1 DEVELOPMENT OF A TOLEROGENIC , HYDROGEL MICROPARTIC LE MATRIX FOR THE PREVENTION O F TYPE 1 DIABETES IN NOD MICE By YOUNG MEE YOON A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQ UIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 201 4
2 Â© 201 4 Young Mee Yoon
3 To my parents and my fiancÃ©, for their endless and unconditional support .
4 ACKNOWLEDGMENTS This research work would not have bee n possible without the help and support of many people. First of all, I would like to express my great appreciation to my mentor, Dr. Mark Atkinson, for giving me the opportunity to learn about and research Type 1 Diabetes in his lab for my doctoral study . He has given me encouragement, motivation, and enduring support. I can truly say i t has been a great honor to learn from someone who is so passionate in the field of Type 1 Diabetes research . I would also like to express my sincere gratitude to Clive Wass erfall for his invaluable guidance and help. He taught me how to enjoy doing research even if there are obstacles and frustrations. Under his guidance, I have grown to be a better scientist. Next, I must thank my supervisory committee members, Dr. Michael Haller, Dr. Benjamin Keselowsky, Dr. Clayton Mathews, and Dr. Sihong Song. I truly appreciate their outstanding guidance and inspiring advice. My work would not have been productive without these faculty members and their lab members . Firstly, I would like to express my gratitude to Jamal Lewis who has made substantial contributions to my work. I would also like to thank Dr. Laurence Morel and one of her lab members, Ramya Sivakumar. I would like to extend my thanks to Dr. Tod d Brusko and all of his lab mem bers, as well as, Dr. Martha Campbell Thompson and the Molecular Pathology Core. McGrai l, Sean McGrail, Amanda Posgai, Alicia Chechele, Priyanka Ingle, Ther e sa Sumrall, Blair Eckman , and Robert Camacho. I also would like to express my appreciation to our former lab members including Maigan Hulme, Patrick Rowe,
5 Courtney Myhr, Dustin Blanton, Zhao Han, and Peter Hong. I must thank my fellow IDPers including Sindhu Arivazhagan, Yun Jong Park, and Kyungah Maeng. My family has been an incredible source of support and motivation for me throughout this process and I would like to thank to my parents, Mu Sun Min and Wonkyung Yoon. I also want to thank my little brother, Jeo ng Wook Yoon. Last, but not least, I would like to send special appreciation and love to my fiancÃ©, who has patiently waited and continuously motivated me during this journey.
6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 11 ABSTRACT ................................ ................................ ................................ ................... 13 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 15 Type 1 Diabetes General Overview ................................ ................................ ..... 15 Therapeutic Approaches for Type 1 Diabetes ................................ ......................... 16 Exo genous Insulin Replacement Therapy ................................ ........................ 17 Islet Cell Transplantation ................................ ................................ .................. 17 Immune Based Strategies ................................ ................................ ................ 18 Nonautoantigen specific primary and secondary prevention ..................... 18 Nonautoantigen specific intervention ................................ ......................... 20 Autoantigen specific immunotherapy ................................ ......................... 22 Autoantigen specific primary and secondary prevention ............................ 22 Autoantigen specific intervent ion ................................ ............................... 23 Antigen Specific Tolerance ................................ ................................ ..................... 24 Non Obese Mouse Model for Type 1 Diabetes ................................ ....................... 25 Biomaterials for Immunological Applications ................................ ........................... 27 PLGA as a Vaccine Delivery Vehicle ................................ ................................ 27 Injectable PuraMatrix Pep tide Hydrogel ................................ ........................... 28 Vaccine Adjuvant ................................ ................................ ................................ .... 29 GM CSF ................................ ................................ ................................ ........... 30 CpG ODN ................................ ................................ ................................ ......... 30 Hemoglobin, Haptoglobin, and Heme Oxygenase 1 ................................ ........ 31 2 GENERAL METHODS ................................ ................................ ............................ 33 Mouse Strains ................................ ................................ ................................ ......... 33 Determination of Hyperglycemia ................................ ................................ ............. 33 Mouse Anesthesia ................................ ................................ ................................ .. 34 Animal Microchipping ................................ ................................ .............................. 34 Spleen and Bone Marrow Cell Purification ................................ .............................. 34 Preparation of Reagents ................................ ................................ ......................... 36 Flow Cytometry Analysis ................................ ................................ ......................... 36 Staining Cell Surface Antigens ................................ ................................ ......... 37 Staining Intracellular Antige n ................................ ................................ ............ 39 Statistical Analysis ................................ ................................ ................................ .. 40
7 3 DEVELOPMENT OF A VACCINE DELIVERY SYSTEM OF HYDROGEL MICROPARTICLE MATRIX MATERIAL ................................ ................................ . 42 Background ................................ ................................ ................................ ............. 42 Materials and Methods ................................ ................................ ............................ 44 Preparation of Human Denatured Insul in Encapsulated PLGA MP .................. 44 PLGA MP Characterization ................................ ................................ .............. 45 Preparation of PuraMatrix Peptide Hydrogel ................................ .................... 45 Sample Preparation for Scanning Electron Microscopy (SEM) Analysis .......... 46 In Vitro Gel Release Assay ................................ ................................ ............... 46 In Vitro Migration Assay ................................ ................................ .................... 46 Evaluation of Biodistribution and Accumulation of Subcutaneously Injected PLGA MP Hydrogel Matrix ................................ ................................ ............ 47 Results ................................ ................................ ................................ .................... 48 Characterization of Hydrogel PLGA MP Matrix ................................ ................ 48 Characterization of PLGA MP ................................ ................................ .......... 48 Hydrogel Release Profiles ................................ ................................ ................ 48 Biodistribution and Accumulation of Subcutaneously Injected Hydrogel MP Matrix ................................ ................................ ................................ ............ 49 Discussion ................................ ................................ ................................ .............. 50 4 A NOVEL HYDROGEL MICROPARTICLE MATRIX DELIVERING AUTOANTIGEN AND ADJUVANT TO PREVENT TYPE 1 DIABETES IN NOD MICE ................................ ................................ ................................ ....................... 61 Background ................................ ................................ ................................ ............. 61 Materials and Methods ................................ ................................ ............................ 65 Diabetes Prevention Studies ................................ ................................ ............ 65 Study of Granuloma ................................ ................................ ......................... 67 In Vitro Adjuvant Stimulations ................................ ................................ ........... 68 Insulitis Scoring ................................ ................................ ................................ 68 Results ................................ ................................ ................................ .................... 69 In Vivo Survival Curves of NOD Mice Study I ................................ ................ 69 Histological Analysis of Gr anuloma Study I ................................ ................... 69 Cytokine Productions by Adjuvant Stimulations In Vitro Study I .................... 70 Insulitis Scoring Study I ................................ ................................ ................. 70 Flow Cytometry Analysis of Treg Population Study I ................................ ..... 71 In Vivo Survival Curves of NOD Mice Study II ................................ ............... 71 Width of Granuloma Study II ................................ ................................ ......... 72 Insulitis Scoring Study II ................................ ................................ ................ 72 In Vivo Survival Curves of NO D Mice Study III ................................ .............. 73 Granuloma Size Change Study III ................................ ................................ . 74 Flow Cytometry Analysis of T Cell Subpopulations Study III ......................... 74 Discussion ................................ ................................ ................................ .............. 75 5 IMPAIRMENT OF NOD MICE SPLENOCYTE MOBILIZATION IN RESPONSE TO GM CSF AND G CSF ................................ ................................ ....................... 93
8 Background ................................ ................................ ................................ ............. 93 Materials and Methods ................................ ................................ ............................ 94 In Vitro Migration Assay ................................ ................................ .................... 94 In Vitro Stimulation and RNA Isolation ................................ ............................. 94 Gene Expression Analysis ................................ ................................ ................ 95 Results ................................ ................................ ................................ .................... 96 Migration Patterns of Bone Marrow Cells in Response to GM CSF or G CSF ................................ ................................ ................................ ............... 96 Migration Patterns of Splenocytes in Response to GM CSF or G CSF ............ 97 Flow Cytometry Analysis of Migrated Splenocytes ................................ ........... 97 Differences in Gene Expression Levels in Bone Marrow Cells and Splenocytes ................................ ................................ ................................ ... 98 Differences in Gene Expression Levels in BM DCs and Splenic DCs ............ 100 Discussion ................................ ................................ ................................ ............ 101 6 CONCLUSIONS AND FUTURE DIRECTIONS ................................ .................... 114 LIST OF REFERENCES ................................ ................................ ............................. 121 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 136
9 LIST OF FIGURES Figure page 3 1 Three dimensional hydrogel microparticle matrix and scanning electron micro scope (SEM) image of the matrix ................................ ............................... 53 3 2 Microparticle characterization ................................ ................................ ............. 54 3 3 Release profiles of GM CS F and CpG ODN1826 from hydrogel ........................ 55 3 4 Functionality of hydrogel relea sed GM CSF as a chemoattractant .................... 56 3 5 In vivo biodistribution and accumulation of microparticle ................................ .... 57 3 6 In vivo biodistribution and accumulation of hydrogel ................................ .......... 59 4 1 Study I: Kaplan Meier plots of diabetes incidence ................................ .............. 81 4 2 Study I: Histology of granuloma ................................ ................................ .......... 82 4 3 Study I: Effect of adjuvant stimulations on cytokine production in vitro ............... 83 4 4 Study I : Insulitis scoring of pancreatic islets at 13 weeks of age ........................ 84 4 5 Study I: Treg frequency in the spleen of surviving mice ................................ ..... 85 4 6 Study II: Kaplan Meier survival curves of NOD mice ................................ .......... 86 4 7 Study II: Size change of granuloma ................................ ................................ .... 87 4 8 Study II: Insulitis scoring of islets from a midpoint mechanistic study ................. 88 4 9 Study III: Diabetes incidence following the vaccine injections ............................ 89 4 10 Study III: Formation, regression, and regrowth of granuloma ............................. 90 4 11 Study III: Frequency of T cell subpopulations in the spleens of surviving mice .. 91 4 12 Study III: Frequency of T cell subpopulations in the brachial lymph nodes (LNs) of surviving mice ................................ ................................ ....................... 92 5 1 Schematic illustration of in vitro cell migration assay ................................ ........ 104 5 2 Percentage of bone marrow (BM) cell migration at 8 and 12 weeks of age ...... 105 5 3 Percentage of splenocyte migration at 8 and 12 weeks of age ........................ 106 5 4 Fold change of migrated splenocyte populations ................................ .............. 107
10 5 5 Itgam gene expression at 8 and 12 weeks of age ................................ ............ 108 5 6 Socs3 gene expression at 8 and 12 weeks of age ................................ ........... 109 5 7 Csf3r gene expression at 8 and 12 weeks of age ................................ ............. 110 5 8 Csf2ra gene expression at 8 and 12 weeks of age ................................ ........... 111 5 9 The levels of gene expression in BM DCs and s plenic DCs ............................ 112
11 LIST OF ABB REVIATIONS Alum Aluminum hydroxide ANOVA Analysis of variance APCs Antigen presenting cells BM Bone marrow C57BL/6 C57 black 6 CD Cluster of differentiation CO Carbon monoxide CTLA 4 Cytotoxic T lymphocyte antigen 4 o C Degrees C elsius DC Dendritic cell ELISA Enzyme linked immunosorbent assay FBS Fetal bovine serum Foxp3 Forkhead box P3 G CSF Granulocyte colony stimulating factor GAD Glutamic acid decarboxylase GM CSF Granulocyte macrophage colony stimulating factor H&E Hematoxylin and eosin Hb Hemoglobin HBSS HO 1 Heme oxygenase 1 Hp Haptoglobin IA 2 Islet antigen 2 IACUC Institutional animal care and use committee IDD Insulin dependent diabetes
12 IFN Interferon IHC Immunohistochemistry IL Interleukin LN Lymph node LYVE 1 Lymphatic vessel endothelial hyaluronan receptor 1 mAb Monoclonal antibody MHC Major histocompatibility complex MP Microparticle NOD Non obese diabetic NOR Non obese resistant ODNs Oligodeoxynucleotides PBS Phosphate buffered sal ine PLGA Poly(lactide co glycolide) qPCR Quantitative polymerase chain reaction RBC Red blood cell SEM Scanning electron microscopy SPF Specific pathogen free SPL Splenocytes T1D Type 1 diabetes TCR T cell receptor TLR Toll like receptor Tm Melti ng temperature Treg Regulatory T cell ZnT8 Zinc transporter 8
13 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 DE VELOPMENT OF A TOLER OGENIC, HYDROGEL MIC ROPARTICLE MATRIX FOR THE PREVENTION O F TYPE 1 DIABETES IN NOD MICE By Young Mee Yoon August 2014 Chair: Mark A. Atkinson Major: Medical Sciences Immunology and Microbiology Type 1 diabetes (T1D) is an autoimm une disease characterized by the destruction of insulin producing pancreatic beta cells . T o date , t he disorder cannot be prevented or cured . While i mmunosuppressive therap ies have p rovided short term remissions in individuals with T1D , t hese approaches may pose potential risk s associated with non specific immuno suppression. A n tigen specific immunomodulation has emerged as a targeted therapeutic intervention to overcome this limitation . The aim of this approach is to elicit immunological tolerance against beta cell autoantigen s through re education of the immune system. However, difficulties in delivery of antigen s to immune cells and in the education of these cells, has led to an increasing desire to develop an efficient delivery method. To that end, we utilized synthetic controlled release biomaterials: poly (lactide co glycolide); ( PLGA) microparticle s (MP s ) , and a P ura M atrix peptide hydrogel. PLGA MP s were used as a delivery vehicle for denatured human insulin. T he hydrogel provided two critical roles, holding MPs and enabling sustained release of adjuvants at the site of injection. A c ombination of hemoglobin and haptoglobin (Hb:Hp) , and CpG ODN1826 (CpG ) w er e selected as vaccine adjuvants.
