IL28B IL7 CCR2 IL19 GENOMIC3 KITL IL15 LIF CXCL5 IL21R IL1RL1 IL17A CCL7 CXCL12 CXCL1 CCL11 CSF3 IL1B IL3 CCR3 IFNG -70 -10 -5 -2 1 4 17 600 1183fold change C D

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78 Figure 4 5 continued. E p = 0.071

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79 Figure 4 5 continued. Unpulsed DC vs SD-DC CCL12 IL28B LIF IL9 IL19 LIFR CXCL1 IL2 CXCL12 CCR8 IL11 IL9R CXCL10 CXCL9 IL13RA2 IL22RA1 GENOMIC3 KIT CCR4 IL21 IL25 IL1R1 IL23R CCR7 IL7 CCL4 CCL5 CXCR3 IL17A IL27RA IL24 IL21R IL1RL1 IL12RB1 IL6ST IFNG IL23A IL5 KITL IL15RA IL2RB IL2RG CX3CR1 IL1A IL10 IL6RA CXCL2 TNF IL2RA CXCR4 CSF2 CCL3 CCR3 IL1RN IL18R1 IL17RA IL10RB CXCR5 CSF1 IL7R CX3CL1 IL15 CCR5 TGFB1 IL4RA IL5RA IL27 IL11RA1 IL6 CCL11 IL3RA IL12RB2 CCR1 RN18S DARC CCR2 IL12B IL8RB IL4 IL12A CCL2 IFNA1 IFNB1 CCL1 CCL28 CXCL5 CSF3 IL3 CCL7 IL13 PF4 IL1B PPBP IL22 IL17F IL18 -1000000 -500000 -7 -1 5 7 147 287fold change p = 0.056 Unpulsed vs ID-DC CX3CR1 CXCL12 IL3 CCR4 IL1RL1 KITL IL17A IL2 IL9 IL7 IL21 IL4 CXCL9 IL13RA2 IL22RA1 IL25 GENOMIC3 CXCL10 IL13 CCL1 IL1RN CXCR3 CCL11 IL1A IL18R1 IL19 IL3RA IL6 IL28B IL5 LIF CCR8 IL23R IL11RA1 CCL12 IFNG IL21R IL27RA IFNB1 IL2RG CCR5 CSF2 IL6ST CCL3 IL2RB IL10RB IL4RA CSF3 CCR7 CCL5 CXCR5 IL15RA IL1R1 KIT IL12RB1 IL15 IL8RB RN18S IL17RA IL12B TNF IL23A IL6RA CCL4 CXCL2 CXCR4 IL2RA TGFB1 IL10 IL1B CSF1 IL12A IL7R IL9R CCR2 IL5RA LIFR CXCL5 CCL2 IL12RB2 IL24 CCL7 CCR1 CCR3 IL17F CCL28 CXCL1 CX3CL1 IL22 PF4 IL18 PPBP IL27 IL11 DARC IFNA1 -1.51006 -500000.0 -5 0 5 15 300015fold change F G

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80 Figure 4 6. Frequency of BrdU proliferation in organs. Nine week old female NOD m ice were given 3 weekly injections of PBS or DC therapy (N=3/group) then treated with BrdU to assess its incorporation by proliferating cells. Organs were formalin fixed and paraffin embedded, then section ed for staining with anti BrdU antibody. %Brdu indicates proportion of BrdU+ cells within a defined tissue area as determined with image analysis software. Pancreas -Brdu Trmt -Brdu Ab PBS unpulsed DC SD-DC ID-DC 0.00 0.05 0.10 0.15 0.20% BrdU+ Spleen -BrdU Trmt -Brdu Ab PBS unpulsed DC SD-DC ID-DC 0 1 2 3 4% BrdU+ Liver -BrdU Trmt -BrdU Ab PBS unpulsed DC SD-DC ID-DC 0.00 0.05 0.10 0.15 0.20 0.25% BrdU+ABC