14 This v accine formulation was injected subcutaneously in to 8 week old non obese diabetic (NOD) mice . A t otal of five injections of hydrogel loaded with Hb:Hp, CpG, and ins ulin MPs successfully protected nearly 80% of the mice from the development of dia betes , w hereas control groups had 10 20% diabetes free survival . Subsequent in vitro studies revealed that the potential mechanisms involved in the diabetes prevention were enhanced IL 10 production, increased Foxp3+ Treg , and decreased insulin B(9 23) reactive CD4 T cell population s . Another remarkable finding was the detection of granuloma formation , caused by multiple subcutaneous injections of matrix containing insulin MPs. Histological analysis of the granulo ma indicated infiltration of T cell , B cell, and macrophage populations with sign s reminiscent of a tertiary lymphoid organ. We also detected potential evidence of antigen recall response s based on notable size change of the granuloma post injection. These results demonstrate d the feasibility of our system of biomaterial consisting of hydrogel loaded with adjuvants and PLGA MPs encapsulating insulin antigen for prevent ing T1D in NOD mice.
15 CHAPTER 1 INTRODUCTION This introduction p rovides our current knowledge of Type 1 Diabetes (T1D) as an autoimmune disease and describes current efforts to prevent and cure the dis order , particularly with regard to both clinical and preclinical , prophylactic or therapeutic approaches . To that end, selected clinical and preclinical immune based strategies will be discussed in terms of the use of agents, the aim of trials, and their potential imp act on the disease . In particular , antigen specific approaches will be further discussed with respect to th eir goal and ability to induc e antigen specific tol erance. The first immune based therapeutic approach was attempted almost three decades ago , but the disease is still incurable. Intensive efforts to investigate safe and effective therapies for T1D are bei ng made worldwide . Additionally , the development of innovative applications using biomaterials, particularly for immunomodulation, has gained much attention due to their biocompatible and biodegradable features . We will present two types of biomaterials wi th advantageous characteristics previously demonstrated in preclinical settings. It is essential to select proper adjuvants for inducing desirable immunomodulation because a vaccine antigen alone is poorly immunogenic by comparison. To that end, selected i mmunologic adjuvants will also be discussed based on their bioactivity in preclinical settings. Type 1 Diabetes General Overview Type 1 diabetes (T1D) is a chronic autoimmune disorder , which is associated with autoreactive T cell mediated destruction of insulin producing cells in the pancreatic islets of Langerhans  . Common c linical signs and symptoms of this disease are frequent urination ( polyuria ) , increased thirst ( polydipsia ) , and hunger
16 ( polyphagia ) . Lack of insulin production leads to aberrant glucose metabolism and h yperglycemia. I nsufficient insulin production results in excessive ketone body production from fatty acid metabolism , causing ketoacidosis  . Poorly managed blood sugar levels can lead to life threatening complications including cardiomyopa thy, neuropathy, retinopathy , and nephropathy  . Worldwide, T1D patients comprise 5 10% of the total case s of diabetes  . According to the Juvenile Diabetes Research Foundation, as man y as 3 million people in the US may have T1D. Additionally, epidemiologic al studies have demonstrated that the incidence of T1D is increasing annually by 2 5 % worldwide  . An e stimated $14.4 billion in medical costs and incom e are spent in the US each year to cover T1D related expenses  . Intensive efforts have been made to develop an effective therapy to prevent and cure T1D in order to relieve the social, physical, and economic burdens on both patien ts and the health care system caused by T1D. Although genetic and environmental factors are considered to be the cause of T1D , trigger ing and progression of T1D is predominantly a result of aberrant autoimmune responses  . Therefore, i mmuno modulatory approaches are being explored in human clinical trials and animal models in order to correct a berrant immune responses, which may ultimately lead to prevent ion or cure of the disease . Therapeutic Approaches for Type 1 Diabetes Almost a century has passed since the discovery of insulin , and the administration of exogenous insulin is still a crucial therapy for persons with T1D . However, awareness of limitations and a massive effort to cure or prevent the disease has led to attempts to find sa fe r and more effective therapeutic ap proaches. T his section of the introduction will describe both traditional and current strategies, and
17 highlight immune based therapie s that have been attempted in clinical and pre clinical settings . Exogenous Insulin Replacement Therapy In 1921, Frederick blood glucose level reduction effect  . A year later, they su ccessfully treated their first human di abetic patient with insulin . In 1923, t he pharmaceutical company Eli Lilly started to sell a commercial animal insulin product  . To date, lifelon g exogenous insulin therapy is still crucial for patients with T1D in order to manage their blood glucose levels . The r apid acting insulin analog glulisine acts quickly after administration , but the activity only lasts a short amount of time . Additionally, the l ong acting insulin analogue glargine has been developed in order to sat isfy basal insulin requirement s  . Continuous subcutaneous insulin infusion ( insulin pump) therapy, first introduced in 1976, han dles insulin delivery in two ways , providing a basal level of insulin throughout the day and a bolus dose of insulin around mealtime s  . Although the insulin pump mimics the insulin release patterns of normal cells, an i ncreased risk of ketoacidosis and severe hypoglycemia caused by interference in insulin delivery ha ve been reported with use of the insulin pump  . Development is in progr ess for n ewer technologies to provide better means of insulin delivery. However, current insulin replacement therapies are not free from potential side effects and complications. Islet Cell Transplantation Since T1D is characterized by selective destruct ion of insulin producing cells in the islet s of Langerhans, islet cell transplantation from healthy islets was thought to be a promising alternative to exogenous insulin replacement therapy . In 2000, researchers at
18 the University of Alberta in Edmonton, Canada, published the Ed monton Protocol , describing findings and advances in islet transplantation. In this publication, they reported that the first seven patients with T1D who received islet transplants achieved insulin independence  . In a 5 year follow up, 80% of the recipients maintained insulin independence at 1 yea r, but only 10% of the recipients remained in an exogenous insulin free condition in 5 years [14, 15] . Besides having disappointing long term results, the current islet transplantation therapy has many obstacles including the requirement of large number s of islets from donors and the risk of substantial side effects from long term use of imm unosuppressive drugs. Therefore, to date, islet transplantation cannot be the ultimate therapeutic approach for T1D. Immune B ased Strategies I mmune based therapies for T1D have largely been categorized into two different settings based on the purpose of t he therapy, whether prevention or intervention (often termed cure) . Primary prevention is mainly for individuals who have a genetic risk for T1D without the presence of islet autoantibodies. Secondary prevention is for persons who have already developed au toantibodies , with the purpose of delaying T1D onset. I ntervention strategies are for newly diagnosed patien ts in order to preserve remaining cell function. The se immune based approaches , either prevention or intervention, have been subcategorized into e ither nonautoantigen specific or autoantigen specific , depending on the target of the therapy  . Nonautoantigen s pecific p rimary and s econdary p revention Nonautoantigen specific primary prevention has mainly been focused on dietary modulations earlier in life. For example, c onsumption of based infant formula in infancy has been suggested to increase the risk of T1D  . In Finland, 230 infants
19 with genetic susceptibility to T1D and with one or more first degree relative s with T1D were rand omly selected to receive either a casein hydrolysed formula or conventional , during the first 6 8 months of life. Interestingly, t h e casein hydrolysate formula fe d group reduced the development of i slet autoantibodies compared to the conventional formula received group  . Vitamin D , another examp le, is considered a non essential dietary vitamin since it can be synthesized from exposure to sunlight. In a study with NOD mice, it was demonstrated that oral administration of 50ng vitamin D per day , s tart ing at 3 weeks of age , could protect the mice fr om development of diabetes for up to 28 weeks of age  . Interestingly, it has been reported that low vitamin D levels during pregnancy may incr ease th e risk of T1D in children  . In Norway, supplementation with c od liver oil, a source of v itamin D and omega 3 fatty acids , during the first year of life significantly reduced the risk of T1D  . However, this beneficial effect was not observed among children who consumed other type s of vitamin D s upplementation . This study suggested that omega 3 fatty acids might be an important factor in reducing the risk of T1D rather than the vitamin D itself  . Cy closporin e , an immunosuppressive agent , has been widely used for organ transplantation to prevent rejection  . A Japanese research group showed that starting cy closporin e treatments (every 2 days for 35 days) at an earlier age in NOD mice (~4 weeks of age) significantly decreased diabetes incidence for up to 23 weeks of age , compared to a group of mice given the drug at diabetes onset  . Cy clospo rin e has also been tested in first degree relatives of diabetes patients with islet autoantibodies  . The drug was given twice daily for four years ; a ll control subjects
20 developed diabetes within a year, but two subjects who were given the drug delayed diabetes onset until 47 and 57 months after starting th eir first treatment. However, this treatment could not prevent the clinical onset of diabetes  . Bacillus Calmette Guerin (BCG), a vaccine for tuberculosis, has been tested in bo th preclinical and clinical settings for secondary prevention of T1D. A single intravenous BCG administration in 10 week old NOD mice showed efficacy in terms of lower diabetes incidence for up to 30 weeks of age , and less severe signs of insulitis with a possible prevention mechanism, the generation of suppressor immune cell populations  . Promising pr e clinical data led to clinical trials in Germany. T he anticipated effect was reduction of diabetes incidence through BCG vaccination in at risk children  . Unfortunately , there was no significant effect from the vaccination in te rms of diabetes prevention. Strong immunostimulatory effects of the BCG vaccine were thought to be the reason why disease acceleration occurred in the study subjects  . Non a utoantigen s pecific i ntervention This type of approach has primarily focused on the use of immunosuppressive regimens. Anti CD3 monoclonal antibodies (mAb) teplizumab and otelixizumab have been tested extensively in regards to disease intervention in mice as well as human s [27 31] . The purpose of using anti CD3 product s is to achi eve depletion of autoreactive T cells in the immune system . A French research group has demonstrated that intravenous anti CD3 mAb treatments (5 consecutive days) in NOD mice at the time of dia betes onset resulted in a complete remission of the disease and maintained the remission for more than 4 months  . A multicenter clinical trial demonstrated that a group of newly diagnosed patients who were given teplizumab for 6 consecutive days