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81 Figure 4 7 In situ proliferation mar ked by BrdU incorporation. Spleens were fixed with 4% paraformaldehyde, paraffin embedded, and stained for BrdU detection Arrows denote representative BrdU + cells C = capsule. GC = germinal center. MZ = marginal zone. PALS = periarteriolar lymphoid she ath. RP = red pulp. WP = white pulp 7x GC C PALS RP MZ 12x WP

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82 CHAPTER 5 MODULATION OF REGULATORY T CELL POPULATI ON Introduction In a healthy individual, autoreactive cells are present but do not cause autoimmunity due to an effective immunor egulatory mechanism However in T1D the lack of an adequate regulatory response allows autoreactive T cells to become pathogenic, thereby invading and destroying the pancreatic islet cells. Regulatory T cells (Tregs) inhibit immune responses against self antigens by secreting immunosuppressive cytokines or by interacting directly with effector T cells and it has been shown that animal models with absent or defective populations of Treg develop autoimmune disease (108114) Multiple studies have demonstrated that DC therapy can confer protection against autoimmunity through the induction of regulatory T cells that inhibit the pathogenic T cell inflammation (85,87,183189) Our work has shown that Ag pulsed iDC therapy protects NOD mice from T1D, and that DC therapy induces homeostatic proliferation of CD4+ T cells (among other subsets). However, it is unclear if Tregs are being induced and whether they are part of the proliferating cell population. We sought to identify whether there were changes in regulatory T cell frequency an d function following DC therapy by evaluating the proportion of CD4+Foxp3+ cells in DC treated and control mice and examining their ability to suppress proliferation of effector cells. Materials and Methods Flow Cytometry Cells were prepared into single -cell suspensions in FACS buffer (1x PBS / 1% FCS), then stained for surface markers using CD4 and CD25 in FACS buffer in the presence of Fc block (CD16/32) (BD Pharmingen). Cells were then fixed using Cytofix/CytoPerm reage nt (eBioscience) for 15 minutes at room temperature, then washed in PermWash (eBioscience) All

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83 subsequent steps were performed in PermWash to maintain membrane permeability. Nonspecific staining of cells was blocked again using Fc block for 10 minutes, then cells were labeled with Foxp3 antibody. Cells were analyzed by flow cytometer (FACS Calibur, BD Pharmingen). Live cells were gated from dead cells on the basis of forward/side scatter or with 7AAD (amino antimycin D) labeling. Isotype controls inc Results were analyzed using FCS Express (BD Pharmingen). Suppressor Assay Spleen cells were isolated as described previously. Cells were then suspended in MACS buffer and CD4+ cells were enri ched through depletion of unwanted cells using the CD4+CD25+ Regulatory Cell Isolation Kit (Miltenyi Biotec). Next, CD25+ cells were positively selected from the pre -enriched fraction Responder CD4+CD25+ depleted cells (105) were cultured with suppresso r CD4+CD25+ cells at 1:2 and 1:4 titrations in a round bottom 96 well plate Cells were cultured in serum -free media with anti CD3 e (0.05 ug/200 ul well) and ID peptide stimulation (25uM), then proliferation was determined using 3H thymidine incorporation Retinoic Acid Treatment Tretinoin, an all trans retinoic acid gel prescribed for the topical treatment of acne vulgaris, was topically applied to a 1 x 1 shaved region of skin at the nape of the neck of C57BL/ 6 J mice. Mice were placed under isoflurane anesthesia at 4% 5% for induction, 1%2% for maintenance as controlled by an anesthesia machine. Mice were monitored for their state of consciousness using toe/tail pinch or palpebral reflex. RA gel was applied to cover exposed skin several hours before DC injection, once daily for up to 5 days including a pre -conditioning day in which no DC injection occurred.

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84 Results DC Therapy Results in Sustained Expansion o f Regulatory T Cells Evidence from the literature suggests that a possible mechanism for pro tection from DC therapy is the induction of regulatory T cells. We sought to determine whether the DC treatment was able to increase the frequency of CD4+Foxp3+ regulatory T cells. We treated mice with either PBS, unpulsed DC, or ID -pulsed DC at 9 weeks of age, then collected spleens from mice at various ages to assess spleen cell response. Spleen cells were labeled with CFSE and allowed to proliferate in the presence and absence of peptides for 72 84 hours. Cells were then labeled with regulatory T cel l markers and analyzed by flow cytometry. A s shown in Fig. 5 1, w e found that there was an over 2 -fold increase in the frequency of CD4+Foxp3+ T cells in mice rece iving unpulsed DC, and an over 4 -fold increase in frequency of CD4+Foxp3+ T cells in mice re ceiving ID DC demonstrating that DC therapy results in sustained expansion of regulatory T cells, and that the effect is particularly enhanced in mice receiving ID -pulsed DC. This homeostatic expansion of Tregs was independent of in vitro peptide stimula tion, as the pattern was observed in both stimulated (data not shown) and unstimulated cell cultures. Regulatory T Cell Function i s Enhanced Following DC Therapy We also examined whether there were function al differences in regulatory T cells following DC therapy. We performed a suppressor cell assay by co -culturing CD4+CD25+ depleted cells with CD4+CD25+ purified cells at various titrations of 1:2 and 1:4 in the presence of anti CD3 and ID peptide stimulation. As seen in Fig. 5 2, r egulatory cells from b oth unpulsed and peptide pulsed treated mice demonstrated greater suppressive function in a dose dependent manner, with the effect enhanced in the peptide -pulsed DC group. The enhanced suppression was found to be nearly 2 3 fold greater in DC treated mice at 1:2 titration. This effect was magnified when the titration was decreased to 1:4, where up to a 10-fold enhancement in

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85 suppression was observed. These results demonstrate that on a cell to cell level, Tregs isolated from DC treated mice are more pote nt in suppressor function than Tregs isolated from PBS treated mice. R et inoic Acid Increases Frequency o f Regulatory T Cell Population Another aim for our study was to identify how to better engineer tolerogenic DC Studies using retinoic acid (RA), a metabolite of Vitamin A, discovered that its treatment could induce Foxp3 expression in CD4+ cells. We treated the injection site with RA using a topical gel for 3 days to condition the DC microenvironment prior to a single subcutaneous DC injection, then isolated spleen cells from mice 24 and 48 hours after the injection. We found within 24 hours of the DC injection, mice that were pretreated with RA had an increase in the frequency of CD4+ CD25+ Foxp3+ cells from 5.14% (no RA + DC ) to 8. 23% (RA + PBS) and 8.23% (RA + DC) (Fig. 5 3) and this was observed regardless of whether DC were injected. This effect magnified at 48 hours following DC injection, as mice without RA treatment had a CD4+CD25+Foxp3+ cell frequency of 7.07% compared to RA treated mice at 12.71 (RA + PBS) and 13.88% (RA + DC). As RA has been shown to induce the expression of CD103, the gut homing receptor, we speculate that the increase in Tregs in the absence of DC injection is due to the possibility that RA treatment condition ed existing Langerhans cells to migrate to gut LN and interact in the T cell environm ent to induce Foxp3+ expression. Discussion Overall our results suggest that DC treatment increases the frequency and function of regulatory ce lls. We found that mice receiving DC had a 2 3 fold increase in the frequency of CD4+Foxp3+ cells, and that the effect was furthe r enhanced when mice received a peptide pulsed DC. We also assessed the ability of CD4+CD25+ cells to suppress anti -CD3 and p eptide stimulated proliferation of CD4+CD25+ depleted effector cells and found that regulatory cells