21 preserved residual cell functio n for as long as 18 months after the drug administration  . However, the phase III clinical trial for this drug , P rotÃ©gÃ© , ended sooner showing no difference in metabolic responses  . Rituximab, an anti CD20 mAb , which demonstrated targeted depl etion of B cells, has been tested for its efficacy in NOD mice and persons with new onset T1D [32, 33] . Anti human CD20 (hCD20) transgenic NOD mice treated with hCD20 antibody within 6 days of diabetes onset (4 inj ections at 3 days apart) reversed the disease and maintained normal glucose levels for over 130 days  . In TrialNet, selected patients with new onset T1D were treated with either rituximab or placebo on days 1, 8, 1 5 , and 22. At o ne year af ter the treatments , C peptide preservation was observed in the group administered rituximab , but this beneficial effect was no longer detectable after two years  . However, this result was quite astonishing because T1D is mainly considered an autoreactive T cell mediated disease. The study highlighted that in depth study of B cells in T1D pathogenesis is crucial . Cytotoxic T Lymphocyte Antigen 4 (CTLA 4) is a recepto r expressed on the surfa ce of T helper cells and inhibits excessive activation of T cells by blocking CD28 signaling activity  . Association between polymorphisms with in the CTLA 4 gene and susceptibilities to T1D has been reported in both human s and mice [35 37] . Abatacept, which is the fusion protein of CTLA 4 and immunoglobulin IgG1 (CTLA 4 Ig), is already approved for the treatment of rheumatoid arthritis in the US  . In a clinical trial, p ersons with new onset T1D received a total of 27 infusions of CTLA 4 Ig over 2 years. Following the treatment, a d elayed C peptide reduction by 9.6 months was obser ved ,
22 but decreased cell function was noted after 6 months of the treatment and this trend was parallel to that in the placebo group  Autoa ntigen s pecific i mmunotherapy The p resence of autoantibodies against cell autoantigens is one of the major clinical markers of T1D  . Therefore , autoantigen specific approaches h ave gained widespread attention as a preferable and targeted way to prevent or intervene in T1D. The main aim of antigen specific primary prevention is one of achieving protective immune tolerance against autoantigens such as insulin, glutamic acid decarboxylase (GA D), islet antigen 2 (IA 2), or zinc transporter 8 (ZnT8) and eliciting autoantigen specific regulatory immune response s [16, 41] . Autoantigen s pecific p rimary and s econdary p revention Insulin therapy has been studied under multiple routes of administration including parentera l, intranasal , and oral in b oth pre clinical and clinical settings. [42 47] . Hutchings et al . demonstrated that NOD mice which were given intravenous ovine insulin injection starting at 4 weeks of age exhibited diab etes prevention for up to 30 weeks of age compared to a control group , which was given hen egg lysozyme  . In 1994, The Diabetes Prevention Trial 1 ( DPT 1 ) of parenteral in sulin administration in human s started in the US. Daily subcutaneous injections along with annu al intravenous insulin infusion s were given to individuals who w ere autoantibody positive first degree relatives of patients with T1D. Disappointingly , the treat ment turned out to have no effect on diabetes incidence  . Zhang et al . investigated that oral insulin treatments in NOD mice had efficacy in terms of delaying the onset and decreasing th e incidence of diabetes  . In contrast to the promising pre clinical data, the DPT 1 oral insulin study
23 ended up with no clinical effect on di abetes development. However, a post hoc analysis identified that a subgroup of participants with insulin autoantibodies delayed onset of diabetes more than 4 years  . In general, oral ingestion of protein based drug s leads to extensive degradation in the gut, thereby efforts to find alternative routes of drug administration have been made. Daniel and Wegmann demonstrated that 7 doses of intranasal insulin peptide B (9 23), a major epitope for initiation of diabetes i n NOD mice, protected the mice from diabetes for up to 32 weeks of age  . In Australia, the Intranasal Insulin Trial was conducted with 38 individuals who have first degree relatives with T1D and positivity of one or more islet autoanti bodies  . The subjects were randomly assigned to treat ment with either 1.6 mg of intranasal insulin or a carri er solution, daily for 10 days and then followed by 2 days per week for 6 months. As a result, there were no significant effects on cell function by the intranasal insulin treatments, but enhanced mucosal tolerance to insulin was observed  . Primary Intervention with Oral Insulin for Prevention of T1D (Pre POINT) is an ongoing study for chil dren who have high genetic risk and at least two affected first degree relatives or a full sibling with T1D. Selected study participants will be given insulin either via an oral or intranasal route with the aim of inducing insulin autoant igen specific immune tolerance and prevent ing insulin autoantibody developm ent  . Autoantigen s pecific i ntervention GAD is an enzyme that catalyzes conversion of glu tamic acid into gamma aminobutyric acid and is considered another major autoantigen in T1D  . Li et al . reported that intraperitoneal treatments of GAD65 217 230 peptide and soluble MHC class II antibody dimer at a relatively late preclinical stage (12 weeks of age) could prevent
24 diabetes in NOD mice for up to 30 weeks of age with sign s of IL 10 mediated suppression  . I nitial clinical trial s with a subcutaneous GAD65 aluminum hydroxide (alum) vaccine also showe d promising results including delaying loss of C peptide secretion in individuals who were given this vaccine formulati on within 6 months of diagnosis  . B e yond this , a report noted that GAD alum vaccine induced GAD65 specific regulatory T cell (Treg) population s in T1D patients [53, 54 ] . Despite high hopes for this vaccine , a recent phase III trial in Sweden showed discouraging results that GAD alum was ineffective in recent onset of diabetes patients  . An altered peptide ligand of insulin peptide B ( 9 23 ) , NBI 6024 , with peptide residues 16 and 19 substituted to alanine, was subcutaneously administered in recent onset NOD mice. As a result, the peptide treated mice exhibited great reduction in diabetes incidence with a sign of regulatory Th 2 cytokine production  . In a phase I clinical trial, t reatments with NBI 6024 in r ecent onset patients showed immunological ly effective responses such as a switch from pathogenic Th1 to protective Th2  . Disappointingly , no change in cell function from NBI 6024 treatment was observed in a phase II clinical trial  . Antigen Specific Tolerance In the previous section, we discussed therapeutic approaches for T1D including nonauto ant igen or autoantigen specific prevention and intervention strategies in both preclinical and clinical settings . Nonspecific immunosuppression has been demonstrated partially effective with temporary remission of the disease, but long term treatments carry s erious risks as a result of a weakened immune system  . As such , t argeted a ntigen specific immunomodulation has gained a lot of attention. The main
25 goal of this type of therapy is to induce antigen specific tolerance followed by targeted blockage of un desirable self reactive immune cell activities  . In a normal setting, T cell progenitors with strong affinity T cell receptor s (TCR) for self peptides can be selected and deleted in the thymus  . However , e ven if autoreactive T cell clones escape the thymic negative selection of central tolerance , there is another backup mechanism, which is known as peripheral tolerance . The peripheral tolerance includes deletion, induction of anergy , and ignorance  . However, these mechanisms can still fail the r egulation of autoreactive T cells, and this failure ultimately leads to destructive autoimmune responses. Therefore, it is critical to develop therapeutic strategies , which can induce antigen specific tolerance . N aÃ¯ve CD4+ T cells need to receive two signa ls th rough TCR in order to be activated. The TCR interacts with MHC class II molecules expressed on antigen presenting cells (APCs) . In addition, CD28, a co stimulatory receptor expressed on T cells , binds to co stimulatory molecules CD80 and /or CD86 expre ssed on the surface of APCs . If one of the signals is not deliver ed properly, the naÃ¯ve T cells cannot be activated, and eventually experience anergy or apoptosis  . Chang et al . demonstrated that immunization s with a DNA vector encoding pre pro insulin with the addition of mutant CD80/CD40L fusion protein, which is considered a negative regulator of reactive T cells , significantly reduced incidence of diabetes in NOD mice for up to 35 weeks of age  . Non Obese Mouse Model for Type 1 Diabetes The Non Obese Diabetic (NOD) strain is the most appreciated and useful mouse model in T1D research. This strain of mouse developed in Japan by Makino and colleagues during the selection of the Cataract Shionogi strain was originally derived from outbred Jcl:ICR mice  . Through a repetitive process of inbreeding , a
26 normoglycemic female mouse spontaneously developed diabetes and this was the progeny of the current NOD strain. The reason why this strain is widely used in diabetes research is that there are numerous similarities between human and NOD mouse diabetes pathogenesis. A limited number of immune cells begin to surround and infiltrate is lets around 3 to 4 weeks of age. Immune cell invasions of islets become more severe at later ages . The t ypes of is let infiltrated immune cells include CD4+ and CD8+ T cells, B cells, macrophages , and dendritic cells (DC s ). NOD female mice start to exhibit overt diabetes around 12 to 14 weeks of age. Interestingly, there is a gender bias in terms of diabetes incidence in NOD strain . A round 60% to 80% of female NOD mice exhibit overt diabetes by 30 weeks of age, but only 20% to 30% of male NOD mice spontaneously develop the disease [64, 65] . Since T1D is known as an autorea c tive T cell mediated autoimmune disease, the importance of understanding T cells in NOD mice has been emphasized. Multiple g enetic susceptibilities also cont ribute to diabetes devel opment. The NOD mouse has a unique MHC haplotype H2 g7 and this haplotype is known as a weak peptide binder, which has been hypothesized to contribute to autoimmunity  . Interestingly, t his haplotype allele has similarities to the human T1D susceptibility locus HLA DQB1  . Extensive genetic analysis of NOD mice has identified nearly 50 diabetes relate d loci  . In addition to such genetic defects, NOD mice have a number of diabetes associated immunological defects including aberrant central and peripheral tolerance, defective natural killer cell and macrophage function, and potential defects in suppressive function s of Treg s [69, 70] . Given these features , particularly the autoimmune related susceptibilities , have made the NO D mouse strain a predominant animal model in T1D research.