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86 isolated from DC treated mice has superior function in suppression exhibited by an 2 10 fold enhancement in suppression compared to cells isolated from PBS treated mice. These findings were consistent in all mice that received DC therapy, and were enhanced when the DC was loaded with an antigen. Of interest, the results of the proliferation studies were verified in both diabetic and non -diabetic mice. As Tregs are expected to promote tolerance, i t is unclear why the observed expansion in Tregs was not consistently associated with protection from T1D. It is possible that the increased frequency and function of Tregs w ere not sufficient in scale to override the inflammatory activity of effector cells. A balance in favor of Tregs over pathogenic effector cells must be achieved to yield immune deviation to tolerance. Another potential explanation was proposed by work from Diane Mathiss group, which demonstr ated that while defects in NOD Treg contribute to T1D, it may be an effect of over responsive effector T cells to self antigen (or monoclonal Ab stimulation ) that truly drive the immunopathology (190) Thus the loss of tolerance may be related not to impaired function or decreased frequency of NOD T regs, but rather a decline in the ability of NOD T cell effectors to re spond to fully competent Tregs. However, our studies of Treg function examine PBS vs. DC t reated Tregs against NOD effectors, which in concept should be similarly impaired, so our observation of functional differences between the treatment groups can be attributed to a true variation between PBS and DC -treated Tregs. To investigate other metho ds to improve Treg frequency, we examined whether topical retinoic acid treatment, which has been shown to induce Foxp3 expression, can induce Foxp3+ Tregs. We also found that RA conditioning of the injection microenvironment alone, with or wit hout additi onal administration of DC, was able to increase the frequency of splenic CD 4+ CD25+ Foxp3+ regulatory T cells by over 2 fold. This effect was immediately observed

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87 with just 3 daily RA treatment s. An increase in the frequency of RA -induced Foxp3+ Tregs in c onjunction with DC therapy induced Tregs may together be able to dampen the potent inflammatory response from increased or over responsive effector T cells. T aken together, t hese findings suggest that DC the rapy effectively improves both the freque ncy and function of regulatory T cells that are imperative to the suppression of autoreactive T cells Importantly, the homeostatic expansion of Tregs continues after the cessation of DC treatment. We also demonstrate that the protective effects of DC th erapy can be improved using topical RA conditioning of the injection site microenvironment located away from the autoinflammatory site where steady -state resident DC in the skin can be recruited to participate in immune modulation. Collectively, these res ults provide evidence that DC therapy induces regulatory T cells that are more potent suppressors compared to Tregs isolated from a PBS treated mouse, and that these cells exist in greater proportions as they continue to expand following DC therapy.

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88 Figu re 5 1. Assessment of regulatory T cell population following DC therapy. CFSE labeled spleen cells from female NOD mice were cultured in serum -free media without stimulation and allowed to proliferate for 72 84h. Cells in right panels are gated on CD4+. Data shown is representative of 3 experiments from mice aging from 13 41 weeks old. CD4+ Foxp3CFSE+ ID DC unpulsed DC PBS

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89 Figure 5 2. Suppressor cell assay. Female 9 wk old NOD mice were treated with 3 weekly injections of DC, then Treg function was assessed at 13 weeks of age. CD4+CD25+ depleted cells from spleen were co -culture d with titrations of CD4+CD25+ purified cells and stimulated with antiCD3 and ID peptide. Proliferation was assessed by 3H thymidine incorporation. Figure 5 3 Retinoic acid increases frequency of reg ulatory T cell population. Female C57BL/6J m ice were treated with a topical application of RA at injection site for 3 days before injection with PBS or DC (N=3/group) Expression of CD4+CD25+Foxp3+ was assessed in spleen cells. 0 10 20 30 40 50 60 70 80 PBS (N=3) unpulsed DC (N=3) ID DC (N=3)% suppression 1:2 1:4

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90 CHAPTER 6 CONCLUSIONS Type 1 diabetes is a metabolic disorder characterized by autoimmune destruction of the insulin -producing pancreatic beta cells. The autoimmune process is driven by T cells reactive to autoantigens expressed in beta cells including GAD, ICA69, IA 2, insulin, and proinsulin in the absence of an effective regulatory mechanism (191195) Dendritic cells, which present antigen and direct T cell responses, are an ideal platform for use in T1D treatment as DC therapy works to correct the specific underlying autoimmune aberrancy in T1D. DC therapy can uniquely control (1) the direction of the immune response through the selection of either immunogenic or tolerogenic classes of DC, as well as (2) dictate the ta rget antigen that the response is directed toward through the presentation of a chosen antigen, reinforcing DC therapy to be an effective and powerful strategy for immune modulation. However, a consensus on what defines a tolerogenic DC and which antigens to select for DC loading remains to be established, as multiple studies in the NOD mouse have reported protection from T1D using a range of DC subsets and beta cell antigens or antigen determinants. Additionally, another confounding factor lies in the re quisite of early intervention. Reports of DC therapy for tolerance induction has been successfully demonstrated when applied before or in the early stages of autoreactivity in animal models of various autoimmune diseases, as well as in studies of transpla nt/graft acceptance (85,135, 196200) However, if treatment is initiated after the autoimmune process is advanced, efficacy in DC -mediated protection declines. While NOD mice have a predictable timeline for T1D onset allowing for intervention to be planned accordingly, the dynam ics of autoreactivity processes in human has not been difficult to define due to multiple variations in subtypes that compound assessment Additionally, the majo rity of subjects susceptible to Type 1 diabetes lack familial history that would otherwise prompt early autoantibody screening, thus t he

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91 opportunity for early intervention in humans is lo w emphasizing the need for therapy that can treat both established and new onset disease. We sought to understand how to better develop DC therapy for translation into the clinical setting. We first addressed the issue of what defines a tolerogenic DC. Classical models of DC subsets classify DC function on the basis of phenotypic characteristics associated with the expression of maturation markers such as MHC cla ss II and T cell costimulatory molecules and production of cytokines. Immature DC, which have low expression of maturation markers, and anti inflammatory biased cytokine production, are believed to be tolerogenic while mature DC, which have high expressio n of maturation markers and a pro inflammatory bias in cytokine production are defined as immunogenic. However, critics of iDC speculate whether they can migrate efficiently to dLN for critical interaction with T cells and whether a new subset of DC term ed semi -mature DC with low production of inflammatory cytokines and high expression of T cell co stimulatory molecules may be better suited to stimulate nave T cell s into regulation. We first examined iDC migration and found that a substantial proportion o f injected DC traveled to the dLN. Then we compared the efficacy of iDC and smDC therapy for the prevention of T1D in 9 week old NOD mice with advanced insulitis and found that iDC were superior to smDC in delay ing of T1D onset, though neither treatment conferred significant protection. To create DC for therapy with more durable protection, we considered a nother aspect of DC therapy : selection of antigen for loading prior to infusion. We and others have demonstrated that the administration of beta cell a utoantigens in a tolerogenic modality is highly effective in preventing T1D in the NOD mouse (61,64,65,125,129,141,201) However, uncertainties in extrapolating appropriate Ag doses and correlating treatment timeline, as well as limitations in identifying epitopes in human has hinder ed its translation into the clinical setting particularly

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92 since studies have shown that the immune response can pivot toward immunity or tolerance depending on antigen dose. Fortunately, a ntigen presentation in the context of a tolerogenic DC may circumvent the issue of ambiguous immune deviation associated with antigen treatment alone Traditionally, antigens were selected for DC therapy by focusing on autoantigens or peptide epitopes within self -Ag that are dominantly -expressed and are primary targets of the autoimmune response. These whole antigens or domina nt determinants (DD) were then administered in modalities that induce tolerance, and has been shown to prevent T1D as well as other autoimmune diseases in mice when applied early in the autoimmune process (61,64,124,144) However, this strategy becomes ineffective when administered at advanced stages of T1D (138,144) We speculated that the decline in protection is associated with an altered immune repertoire with time as dominant determinants (DD) continuously recruit nave T cells into spontaneous autoimmune attack while subdominant determinants ( SD ), which do not compete as effectively as DD, and ignored determinants (ID), which are not pro cessed and presented at all, have a minimal effect on the nave T cell pool (144,145) Thus, the re administration of DD may simply reactivate cells that are preprogrammed to respond pathogenically, while SD or ID can recruit the available pool of nave T cells for priming into tolerance when natural antigen processing is bypassed experimentally to present these previously unseen epitopes. We compared the efficacy of DD, SD, and ID peptide classes in DC therapy to protect 9 week old NOD mice and found that only SD and ID -pulsed DC were able to protect mice when the treatment was applied in the presence of advanced autoimmune processes. Specifically, just t hree weekly injections of SD DC protected NOD from T1D with a significant delay in the onset of T1D, though complete protection was not achieved. Consistent with this finding, we saw that