27 Biomaterials for Immunological Applications Over a half century ago, British ophthalmologist Harold Ridley found that unintentionally implanted canopy plastic, which was composed of poly (methyl m ethacrylate), did not cause severe adverse effects in the eyes of Spitfire fighter pilots who were attacked by gunfire during World War II . This observation contributed to the develop ment of implantable intraocular lenses that are still in use for persons with cataracts  . Growing efforts in biomaterial development have broadened the scope of applications from implantable medical devices to drug delivery systems. This section of the introduction will provide an overview of two biomaterials, which were used as vaccine delive ry vehicles in N OD mice for the purpose of prevention of T1D in this dissertation . PLGA as a Vaccine Delivery Vehicle Poly (lactide co glycolide ) ; ( PLGA) is a synthetic biodegradable and biocompatible polymer and is already approved by the US Food and Drug Administration for human use , such as in absorbable suture material s and prosthetic devices  . Since this biomaterial has ma ny desirable features including safety, non immunogenic ity , ease of customization , and the ability to control the release of drugs or protein antigens, PLGA has been intensively studied for the purpose of use in a vaccine delivery system  . This controlled release biomaterial can be processed into any size. Particularly, a diameter of 1 to 7 m micros pheres can be taken up by APCs including DCs and macrophages [74, 75] . Phagocytic activity creates an intracellular p hagosome, and this vesicle fuses with lysosome , followed by formation of a phagolysosome, where P LGA degradation takes place . Encapsulated antigen is released and presented on both class I and class II MHC molecule s [72 74, 76, 77] . Pedraz and colleagues have
28 demonstrated that immunization with PLGA MPs contai ning coencapsulated bovine serum albumin , TLR4 ligand monophosphoryl l ipid A , and galactosylceramide induced stronger humoral and cellular immune responses in Balb/c mice  . Another research group investigated the finding that vaccination with TLR ligands and t umor lysate encaps ulated PLGA MP s could generate strong cytotoxic T cell res ponses and significantly reduce tumor volume in transgenic adenocarcinoma of the mouse prostate model  . Injectable PuraMatrix Peptide Hydrogel PuraMatrix peptide hydrogel (hydrogel) is a biocompatible and b iodegradable self assembling material composed of repeating amino acids arginine alanine aspartic acid alanine. In the presence of physiological level s of salt, t he peptides can form a stable sheet structu re and self assemble into three dimensional (3D) structures , with pore sizes between 5 and 200 nm in diameter [80, 81] . This 3D scaffold can be widely function al from use as a synthetic extracellular matrix for cell growth to as a controlled release biomaterial for drug delivery. Hydrogel has been tested in a wide range of applications, such as ligament regeneration [82, 83] , bone regeneration  , and therapeutic drug delivery [85 87] . Nishimura and colleagues demonstrated that a subcutaneous injection of hydrogel incorporated with insulin into male Wistar rat s maintained lower plasma glucose level for up to 24 h ou rs  . Koutsopoulos et al . evaluated several types of protein release profiles through the hydrogel. Encapsulating proteins were selected with criteria based on molecular weight s, is oelectric points , and structures. As a result, smaller protein (lysozy me, 14.3 kDa) released much faster through the hydrogel pores than larger protein (Immunoglobulin G, ~150 kDa). Hydrogel
29 density also had an influence on the protein release profile . Higher density gel (1.5% wt/vol, 98.5% water) seemed to interfere with prot ein diffusion compared to lower density gel (0.5% wt/vol, 99.5% water). In addition, t he functionality of the released protein was confirmed as well. The released proteins were not biologically or structurally different from the native proteins  . Vaccine Adjuvant The word adjuvant Since vaccine antige n s alone are not enough to elicit strong immunogenicity, most In 1926, Glenny and colleagues discovered that alum precipitated diphtheria toxoid enhanced antibody responses in guinea pigs, compared to treatment with toxoid alone  . This study was the first to demonstrate the adjuvant effect o f aluminium salts , which have been the most widely accepted adjuvant in human vaccines  . However, alum has been criticized due to limitation s , such as fail ing to induce strong cellular immune responses  . MF59, a squalene based oil in water adjuvant , has been initially accepted for use in influenza vaccine for elder ly people in Eu rope  . Wack et al . have demonstrated that MF59 induced stronger an tibody and CD4+ T cell response s compared to other typ es of adjuvants including alum and calcium phosphate in mice  . However, Vesikari and colleagues have reported side effects from the MF59 adjuvanted H5N1 influenza vaccine in younger age group s (from 6 months to 17 years old). The adv erse effects incl uded erythema, s welling, and injection site pain  . Although these symptoms were resolved within a week, a better understanding of the potential side effects and mechanisms of the adjuvant is necessary.
30 GM CSF Granulocyte macrophage colony stimulating factor (GM CSF) is one of the he matopoietic growth factors secreted by numerous types of cells including monocytes, T cells, and endothelial cells  . This c ytokine can pro mote recruitment of circulating monocytes, lymphocytes, and neutrophils. Additionally, GM CSF has the ability to activate and increase the function of APC s , specifically DC and macrophage populations [95, 96] . Thes e biological functions ma ke GM CSF a potent adjuvant. This cytokine has been tested for diabetes prevention in NOD mice [97 99] . Gaudreau et al . demonstrated that multiple intraperitoneal t reatments of GM CSF (100n g/mouse, 3 times/week until 6 week s old, 2 times/week thereafter until 52 week s old), started at 3 weeks of age could protect NOD mice from diabetes for up to 52 weeks of age with sign s of tolerogenic DC induction . The DCs exhibited an increase in expressi on of programmed death ligand 1 and production of IL 10  . Cheatem and colleagues a lso investigated the therapeutic potential of GM CSF in NOD mice. GM CSF (2 g/mL/mouse) treatments started at 7 weeks of age showed efficacy for up to 36 weeks of age along with evidence of i ncreasing Treg population s and production of anti inflammatory cytokines  . T hose two studies demonstrated a great potential of GM CSF in terms of inducing tolerogenic immune responses le a d ing to prevent ion or delay of the disease. However, requirement s of multiple high dose administrations to obtain treatment efficacy can be a financial burden and potential cause of side effects . CpG ODN CpG oligodeoxynucleo tides (ODNs) are synthetic DNA containing unmethylated CpG motifs and which stimulate immune cells through the TLR 9 signaling pathway  . TLR is a type of pattern recognition receptor and detects conser ved molecular
31 sequences expressed on pathogens  . Activation of TLR leads to trigger ing of innate immune response s including production of proinflammatory cytokines, type I interfer ons and antimicrobial peptides in order to protect the host from invaders [102, 103] . In mice, TLR 9 is expressed by macrophages, monocytes, myeloid DCs, and activated T cells, whereas plasmacytoid DCs and B cells are the main cell types that express TLR9 in humans [100, 104] . There are four classes of CpG ODNs ; A , B , C , and P classes have been classified based on difference s of CpG sequence motifs, palindromic sequences, and biological activities  . To date, B class ODN has been widely tested for its adjuvant activity in vaccine studies. Since CpG DNA stimulated immune cells readily induce both cellular and hu moral immune responses , CpG ODN has gained widespread attention as a vaccine adjuvant  . Particularly, B class CpG ODN strongly stimulates B cells but inhibit s B cell apoptosis. In addition, CpG DNA induces IgG class switch DNA recombination , which is important for early IgG resp onses against pathogens in the absence of T cell help [105, 106] . Although CpG ODN appears to be a strong inducer of Th1 type immune response, studies have also show n that CpG activated B cells could induce anti inflammatory IL 10 production [107, 108] . Hemoglobin, Haptoglobin , and Heme Oxygenase 1 Hemoglobin (Hb) is the predominant protein of the red blood cell (RBC) and acts as an oxygen carrier. Hemolysis, the process of RBC breakdown, causes Hb release from the RBC. Free Hb can bind to oxygen a nd this reaction produces free oxygen radicals, which induce cytotoxic oxidative stress [109 111] . Therefore, the Hb needs to be cleared by a scavenger protein haptoglobin (Hp). The Hp is a n acute phase plasma prot ein and increased production is induced by inflammatory responses [110, 112] . Free
32 Hb binds to Hp with high affinity, and this complex is recognized and endocytosed by a receptor protein CD163, expressed on the surface of monocytes and macrophages [109, 110] . This subsequently leads to breakdown of heme subunit of the Hb by enzymatic activity of heme oxygenase 1 (HO 1 ). The degradation of heme produces biliverdin, iron , and carbon monoxide (CO) [109, 110, 113] . The b iliverdin, an antioxidant product, is catalyze d to bi lirubi n by biliverdin reductase and the b ilirubin is transported to the liver, followed by excretion in the bile  . The heme derived iron is stored by ferritin. Interestingly, the CO has anti inflammatory and anti oxidant effects [109, 113] . Otterbein et al . demonstrated that CO inhibited pro i nflammatory cytokine production induced by lipopolysaccharide stimulation, and induced anti inflammatory cytokine IL 10 in mouse peritoneal macrophages  . In ad dition, Philippidis et al . investigated Hb:Hp complex binding to CD163 and leading to induction of IL 10 and HO 1 in human macrophages  . Taken together, the combination of Hb and Hp mig ht be a good candidate for immunological tole rance induction .
33 CHAPTER 2 GENERAL METHODS Mouse Strains In this dissertation, the NOD/ShiLtJ (NOD) strain was used for a biomaterial based tolerogenic vaccine study. Two additional strains including C57 black 6 (C57) and N on O bese R esistant /LtJ (NOR) w ere used for characterizing mobilization patterns in response to cytokines , including granulocyte macrophage colony stimulating factor (GM CSF) an d granulocyte colony stimulating factor (G CSF) . For the vaccine study, NOD mice were ordered at 7 weeks of ag e from The Jackson Laboratory (Bar Harbor, ME, USA) and given a week without interruptions to adjust to their new housing environment . In order to test the age dependence of mobilizing patterns in response to GM CSF or G CSF , all mouse strains were ordered at 8 and 12 weeks of age from the same company. All the mice were housed in SPF conditions in the Biomedical Sciences Building Animal Care Services facility at the University of Florida. In addition, a ll experimental procedures were performed in accordanc e with approved Institutional Animal Care & Use Committee (IACUC) protocols. D etermination of Hyperglycemia Blood glucose level s in NOD mice w ere measured once e very week from 10 weeks of age. A tail bleed was performed using a 1 inch 22 gauge needle (BD Biosciences, NJ, USA) and a drop of blood was applied to the test strip of an AlphaTRAK 2 Glucose meter (Abbott Animal Health , Abbott Park, IL , USA). If the value was higher than 250mg/dL for two consecutive days , the animal was considered to be diabetic .
34 Mouse Anesthesia Anesthetization was performed for the process of implantation of an identification (ID) microchip and for vaccine administration. All animals assigned for the vaccine study were anesthetized by 3 5% Attane isofluorane (Piramal Critical Ca re, Inc., Bethlehem, PA, USA) inhalation through a vaporizer (VetEquip, Inc., Pleasanton, CA, USA) up to five times throughout the study. Each animal was placed in an acrylic induction chamber and ex posed to 5% of isofluorane with oxygen flow . The animal w as then transferred to a nose cone apparatus and inhaled 3% isofluorane with oxygen. Following the procedure , animals were carefully monitored until they became fully conscious. Animal Microchipping Throughout the vaccine study, all animals were individua lly monitored with a unique 9 digit ID number in order to manage each mouse based on its corresponding vaccine treatment . An ID microchip (FriendChip, Avid Identification System, Inc., Norco, CA, USA) was implanted under anesthesia subcutaneous ly in the mi d dorsal area by using a sterile syringe containing the microchip. Following this chip implantation procedure , each mouse obtained an individual ID number, which could be detected by a MiniTracker (Avid Identification System, Inc., Norco, CA, USA). Spleen and Bone Marrow Cell Purification After sacrificing an animal, the spleen was collected usi ng sterile surgical instruments and then transferred into a conical tube containing 5mL Hank Salt Solution (HBSS; Mediatech Inc . , Westwood, M A, USA). For bone marrow (BM) cell purification, t he femurs and tibiae from hind limbs were obtai ned. Attached soft tissues and muscles were carefully removed by using a sterile scalpel. Then, the bones were placed in a 1.5mL microcentrifuge tube containing 1mL HBSS. For splenocyte isolation ,
35 the spleen was placed in a sterile 40 m nylon cell strainer (BD Biosciences, San Jose , CA, USA) on top of a new 50mL conical tube (BD Biosciences, San Jose, CA, USA) and perfused by a n HBSS filled 3mL syringe attached 27 gauge needle (BD Biosciences, San Jose, CA, USA) . Then, the plunger was removed from the syringe and used for mashing the spleen. HBSS was added during the tissue grinding until the total volume reached 30mL. Centrifugation at 300g for 10 minutes at room temperature was performed to obtain a cell pellet. The supernatant was de canted and the pellet was resuspended with 2 mL HBSS . For BM cell isolation, both end s of the bone were removed using a sterile scalpel (BD Biosciences, San Jose, CA, USA) and the marrow was flushed by a n HBSS filled 3mL syringe attached 27 gauge needle (BD Biosciences, San Jose, CA, USA) . All isolated marrows were collected in to sterile 15mL conical tube s . Centrifugation at 300g for 10 minutes at room temperature was performed to acquire a cell pellet. The supernatant was decanted and the pellet was resuspe nded in 10mL HBSS followed by filtration through a sterile 100 m nylon cell strainer (BD Biosciences, San Jose , CA, USA). The cells were then centrifuged at 300g for 10 minutes at room temperature . The supernatant was carefully removed followed by resuspension of t he cell pellet in 2 mL HBSS. The f ollowing steps were identical for both the spleen and BM cell purification, unless otherwise stated . To lyse the RBCs, 3mL of 1x lysing buffer (BD Biosciences, San Jose , CA, USA) w ere added the tube and placed on ice for 5 minutes. Two additional washes and centrifugations w ith 30mL HBSS were performed. Washed c ells were resuspended in 30mL HBSS for splenocytes and 20mL HBSS for BM cells .