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93 SD DC treated mice had a greater number of pancreatic islets compared to unpulsed and DD DC treated mice though the level of islet infiltration remained similar between all groups. Next we examined whether ID DC, which should have a comparatively larger pool of nave T cells to prime into tolerance, would be more effective in conferring protection However, we fou nd that three injections of ID DC were not sufficient to achieve protection as a sudden drop in survival within 6 weeks of the last treatment dampened the treatment success. We speculated that since ID are not constitutively presented, treatment may need to be continued to maintain the regulatory T cell pool. We treated another cohort of mice with repetitive injections of ID DC and found that mice were completely protected from T1D. This total protection was not seen in mice treated with repetitive inje ctions of PBS or unpulsed DC, suggesting that the protection is attributed to the ID. To exclude the possibility that the observed protection from T1D was due to global immunosuppression, we examined whether NOD mice could generate a normal immune respon se to a non T1D related antigen challenge following treatment with DC therapy. We immunized PBS, unpulsed DC, and ID -DC treated mice with KLH and examined their serum antibody responses. All DC treated mice were able to mount antibody responses to KLH in a manner comparable to PBS controls, demonstrating that normal immune responses were intact and the previously observed protection could be attributed to diabetes -specific protection. Much of the current knowledge on how DC therapy affects immune response s has been delineated from studies with a focus on antigen specific immune modulation, as we have shown that Ag -based DC therapy -mediated protection is limited to the suppression of autoreactive processes specific to T1D However, whether DC therapy results in antigen n on-specific immune changes has yet to be studied in detail. The spleen is a major site of immune cell

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94 interactions and antigen processing, with active processes that contribute to the overall immune status (163,164) Thus, we sought to examine the spleen cell response in the absence and presence of T1D peptide stimulation To our surprise, we found robust homeostatic proliferation of spleen cells isolated from DC -treated, but not PBS t reated mice. This effect was immediate and sustained, as the proliferation could be observed as early just two weeks following 2 DC injections, and was durable even 29 weeks after treatment had ended. Characterization of these cell s revealed that the pro liferation could be predominantly attributed to B and T lymphocytes, though there was no evidence for the expansion of memory B or T cells within these subsets. We found that this DC -mediated reprogramming of the immune response extended into changes of c ytokine production as well, as we observed a 2 -fold shift from the production of IFN production of anti inflammatory IL 10. To determine what may be driving this immune modulation, we performed a gene array analysis to identify potential changes in expression of cytokine/chemokines genes and did not find any statistically significant difference between treatment groups, though there was some evidence to suggest that the down -regulation of IL 17f expression seen in DC treated mice may have contribu ted to the proliferation of a previously inhibited anti inflammatory cell subset. This data agrees with our previous results in which the proliferating cells of DC treated mice had a cytokine shift biased toward the production of anti inflammatory IL 10. We also observed gene expression that may not delineate the cause of the spleen cell proliferation but still contributes to our understanding of the effect of DC therapy on disease outcome. We identified an increase in CXCL12 expression in the spleen cell s of Ag -pulsed DC treated mice compared to unpulsed DC treated mice. CXCL12 has a role in the generation and proliferation of early B cell progenitors and has also been found to be over -expressed in NOD

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95 BM thereby promoting retention of HSC and nave and regulatory T cells within the BM compartment resulting in a deficit of these cells in the peripheral lymphoid tissues where their regulatory function is needed (173) The elevated expression of CXCL12 in the spleen cells of Ag pulsed DC treated mice may compete with this aberrant signaling found in NOD bone marrow and recruit cells into the spleen for interactions leading to tolerance induction A dditionally, CXCL12 and its ligand CXCR4 are also expressed on beta cells and the proliferating ductal epithelium of pancreas that gives rise to regenerating beta cells and its over expression results in protection from streptozotocin induced T1D (180,181) While our studies only examined gene expre ssion in spleen cells, it is possible that this increased expression may occur in other cell types including beta cells. Our earlier findings of increased islet survival in SD treated mice would support this notion. We also found that spleen cells of SD DC treated mice had an increase in the expression of CCL12, which has been shown to be upregulated following priming with antigen (182) It is possib le that the SD -pulsed DC treatmen t increased trafficking of both CCL12 responsive Ag -specific Tregs and DC presenting cognate SD into the spleen promoting their interaction to induce proliferation of these Treg. This may elucidate why we observe proliferation of Tregs in the absence of in culture peptide stimulation, and why this increased expression is only observed in SD but not ID groups as there should not be continued constitutive presentation of ID following cessation of treatment to induce the up regulation of CCL12. Proliferation observed in splenic cultures derived from ID treated mice may also benefit in a similar manner from infectious tolerance (202,203) However, in vivo BrdU studies provided no evidence of homeostatic proliferation suggesting that this phenomenon is only occ urring in vitro Whether this is due to an accumulation of cytokines that may drive proliferation in culture remains to be determined.

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96 Because NOD mice have a defect in DC phenotype and function, we evaluated whether this homeostatic proliferation was a t rue effect of DC therapy or an only an effect associated with therapy using DC with an aberrant phenotype. We treated the nonautoimmune prone mouse strains Balb/c and C57BL/6J mice with PBS or DC and observed a similar enhancement in homeostatic prolifer ation in mice receiving DC therapy, confirming that the effect is a true immune response to DC therapy. Dendritic cells play an important role in the induction and maintenance of regulatory T cells that may confer tolerance (75,118121,123,184) Thus, we evaluated the effect of DC therapy on the regulatory cell subset. We found that DC therapy resulted in the durable expansion of CD4+Foxp3+ regulatory T cells, and this effect was further enhanced using Agpulsed DC. DC therapy also improved the function of Tregs, as CD4+CD25+ regu latory cells from DC -treated mice were able to more po tently suppress CD3 and peptide -stimulated proliferation of CD4+CD25 -depleted responder cells compared to regulatory cells from PBS controls. This Treg expansion can be further enhanced with retinoic a cid, as we found that RA pre conditioning of the DC microenvironment was able to increase the frequency of Foxp3+ Tregs in the spleen with or without co -injection with DC. This may be due to the RA -induced up regulation of CD103 expression on resident Langerhans cells that home to the gut and induce Foxp3+ expression. While we observed an increase in both frequency and function of Tregs with DC treatment, we did not observe a correlation in protection from T1D. It is possible that the improvement was not sufficient to pivot the balance in favor of regulation in the presence of a potent inflammatory effector T cells response that has been shown to grow with age (190) This may be supported by our observation of increased islet survival in Ag -pulsed DC treated mice, but not unpulsed DC treated mice that had similar levels of lymphocyte infiltrate, as the