36 Following this step, t he cells were ready for cell counting. To confirm the number of live cells , 10 L of the resuspended cells was mixed with 90 L t rypan b lue dye (Corning Inc., Tewksbury, MA, USA). Then, 10 L of the mixture was placed under the cover glass of a hemocytometer and the cells within the four corner squares were counted. The o btained t otal number of live cells was divided by 4 and t he average value was multiplied by 10 5 to determine the number of cells per mL . Preparation of Reagents Recombinant mouse GM CSF and G CSF were purchased from R&D Systems (Minneapolis, MN, USA). GM CSF was dissolv ed in sterile phosphate buffer ed saline (PBS; Hyclone, Logan, UT, USA ) at final concentration 20ng/ L. G CSF was reconstituted at 10ng/ L in sterile water ( Fisher Scientific, Waltham, MA, USA). Both cytokines were stored at 20 o C until used. Human hemoglobin (Sigma Aldrich, St. Louis, MO, USA) was freshly prepared for each experiment and dissolved in st erile water at a final concentration of 20 g/ L without vigorous agitation. Human haptoglobin, phenotype 1 1 (Sigma Aldrich, St. Louis, MO, USA) , was reconstituted at 20 g/ L in sterile water and stored at 20 o C until used . CpG ODN1826 was purchased from I nvivoGen (San Diego, CA, USA) and reconstituted at 5 g/ L in the manufacture r provided endotoxin free water. Flow Cytometry Analysis To investigate specific immune cell populations, flow cytometry experiments were performed. Ten million cells from each sa mple were prepared and followed by resuspension with 1mL of staining buffer in order to dilute the cells to the final concentration of 1x10 6 / 100 L. In addition to test samples, unstain ed, corresponding
37 isotype control s , and compensation control samples wer e prepared for each set of experiment s . Each sample containing 1x10 6 cells was placed in a 5mL Falcon round bottom tube (Corning Inc . , Tewksbury, MA, USA). Staining Cell Surface Antigens Before surface staining, all samples were incubated with 0.63 g of p urified anti mouse CD16/32 (eBioscience , Inc . , San Diego, CA, USA) for 15 minutes at 4 o C in order to block non specific Fc mediated interactions. Without washing, antibody cocktails were added to the corresponding tubes. There were three different sets of surface antibody cocktails, tested in this dissertation. The first set was for immunophenotyping of profess ional APCs , and consisted of four different antibodies including : 0.5 g anti mouse CD11c FITC, clone N418 (eBioscience , Inc . , San Diego, CA, USA) ; 0.1 g anti mouse I A d PE, clone AMS 32.1 (BD Biosciences, San Jose, CA, USA) ; 0.25 g anti mouse CD19 PE Cy7, clone eBio1D3 (eBioscience , Inc . , San Diego, CA, USA) ; and 0.2 g anti mouse CD11b APC, clone M1/70 (eBioscience , Inc . , San Diego, CA, USA). The corresponding isotype control antibodies for this set were : Armenian Hamster IgG FITC (eBioscience , Inc . , San Diego, CA, USA) ; mouse IgG2 b PE (BD Biosciences, San Jose, CA, USA) ; r at IgG2a PE Cy 7 (eBioscience , Inc . , San Diego, CA, USA) ; and rat IgG2 b APC (eBioscience , Inc . , San Diego, CA, USA) . It must be pointed out that anti mouse I A d PE antibody was used for detection of MHC Class II antigen expressed on the immune cells of NO D and NOR mouse strains. If the test sample was derived from a C57 mouse, 0.1 g anti mouse I A/I E PE, clone M5/114.15.2 (eBioscience, Inc., San Diego, CA, USA) was used instead. The corresponding isotype control for this antibody was rat IgG2b ( eBioscie nce, Inc., San Diego, CA, USA). The second set of
38 antibodies was for detecting memory T cell populations. There were also four different antibodies including : 0.25 g anti mouse CD8b FITC, clone eBioH35 17.2 ; 0. 1 25 g anti human/mouse CD44 PE, clone IM7 ; 0. 2 5 g anti mouse CD4 PE Cy7, clone GK1.5 ; and 0.06 g anti mouse CD62L APC, clone MEL 14 (eBioscience , Inc . , San Diego, CA, USA). The corresponding isotype control antibodies were : rat IgG2 b FITC ; rat IgG2 b PE ; rat IgG2 b PE Cy7 ; and rat IgG2a APC (eBiosci ence , Inc . , San Diego, CA, USA). The third set included antibodi es for insulin B(9 23) tetramer reactive CD4+ T cell detection. The appropriate antibodies were : 0.25 g anti mouse CD3e FITC, clone 145 2C11 ; 0.25 g anti mouse CD8b PE, clone eBioH35 17.2 ; 0.2 5 g anti mouse CD4 PE Cy7, clone GK1.5 (eBioscience , Inc . , San Diego, CA, USA) ; and 1.4 g anti mouse I A g7 Insulin B(9 23) tetramer APC ( Peptide sequence HLVERLYLVAGEEG reg3 C19A ; NIH Tetramer Core Facility, Atlanta, GA, USA). The corresponding isotype con trol antibodies were: Armenian Hamster IgG FITC ; rat IgG2 b PE ; rat IgG2 b PE Cy7 (eBioscience , Inc . , San Diego, CA, USA) ; and human I A g7 CLIP 87 101 APC ( Peptide sequence PVSKMRMATPLLMQA ; NIH Tetramer Core Facility, Atlanta, GA, USA). A set of compensati on control samples was also prepared to correct for fluorescence spillover. Bright and strong positive signals were desired, so anti mouse CD4 antibo dies conjugated to FITC, PE, PE Cy7, and APC were select ed. All samples with the appropriate antibody cockt ails were incubated for 30 minutes in the dark at 4 o C. After the incubation, 2mL of stain buffer was added to each tube , followed by centrifugation at 350g for 5 minutes at 4 o C. The supernatant was aspirated with minimal disturbance of cell pellet. The pel let was then resuspended in 2mL of stain buffer followed by another centrifugation and aspiration . Following this washing step, 250 L BD Cytofix fixation
39 buffer (San Jose, CA, USA) , containing 4% paraformaldehyde was added to the cell pellet while vortexin g , and the samples were incubated for 15 minutes in the dark at 4 o C. After the incubation, one more washing step was performed. The stained cells were resuspended in 250 L of stain buffer the n placed in a refrigerator until analysis by flow cytomet r y . Sta ining Intracellular Antigen F orkhead box P3 (Foxp3 ) staining is necessary in order to detect Treg population s . Since Foxp3 is a nuclear antigen, additional permeabilization step s w ere required after surface antigen staining. Staining for Treg s started with three surface antigens including : 0.25 g anti mouse CD8b FITC, clone eBioH35 17.2 ; 0.25 g anti mouse CD4 PE Cy7, clone GK1.5 ; and 0.125 g anti mouse CD25 APC, clone PC61.5 (eBioscience , Inc . , San Diego, CA, USA). The corresponding isotype control antibodies were: rat IgG2 b FITC ; rat IgG2 b PE Cy 7; and rat IgG1 APC (eBioscience , Inc . , San Diego, CA, USA). The samples mixed with antibody cocktails were incubated for 30 minutes in the dark at 4 o C . Following the incubation, 2mL of stain buffer w ere added into each tube , followed by centrifugation and aspiration. One more wash with 2mL stain buffer was performed , followed by another centrifugation and aspiration. The cell pellet was thoroughly resuspended in 1mL eBioscience fixation/permeabilization buffer (San Diego, CA, USA). The samples were the n incubated for 60 minutes in the dark at 4 o C. Following this incubation, 2mL of 1x eBioscience permeabilization buffer (San Diego, CA , USA) was added to each tube. The tubes were centrifuged at 350g for 5 minutes at 4 o C, decanted, and resuspended in 2mL 1 x permeabilization buffer followed by centrifugation and aspiration . The washed cell pellet was resuspended with 100 L
40 permeabilization buffer, then 0.63 g of purified anti mouse CD16/32 (eBioscience , Inc . , San Diego, CA, USA) was added to each tube. The s amples were incubated for 15 minutes in the dark at 4 o C. Without washing, 0.25 g anti mouse Foxp3 PE, clone NRRF 30 (eBioscience , Inc . , San Diego, CA, USA) was added to the test tubes. The corresponding isotype control rat IgG2a PE (eBioscience , Inc . , San Diego, CA, USA) antibody was added to the isotype control tube. All tubes were incubated for 30 minutes in the dark at 4 o C. Following this incubation, 2mL 1x permeabilization buffer was added to each tube. The tubes were centrifuged at 350g for 5 minutes at 4 o C, aspirated, and resuspended in 2mL stain buffer followed by another centrifugation and aspiration. The stained cells were resuspended in 250 L stain buffer and placed in the refrigerator until time of analysis . Stained cells were analyzed on a BD Accuri C6 flow cytometer with BD CSampler software (San Jose, CA, USA). Fluorescence spillovers were corrected based on the values given by the C6 Co mpensation Calculator (San Jose, CA, USA). Following the compensation step, all data were exported as FCS Express file s for further analysis. The data were analyzed using the FCS Express 4 Flow Cytometry program (De NoVo Software, Los Angeles, CA, USA). S tatistical Analysis Data analyses were performed using GraphPad Prism v5.0 software (La Jolla, CA, USA) . Kaplan Meier survival curves presented diabetes incidence over the course of time. Comparing survival curves by Mantel Cox test determined the P value and significance. Statistical significance of insulitis scoring data was obtained by two tailed Other data were analyzed by either two tailed unpaired t tests or
41 one multiple comparison tests. Statistical si gnificance was defined if the P value was less than 0.05. Error bars represented mean Â± s tandard e rror of the mean (SEM) , unless otherwise indicated .
42 CHAPTER 3 DEVELOPMENT OF A VACCINE DELIVERY SYSTEM OF HYDROGEL MICROPARTICLE MATRIX MATERIAL Background One of the most challenging requirements for a successful vaccine design is to select the proper antigen and its delivery method . In general, the s oluble form of an antigen or adjuvant tends to be rapidly cleared by the immune system without having a chan ce to encounter targeted immune cell populations or induce desired immune responses . To overcome this drawback, the development of controlled and sustained release s ynthetic biomaterials have been intensively investigated [74, 115, 116] . Peptide hydrogel has been recognized as a biodegradable and injectable synthetic biomaterial. In the presence of physiological level s of salt, the hydrogel can self assemble into 3D structures and incorporate drug s with in the gel scaffold . This feature ma kes the hydrogel a controlled and sustained release material [80, 81] . Since the hydrogel is composed of 99% water and 1% ( w/v ) natural amino acids including arginine, alanin e, and aspar tic acid, degradation of the gel does not produce any toxic chemical  . Roy et al. demonstrated advantage s o f the hydrogel as a vaccine delivery system. They found that the delivery of DC chemoattractant MIP3 along with a plasmid DNA IL 10 small interfering RNA encapsulated PLGA MPs via in situ crosslinkable hydrogel could significantly protect vaccine immunize d mice from a lethal dose challenge of A20 B cell lymphoma in comparison to naked DNA vaccine injected mice  . Thus, hydrogel can be a good candidate for vaccine delivery . The widely accepted r ationa le of tolerance inducing vaccine s is that administration of autoantigen lead s to educat ion of APCs , thereby elicit ing antigen specific tolerance via inducing anti inflammatory cytokine production or antigen specific
43 T cell anergy , which is the functionally inactivated status of T cells against the antigen [118, 119] . To that end, vaccine antigen needs to be delivered with in a range of phagocytosable size s in order to be taken up by APCs. PLGA particles from nanomete r to micron size have been widely studied in immunomodulation effects by improved delivery of DNA, adjuvant, or antigen to the immune system [74, 120 122] . Nanoparticles are inter which is not limited to APC s  . However, MPs of a size 1 7 m can be taken up by APCs followed by intracellular transport of their encapsulated agents [74, 75] . Wang and colleagues demonstrated that immunization of C57BL/6 mice with ovalbumin (OVA) p rotein antigen and CpG adjuvant encapsulated PLGA MPs resulted in the highest titers of IgG2b and IgG2c antibodies in serum, and significant induction of OVA antigen specific IFN in splenocytes compared to control groups including no treatment and soluble OVA and CpG treatment s  . Thus , vaccine antigen delivery via PLGA MPs would improve immun ogenicity [74, 75] . The r oute of administration of vaccine s has an impact on the i mmunogenicity of the vaccine  . Studies have demonstrated in vivo translocation and accumulation of MPs [126 128] . Newman and colleagues tested cellular uptake of fluorescently lab eled phagocytosable MPs at two injection sites intraperitoneal and intradermal. Fluorescent signal from intraperitoneally injected MPs was detected in CD14+ macrophages located in the intraperitoneal cavity . In contrast, intradermally delivered MPs were discovered primarily in DCs , which were isolated from inguinal LNs  . Another study done by Lewis et al . revealed that subcutaneous ly inject ed phagocytosable MPs were traff icked by DCs in popliteal LNs  . Additionally, Phillips et al . emphasized that administration
44 of particulate vaccine needs to be performed in a site anatomically prox imal to the target organ in order to deliver and accumulate the injected MPs in the targeted organ  . This chapter describes and discusses the characteriz ation of both denatured insulin encapsulated PLGA MP and vaccine formulation incorporated hydrogel as a vaccine delivery system in in vitro settings. In addition, in vivo live imaging of mice , given a subcutaneous injection of fluorescently labeled MPs and hydrogel matrix , will exhibit biodistribution and ac cumulation of the biomaterials. Materials and Methods Preparation of H uman D enatured I nsulin E ncapsulated PLGA MP Recombinant human insuli n powder ( SAFC Biosciences, Inc . , Lenexa, KS, USA ) was dissolved in sterile water and adjusted to pH 2.5 with 0.1N HCl. The n, 10mM of 2 mercaptoethanol (Sigma Aldrich, St. Louis, MO, USA) was added to the insulin solution in order to reduce disulfide bonds , followed by incubation of the solution at 95 o C for 5 min utes for further denaturing . The heated solution was cooled on ice . Then, 1N NaOH was added to the solution for pH neutralization, followed by fil tration ( Amicon Ultra Centrifugal Filter Unit; Milli pore, Billerica, MA , USA). Final concentration was measured by Bradford assay (BioRad, Hercules, CA, USA). PLGA MP s w ere prepared using a standard water oil water solvent evaporation technique . A 50:50 polymer composition of PLGA (MW ~ 44,000 g/mol in methy lene chloride; Purac , Lenexa, KS, USA ) was used to generate MP s . Poly vi nyl alcohol (PVA) (MW ~ 100,000 g/mol ; Fisher Scientific, Waltham, MA, USA ) was used as an emulsion stabilizer. Distilled water (DiH 2 O) was used as the aqueous phase to form the emulsio ns while methylene chloride (Fisher Scientific, Waltham, MA, USA ) was used as the organic solvent to dissolve PLGA polymer. To inco rporate the insulin and PLGA MP s , 100 mg of PLGA polymer was
45 dissolved in methylen e chloride at 5% w/v ratio. Then, 0.1mL of t he denatured i nsulin solution (1 mg/ mL) was added to the 5% PLGA sol ution and homogenized at 35,000 rpm for 120 s econds to form a primary emulsio n. This emulsion was added to 2 mL of 5% PVA solution and homogenized again at 19,500 rpm for 60 s econds to form th e secondary emulsion which was transf erred to a beaker containing 30 mL of 1% PVA. The particles thus formed were agitated using a magnetic stirrer (Fisher Scientific, Waltham, MA, USA ) for 24 h ou rs to evaporate residual methylene chloride. The remaining so l ution was centrifuged at 10,000 g for 10 min utes to collect MP s , which were subsequently washed three times with DiH 2 O. The water was as pirated from the centrifuged MP s , which were then flash frozen in liquid nitrogen and kept under vacu um in dry ice overn ight. The MP s were stored at 20 o C until used. PLGA M P C haracterization The size dist ributions of MP were measured using the Microtrac Nanotrac Dynamic Light Scattering Particle Analyser (Microtrac, Montgomery, PA , USA ). The MP diameter is reported as mean Â± standard deviation (SD). The l oading efficiency of insulin MP s was measured using a solvent extraction technique followed by spectrophotometric analysis. Preparation of PuraM atrix Peptide Hydrogel Since t he pH of hydrogel in 1.0% ( w/v ) solution (BD Bios ciences, San Jose , CA, USA) is 2 2.5, the gel was mixed with 10% sterile sucrose ( Sigma Aldrich, St. Louis, MO, USA) in order to stabilize the gel incorporated vaccine formulations. T he solutio n is converted into gel in the presence of physiological soluti on s , therefore addition of the vaccine formulation was performed shortly before gelation was desired .
46 Sample Preparation for Scanning E lectron M icroscopy (SEM) A n alysis A t otal of 0.1 mL hydro gel (1.0% w/v ) was mixed with 5 mg empty MP s . The mixture was plac ed on a plastic wrap covered 100mm x 15 mm polystyrene petri dish ( Fisher Scientific, Waltham, MA, USA ) at room temperature until gelation was confirmed. Then, t he scaffold was transferred to a vacuum desiccator containing dry ice for further dehydration , a nd incubated overnight. Following this drying process, the s ample was mounted on an aluminum stud and coated with gold and palladium. SEM imaging was performed using JEOL JSM 76 0F ( JEOL USA, Inc. , Peabody, MA, USA) at the University of Florida Major Analyt ical Instrumentation Center. In V itro G el R elease A ssay To determine the release profile of mouse GM CSF (80ng/mL) or CpG ODN1826 (45 g/mL) from the peptide hydrogel, each agent was incorporated into hydrogel. The t otal volume of the mixture was 100 L. Th e triplic ate samples were plated in each well of 12 well cell culture plate ( Corning Inc., Tewksbury, MA, USA ). After the gelation was confirmed, PBS was added carefully into each well . The plate was placed in a 37 o C CO 2 incubator. S upernatant was collecte d and the same volume of fresh PBS was replenished at each time point (0, 0.5 , 1, 3, 6, 12, 18, 24, and 48 h ou rs). The in vitro release kinetics of GM CSF or CpG from the hydrogel was measured using a mouse GM CSF ELISA kit (eBioscien ce, Inc., San Diego, C A, USA) or NanoVue Plus Spectrophotometer (GE Healthcare Life Sciences, Pittsburgh, PA, USA) , respectively . In Vitro Migration A ssay Female NOD mice were sacrificed at 8 weeks of age (n=3) and the BM cells were isolated for an in vitro migration assay. Th ree millions of whole BM cells in 300 L
47 RPMI1640 cell culture medium were placed in the upper chamber of a sterile 8 m po lycarbonate cell culture insert (Millipore, Billerica, MA, USA). The individual insert s w ere placed in each well of 12 well cell culture plate. Each b elow chamber was conta ined : cell culture medium alone ; 50 L hydrogel with 0.5 mg empty MP s ; 50 L hydro gel with 0.5mg empty MP s and 200ng/mL GM CSF ; or soluble form GM CSF (200ng/mL) . Each bottom chamber was filled with 500 L of the cell culture medium. The cell culture plate was placed in 37 o C CO2 incubator for 24 h ou rs. After this incubation, migrated cells were collected and the number of live cells counted . Evaluation of Biodistribution and A ccumulation of Subcutaneously Injected PLGA M P Hydrogel Matrix Total three female NOD mice were ordered at 5 weeks of age and were fed a low alfalfa chow diet for 3 weeks in order to decrease autofluorescence , which result s from ingestion of high alfalfa contain ing normal mouse chow . I nsulin MPs and hydrogel were fluorescently labeled with IRDye 800RS and 700DX Infrared Dye ( MW 962.1 and 1954.22 g/mol, respectively; Li COR Biosciences, Lincoln, NE, USA) , respectively . The mice were subcutaneously injected with h ydrogel (IRDye 700DX In frared Dye/CpG) + fluorescently labeled insulin MPs at 8 we eks of age . During in vivo imaging , mice were under anesthesia with isoflurane delivered by a Gas Anesthesia System. The i nitial isoflurane concentration was set to 3% and reduced to 2% once the animal was anesthetized. Mice were imaged 3, 2 7 , 51 , and 7 5 h ours post injection. The imaging was performed using a Perkin Elmer IVIS Spectrum In Vivo System (Waltham, MA, USA) in the McKnight Brain Institute Cell & Tissue Analysis Core at the University of Flor ida .
48 Results Characterization of Hydrogel PLGA MP Matri x Both t he hydrogel and PLGA MP s ha ve been tested for their ability to function as drug delivery vehicle s by other groups in several different settings. However , it is important to examine and characterize the two biomaterials in our own setting before uti lizing the system in vivo . We first checked whether the hydrogel could incorporate with PLGA MPs and whether this mixture could be formed into a 3D structure. A t otal of 100 L of hydrogel was added to 5mg PLGA MP s , and the combination of the two biomateria ls formed a 3D scaffold (Figure 3 1 A ) . SEM imaging showed the surface of the scaffold and it seemed that the hydrogel well incorporated the MP s (Figure 3 1 B ). In addition, the hydrogel network , which was holding MPs, was detected inside of the scaffold (Fi gure 3 1 C ) . Characterization of PLGA MP Following the confirmation of incorporati on of the two biomaterials, the i ndividual biomaterial s were then characterized . Firstly, MP s encapsulated with human denatured insulin appeared to have an average diameter of 5.01 m (calculated by volume) , confirmed by Dynamic Light Scattering Particle Analyser (Figure 3 2 A ). Denatured insulin loading efficiencies of the MP s used for our in vivo prevention stud ies , which will be discussed in the next chapter, were measured by spectrophotometer. The e ncapsulation efficiency of insulin MPs using each in vivo study was 9.9 Â± 0.7, 5.3 Â± 0.4, and 9.37, respectively (Figure 3 2 B ). Hydrogel Release Profiles To confirm the release profile of GM CSF or CpG through the hydrogel , in vitro gel release assays were performed. As shown in Figure 3 3 , 20% of GM CSF ( Panel A )
4 9 was released during the first 12 hours . Then, the release of GM CSF reached almost 80% after 24 h ou rs . CpG release from hydrogel ( Panel B ) gradually increased over the time course and had already reached 60% after 12 h ou rs. Both agents were almost completely released after 48 h ou rs. To determine biological activity of released cytokine through the hydrogel, an i n vitro migration assay was performed with 3x10 6 whole BM cells isolated from 8 week old female NOD mice (n=3) . It was shown that BM cells were mobilized by soluble GM CSF with statistical significance (p<0.001) . Hydrogel released GM CSF could attract BM cells as well (p<0.05) . There was no statistical difference in te rms of percentage of migration between soluble and hydro gel released GM CSF (Figure 3 4 ) . Biodistribution and Accumulation of Subcutaneou sly Injected Hydrogel MP Matrix Finally, we investigated in vivo biodistribution and accumulation of PLGA MPs and hydro gel in order to trace the biomaterials post injection (n=3) . Imaging at 3 hours post injection revealed strong fluorescence signals from both MPs (Figure 3 5A) and hydrogel (Figure 3 6A) at the dorsal neck area , where the injection was given . Interestingly , the fluorescence signal released from the hydrogel was detected in the ventral area as well (Figure 3 6B ) . Three additional imaging s performed at 27, 51, and 75 hours post injection exhibited similar patterns to those that were observed 3 hours post inje ction. Following the in vivo imaging at 75 hours post injection, imaging of d issected organs directly revealed accumulation of MPs in the spleen, pancreas, and LNs including ce rvical, axillary, and brachial (Figure 3 5 B and 3 5 C ) . Additionally, fluorescenc e signal released from the hydrogel was discovered in the aforementioned tissues (Figure 3 6 C and 3 6 D) .