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97 undefined lymphocyte population may potentially be an influx of both pathogenic and regulatory T cells. A balance in favor of re gulatory T cells versus immunogenic T cells may be able to push the overall response toward tolerance, but perhaps the DC therapy -mediated increase in regulatory T cells was not enough to completely quench the inflammation generated from the pathogenic T cells However, RA -treatment induced Foxp3+ Tregs in conjunction with DC therapyinduced Tregs may together be able to dampen the potent inflammatory response from increased or over -responsive effector T cells. Nonetheless, the latter stages of advanced autoimmunity immediately prior to T1D onset may simply not be amenable to a one armed intervention; Tregs alone may not be sufficient to rescue beta cell death and the requirement for combinatorial strategies to treat both autoimmunity and regenerate beta cell mass may become necessary. Collectively, these results suggest that DC therapy results in antigen -dependent and antigen independent effects on immune modulation. We find that the choice of peptide d eterminants for DC pulsing has a profound effec t on the efficacy of DC therapy. SD or ID pulsed DC but not DD -pulsed DC, w ere able to protect NOD mice with advanced autoimmunity from development of T1D, demonstrating a role for antigen in T1D prevention. This fundamental principle of altering native d eterminant presentation to accommodate a changing T cell repertoire can be extend ed to the treatment of any dy namic autoantigen -based disease We also demonstrate that DC therapy augments the immune respons e in an antigen -dependent and independent manner. Spleen cells from both unpulsed and Ag -pulsed DC treated mice, but not PBS controls, exhibited robust homeostatic proliferation of B and T cell lymphocytes in vitro with the effect further enhanced in Ag DC recipients. However, t he immune modulation was non antigen specific, as the addition of peptide stimulation in culture did not alter the response.

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98 Importantly, the proliferation appears to be a protective and not pathogenic response as the expanding cells exhibit a deviation in cytokine production from pro inflammatory to anti inflammatory. Furthermore, w e find that DC therapy increases the frequency and enhances the function of Tregs and that the chronic expansion of these cell populations did not require recall Ag peptide stimulation. This reprogrammed Treg response can potentially re -establish the balance between effector and regulatory cells back in favor of tolerance. Together, these findings demonstrate the durable potency of DC therapy in th e modulation of antigen specific and non -specific immune responses and provide an important step toward translation into the clinic as other peptide -based therapies for T1D have been limited to early interv ention. These discoveries will guide the engineer ing of DC therapies for T1D and other dynamic Ag -based disorders .

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117 BIOGRAPHICAL SKETCH Jeannette Lo -Dauer was born in Hong Kong where she lived with her parents and older sister Paulette before moving to Flor ida at the age of 5. She lived in Fort Lauderdale, FL where she at tended and graduated Nova High S chool in 1997. Jeannette began her undergraduate studies at the University of Florida with double majors in microbiology and cell s cienc e, and p sychology. She developed an interested for biomedical research while performing studies examining the action of anorectic drugs in rats with selec t serotonin receptor depletion in the laboratory of Dr. Neil E. Rowland. Following graduation from U F in 2001 with a B achelors of Science in m icrobiology and c ell s cience and p sychology, she continued neuroscience research for one year in the laboratory o f Dr. John M. Petitto where she investigated the role of IL 2 signaling in the immune trafficking of lymphocytes to injured neuronal environment s Jeannette then continued on to obtain a Master of Science in p ublic h ealth in 2004 from the University of So uth Florida, where her graduate research focused on the development of novel nitroplatinum complexes for the treatment of cancer. Jeannette entered the Interdisciplinary Program in Biomedical Sciences Ph.D. at the University of Florida in 2004, and joined the laboratory of Dr. Michael Clare Salzler in 2005 where she studied dendritic cell biology (DC) in the NOD mouse, the autoimmune model of Type 1 diabetes (T1D). Jeannette studied factors associated with the antigen -driven process of autoimmune activati on and immune tolerance and accordingly engineered DC th erapy for the induction of tolerance. Following the completion of her doctoral work, Jeannette plans to continue research in DC biology wi th Dr. Clare -Salzler, and will subsequently relocate to Birmi ngham, AL with her husband Daniel Dauer as he begins his residency training at the University of Alabama, Birmingham. Jeannette plans to remain in the field of immunotherapy with a career in academic research