50 Discussion This study characterized two biomaterials, hydrogel and PLGA MP in vitro . To date, such biomaterials have been widely examined for the ir fea tures as a drug delivery system [74, 85, 88, 121, 129] , including attempts to deliver antigen via biomaterial in order to immunize animal models first initiated more than three decades ago  . However, studies utilizing biomaterials to prevent or cure diabetes are relatively new and are currently ongoing by a number of groups . To design a biomaterial based vaccine, w e first confirmed whether hydrogel could properly mingle with MPs. Each biomaterial has already been well defined as a delivery vehicle , but the combination of PLGA MP and P ura M atrix peptide hydro gel has not been fully examined as a combined delivery system . Our purpose of using hydrogel was to take advantage of t he self assembling peptide scaffold, which is theoretically capable of prolonged release of MPs and adjuvants at the site of injection. T herefore, it was critic al to test whether this particular feature was still reproducible in conjunction with PLGA MPs . First , we confirmed that a t otal volume of 100 L of hydrogel mixed with 5mg of MPs readily created a 3D scaffold at room temperature withi n 30 minutes . Without mechanical disruption (e.g., vigorous pipetting or vortexing), the MP hydrogel matrix could maintain the 3D structure (data not shown). T he total injection volume was selected based on the current recommendation of subcutaneous inject ion volume for m ice, 10 L per gram , in order to minimize adverse effects caused by overdose  . Next , SEM analysis revealed that MPs were well incorporated into the hydrogel . Based on the data , we believe that a hydrogel MP matrix could be used together as a vaccine delivery system.
51 Hydrogel release kinetics are mainly affected by the size of the incorporating agent  . The m olecular mass of GM CSF and CpG ODN1826 is 14.3kDa and 6.6kDa , respectively. The size differences may explain the discrepancy of the initial r elease profiles , the more rapid release of CpG compared to GM CSF . Further evaluation of the hydrogel was performed by an in vitro migration assay using hydrogel loaded with empty MPs and GM CSF. This assay was to test if the GM CSF released through the hy drogel MP matrix could function as a chemoattractant for whole BM cells. Although hydrogel released GM CSF recruited fewer BM cells than soluble GM CSF in 24 hours , it was not statistically different. This result suggested that the hydrogel encapsulation a nd release process did not affect the function of GM CSF. Understanding in vivo biodistribution and localization of injected biomaterial is critical for develop ing an effective and efficient biomaterial base d vaccine delivery system , since unforeseen circu mstances could possibly occur in an in vivo setting , such as accumulation of the biomaterial in an unintended site of organ , or inaccessibility of the biomaterial for the target ed tissue  . To confirm in vivo b iodistribution and accumulation of our vaccine formulation , a single injection consisting of fluorescent labeled MPs and hydrogel was given at the subcutaneous dorsal neck area of 8 week old NOD mice. We detected substantial amount s of fluorescence signals from hydrogel and MPs at the injection site. I nterestingly, some of the signals w ere also observed in the ventral a rea, specifically near the fore limbs , at 3 hours post injection. This was a more rapid response than we expected. However, hydrogel composed of 99% water and 1% (w/v) RADA peptide could be degraded by proteolytic enzymes in vivo  . This process cou ld facilitate the release of encapsulating agents. T herefore early
52 detection of the fluorescence signals at the distal site of the primary injection area was plausible . After 27 and 51 hours post injection , more signals were observed in the ventral area. T he most noteworthy finding in this in vivo imaging analysis was localization of MPs in the organs including cervical, auxiliary, and brachial LNs, spleen, and pancreas at 75 hours post injection. Given these observations, we believe the injected vaccine fo rmulation was taken up by resident and/or migrated APCs including DCs and macrophages, and trafficked to the aforementioned tissues. Although additional evidence is need ed , we believe that the insulin MPs were internalized and processed within the APCs , fo llowed by presentation of the insulin ant igen to T cells in the tissues. In sum, t hese promising results imply that the use of PLGA MP hydrogel matrix would be suitable as a vaccine delivery system particularly targeting APCs in order to educate them via a utoantigen delivery and elicit tolerogenic responses , which will ultima tely lead to prevention of T1D.
53 Figure 3 1. Three dimensional hydrogel microparticle matrix and scanning electron microscope (SEM) image of the matrix. A) Total volume of 100 L v accine formulation consisting of hydrogel and MP readily formed a 1cm diameter 3D scaffold in vitro . SEM revealed B ) well incorporated surface and C ) hydrogel network of the matrix.
54 Figure 3 2. M icroparticle characterization. A) Insulin MP size distr ibution was confirmed by dynamic light scattering particle size analysis. Average diameter of the MP was 5.01 m. As shown in panel B), encapsulation efficiency of insulin MPs used for in vivo studies was also examined.
55 Figure 3 3. Release profiles of GM CSF and CpG ODN1826 from hydrogel. A) Initial release of hydrogel incorporating GM CSF (80ng/mL) was not rapid. Less than 20% of GM CSF was released through the gel for 12 hours. However, nearly 60% more GM CSF escaped the hydrogel within 24 hours. B) While a rapid initial release, almost 60% of CpG (45 g/mL) was detected within 12 hours. Release of both GM C SF and CpG reached almost 90% within 48 hours.
56 Figure 3 4. Functionality of hydrogel released GM CSF as a chemoattractant. In vitro migration assay revealed that the hydrogel released GM CSF could recruit NOD mouse BM cells (8 weeks old, n=3). Alt hough soluble GM CSF (200ng/mL) attracted more BM cells than the gel released GM CSF, the difference in percentage of migrated cell numbers was not statistically different. (ns not significant, * p<0.05, ** p<0.01, and *** p<0.001)
57 Figure 3 5. In vivo biodistribution and accumulation of microparticle. Three NOD mice were subcutaneously injected with a total volume of 100 L hydrogel encapsulated IRDye 700DX (60 g), CpG (10 g), and IRDye 800RS labeled insulin MPs (5mg) at 8 weeks of age. Insulin MPs were mostly located at A) the site of injection. Insulin MPs were detected in both A) the injection site and B) ventral area. Dissection of the mice revealed that the MPs were localized in C) the LNs (top; cervical, middle; axillary, and bottom; brachial), panc reas, as well as spleen.
59 Figure 3 6. In vivo biodistribution and accumulation of hydrogel. Fluorescence signal from the hydrogel was detected at A) the site of injection as well as B) the ventral side of the body at 3 hours post injection. Similarly , most fluorescence signal released from the hydrogel was observed at the site of injection as well as the ventral side at 27 and 51 hours post injection, respectively. Interestingly, less intense fluorescence signal from hydrogel was detected in the site of injection at 75 hours post injection. C) Dissection of the mice revealed that fluorescence signal released from the hydrogel was localized in D) the LNs (top; cervical, middle; axillary, and bottom; brachial), pancreas, as well as spleen.
61 CHAP TER 4 A NOVEL HYDROGEL MICROPAR T ICLE MATRIX DELIVER ING AUTOANTIGEN AND ADJUVANT TO PREVENT TYPE 1 DIABET E S IN NOD MICE Background Recognition of self antigens via T cells followed by destruction of insulin producing cells is generally acce pted to be one of criti cal pathogenic mechanisms of T1D  . In adherence with this notion, targeting specific autoantigens as venu es for therapeutic intervention has become one of the more promising theo retical approaches to curing T1D  . The main goal of a ntigen specific immunotherapy is to achieve induction of regulatory immune responses or elimination of harmful autoantigen specific effector responses , but without inducing non specific immunosuppression, which may increase susceptibility to infe ctions and even cancer s [60, 134 136] . To develop a n efficacious autoantigen specific tolerogenic vaccine, several factors need to be considered , including selection of suitable adjuvant, antigen, dose, timing, fre quency , and route of administration [134, 135] . It has been reported that antigen specific vaccination with multiple high dosages could not achieve immune regulatory or tolerogenic responses. Although h igher dosage has been preferable for i n ducing cell death , this may lead to delet ion o f the Treg population  . In general, r epeated vaccine administrations are considered to be necessary in order to obtain vaccine efficacy. How ever, m ultiple , high dose s of antigenic peptides could induce undesirable effects , such as hypersensitivity and fatal anaphylaxis [134, 135] . Liu et al . reported that NOD mice given 100 g of insulin peptides B(9 23) subcutaneously in 12 doses, died from fatal anaphylaxis with signs of
62 vigorous humoral response  . Another important consideration for developing a suc cessful vaccine is timing of administration. In preclinical settings, a ntigen specific therapies starting at an earlier age yielded better results in terms of prevention of T1D , but this type of approach w as hardly effective at a later age or progressed di sease status  . Taken together, the selection of a vaccine dosage needs to be high enough to induce Treg population and deletion of effector T cell population, but must c arefully consider injection schedule and frequency in order to elicit vaccine efficacy without unintended effects . Several T1D associated autoantigens including insulin, GAD65, Znt8, and IA 2 have been discovered [16, 41] . However, the question of which autoantigen in T1D form s a primary autoantigen in the disease remains unclear. It is also questionable whether a single autoantigen i s enough to induce the disease [41, 134] . One interesting study revealed that intrathymic administration of either insulin B chain or GAD65 at 4 weeks old NOD mice significantly delayed T1D onset for up to 24 week s of age , whereas administration of GAD65 derived peptides p34 and p35 accelerated the disease onset in NOD mice  . Additionally , the authors mentioned that the use of whole intact GAD65 as a vaccine antigen could not induce either of the GAD65 derived antigenic peptide reactiv e CD4 T cell pop ulations. This re calls that p34 and p35 peptides are cryptic epitopes , which are not naturally processed and presented by APC s in NOD mice  . In fact, i nsulin B(9 23) is considered by many to be a major epitope for initiation of diabetes in NOD mice. Immunization with this dominant epitope has successfully demonstrated prevention of T1D in NOD mice. However, this only worked when the treatment started at an earlier age, around 4 to 9 weeks of age [47,
63 140, 141] . If the immune system already has autoanti gen reactive effector T cell populations, then the antigen may not be suitable for eliciting a tolerogenic or regulatory response . T his could be the reason why immunization with the pr edominant epitope has not been successful in the later stage s of the dis ease  . One more important factor to be cons idered is the epitope spreading effect, which is the de novo activation of autoreactive T cells by autoantigens released from bystander tissue damage  . If the immune cell response is to s everal different epitopes, the cells will then be tolerized against the epitopes  . Taken together, use of whole antigen would b e a more effective way to elicit the antig en specific toleranc e than using a single dominant epitope . Since antigen by itself is poorly immunogenic, selection of a proper adjuvant is considered the key in developing highly efficacious vaccine s . Requirements of a good adjuvant include safety and the ability to help ac tivat e the immune system against the vaccine antigen  . However, the importance of adjuvant in immunomodulation has been underappreciated, particularly in the setting of tolerance induction. While alum based adjuvants have been widely ac cepted for use in clinical vaccine s , this type of adjuvant is relatively weak and hardly stimulates cellular immune responses  . Therefore, efforts to find more effective and suitable adjuvants are being made . CpG ODNs , a synthetic DNA containing one or more CpG motifs, are recognized by TLR9 , which is expressed mai nly on plasmacytoid DCs and B cells in human s , and is expressed by macrophages, DCs, and monocytes in mice [100, 104] . In fact , recognition of CpG motif s by TLR9 induces the innate immune response , which triggers p roduction of pro inflammatory Th1 cytokines and type I interferons  . This synthetic
64 DNA has gained widespread attention due to its distinctive features as a vaccine adjuvant. CpG adjuvanted vaccines have been tested in the contexts of viral , bacterial, and parasitic infectious diseases, and cancer s in preclinical settings [143 146] . Although CpG ODNs were mostly evaluated as a strong pro i nflammatory adjuvant, several studies including autoimmune arthritis and diabetes in animal models demonstrated that CpG ODNs can influence induction of anti inflammatory response s characterized by increased IL 10 production and Treg population [147, 148] . Another adjuvant candidate is the Hb and Hp complex. F ree circulating Hb can induce cytotoxic oxidative stress, however , Hp, a plasma glycoprotein , binds to the free Hb. T his complex can be recognized by its recep tor, CD163 , expressed on monocytes and macrophages. HO 1 catabolizes the heme subunit of the Hb and this enzymatic reaction produces biliverdin, iron, and CO [109 113] . Hu et al . demonstrated that a single intraven ous administration of a recombinant adeno associated virus containing HO 1 gene in 9 week old NOD mice could delay the development of diabetes for up to 24 weeks of age  . The authors pointed out that overexpression of HO 1 could suppress Th1 effector function and CD11c+ MHC Class II+ DC maturation  . Given this information, a combination of CpG and Hb:Hp appeared to be suitable as tolerogenic vaccine adjuvants to prevent diabetes in NOD mice. D enatured human insulin was chosen as a vaccine antigen in this study. The goal of this study is to optimize a vaccine like injectable matrix material containing a combination of adjuvants and denatured i nsulin autoantigen, capable of eliciting antigen specific tolerance, therefore affording safe and effective means for the prevention of T1D. To achieve this
65 goal, we hypothesized that the preventio n of T1D via denatured insulin would be improved through the use of a combination of CpG and Hb:Hp as adjuvants . In the previous chapter, we demonstrated tha t hydrogel PLGA MP matrix is an efficient vaccine delivery system. This chapter presents and discusses results of three different sets of T1D prevention stud ies utilizing the biomaterial based tolerogenic vaccine in NOD mice . All studies started at 8 weeks of age and were monitored until 28 weeks of age. The impact of the administered vaccine upon blood glucose level s , T ce ll subpopulations, and degree of insulitis was examined in each study . In addition, w e modified and optimized the vaccine formulation in each attempted study based upon the results already obtained. Finally , a granuloma formation, an unexpected yet meaningful observation, result ing from multiple subcutaneous administrations of insulin MPs , will be presented and discussed in a histological and physical manner. Materials and Methods Diabetes Prevention Studies Study I : A batch of 8 week old female NOD mice was divided in to 5 treatment groups (n=13/group). Vaccine formulations given to the groups of mice were as follows: Group A Hydrogel (GM CSF) + Empty MP s ; Group B Hydrogel (GM CSF/CpG/Hb:Hp) + Empty MP s ; Group C Hydrogel (GM CSF/CpG) + Insulin MP s ; Group D Hydrogel (GM CSF/Hb:Hp) + Insulin MP s ; Group E Hydrogel (GM CSF/CpG/Hb:Hp) + Insulin MP s . The vaccine formulations were adminis tered at 8, 10, and 12 weeks of age. Doses of each formulation per injection were as follows : GM CSF (40ng/mouse); CpG (10 g/mouse); Hb:Hp (50 g each/mouse); empty or insulin MP s
66 (5mg/mouse) ; h ydrogel (91 L/mouse). Total injection volume was 100 L . Mice were injected at the subcutaneous dorsal neck area . Study II: A batch of 8 week old female NOD mice was divided in to 6 treat ment groups (n=13/group). Vaccine formulations were as follows: Group A Hydrogel ( e mpty) + Empty MP s ; Group B Hydrogel (CpG/Hb:Hp) + Insulin MP s ; Group C Hydrogel (CpG) + Insulin MP s ; Group D Hydrogel (CpG/Hb:Hp) + Insulin MP s ; Group E Soluble eq uivalent doses of all factors ( CpG/Hb:Hp/denatured insulin ) ; Group F No treatment control. The vaccine formulations were injected at 8, 10, and 12 weeks of age, but two additional injections at 15 and 18 weeks of age were given to the mice of G roup D. Do ses of each formulation per injection were as follows: CpG (10 g/mouse); Hb:Hp (50 g each/mouse); empty or insulin MP s (5mg/mouse); h ydrogel (91 L/mouse). Total injection volume was 100 L . Mice were injected at the subcutaneous dorsal neck area. Study III: A batch of 8 week old female NOD mice was divided in to 5 treatment groups (n=23/group). The vaccine formulations were as follows: Group A Hydrogel ( e mpty) + Empty MP s ; Group B Hydrogel (CpG) + Insulin MP s ; Group C Hydrogel (CpG) + Insulin MP s ; Group D Soluble equivalent doses of all factors ( CpG/denatured insulin ) ; Group E No treatment control. Mice of g roup A and B were given a total of 4 injections, occurr ing at 8, 10, 12, and 15 weeks of age. One more injection at 18 weeks of age was given to the mice of Group C and D. Doses of vaccine formulation and site s of injection were identical to the Study II.
67 Study of Granuloma In the Study I , granuloma formation was firstly detected in the mice of Group s C, D, and E. The lumps were palpable around the injection area a week a fter the second injection . Interestingly, the granuloma was only formed in the groups of mice given insulin MP s while the empty MP injected mice did not form the lumps. In Study I , we examined the histological features of the granul oma. In Study II and III, we focused on measuring the size of the granuloma each week. The following methods describe the details. Study I: G ranuloma was excised one week after the third injection ( 13 weeks of age ) and placed on a tissue cassette (VWR Corp orate, Radnor, PA, USA) followed by fixation in 10% formalin solution (Fisher Scientific, Waltham, MA, USA) overnight at room temperature. Following this fixation step, the tissue contain ing cassette was immersed in fresh 1xPBS ( Hyclone, Logan, UT, USA ). T he fixed tissues were directly sent to the Molecular Pathology Core at the University of Florida for further hi stological analysis. Paraffin embedded tissue sections were individually stained with H&E , anti mouse CD45R/ B220 , clone RA3 6B2 ( BD Biosciences, San Jose , CA, USA ) , anti human CD3 , clone CD3 12 (Serotec, Raleigh, NC, USA), anti mouse F4/80, clone BM8 (Caltag Medsystems, Buckingham, UK) , or anti LYVE 1 ( Lymphatic vessel endothelial hyaluronan receptor 1; Abcam, Cambridge, MA, USA) antibody for immun ohistochemistry (IHC) . Study II: S ize of granuloma was measured once a week from 11 to 28 weeks of age. Briefly, palpation of subcutaneous neck area was perfo rmed on each mouse. If a lump was detected i n the area, the width of the lump was measured using a carbon fiber
68 composite digital caliper ( Â± USA). Study III: The method to obtain the size of the granuloma was modified in this study. Skin thickness of subcutaneous dorsal neck area was measured using the digital caliper in all groups of mice beginning at 8 weeks of age. Mean value of skin thickness of no treatment control mice was calculated each week, and then the mean value was subtracted from measured granuloma size of the corresponding week in orde r to obtain more precise width of granuloma. In Vitro Adjuvant Stimulations To determine the effect of adjuvant stimulations, 8 or 10 week old female NOD mice (n=3/age) were sacrificed and the spleen was excised in order to purify s plenoctyes . In v itro sti mulation settings were as foll o ws: Cell culture m edium alone; CpG (20 g/mL); CpG (20 g/mL) + Hb:Hp (30 g/mL, respectively); GM CSF (200ng/mL) + CpG (20 g/mL); GM CSF (200ng/mL) + CpG (20 g/mL) + Hb:Hp (30 g/mL, respectively). Isolated splenocytes were plat ed at 1x10 5 cells per well in a round bottom 96 well cell culture plate ( Corning Inc., Tewksbury, MA, USA ) . The cell culture plate was placed in 37 o C CO 2 incubator . Supernatants were carefully collected after 24 and 48 hours and stored at 20 o C until used. IL 10 and IFN levels in the supernatants were analyzed by ELISA ( eBiosci en ce, Inc., San Diego, CA, USA ) . Insulitis Scoring Digital image s of H&E s tained pancreas section s from individual mice w ere uploaded on Aperio eSlide Manager (Version 220.127.116.1129; Leica Biosyste m s, Buffalo Grove, IL, USA). Insulitis scoring was manually performed on the digital image s . The
69 degree of insulitis was scored based on the following grading: 0 no insulitis; 1 peri insulitis; 2 infiltrating more than 25% of islets; 3 more than 75% to complete islet infiltration. Results In Vivo Survival Curves of NOD Mice Study I To determine the vaccine efficacy, the following vaccine formulations were subcutaneously injected in 8 week old female NOD mice (n=10/group) ; Group A Hydrogel (GM CSF ) + Empty MPs; Group B Hydrogel (GM CSF/CpG/Hb:Hp) + Empty MPs; Group C Hydrogel (GM CSF/CpG) + Insulin MPs; Group D Hydrogel (GM CSF/Hb:Hp) + Insulin MPs; Group E Hydrogel (GM CSF/CpG/Hb:Hp) + Insulin MPs. Two additional injections were given at 1 0 and 12 weeks of age in order to boost the vaccine efficacy . As a result, the group given 3 doses of hydrogel (GM CSF/CpG/Hb:Hp) + i nsulin MPs showed the best survival proportion at the end of the study ( Figure 4 1; vs control group A p=0.0 437 ) . The secon d best survival proportion was 40% of the group of mice treated with hydrogel (GM CSF/CpG) + i nsulin MPs ( Figure 4 1; vs group A p=0.0263). Histological Analys i s of Granuloma Study I Interestingly, mice injected with insulin MP s formed granuloma like les ion s around the neck area a week after the second injection . However, none of the mice given empty MPs with adjuvant(s) form ed the granuloma at the injection site . After sacrificing the animal, the granuloma was carefully excised and then prepared for furt her histological analys i s. We initially performed H&E staining and the staining showed that enormous numbers of nucleated cells were infiltrated by the granuloma (Figure 4 2 A and 4 2 B ). In addition, the H&E stain revealed the existence of adipose
70 tissue an d invasion of blood vessel s in to the granuloma (Figure 4 2 A and 4 2 B ). Next , further immunohistochemical analysi s was performed to determine phen otypes of the nucleated cells. The experiments clearly showed the presence of CD3+ T cells ( Figure 4 2 D ) , B220+ B cells (Figure 4 2 E ) , and F4/80+ macrophages ( Figure 4 2 F ) in the granuloma. Additionally , the surrounding area of the granuloma was positively stained with a ly mphatic vessel marker LYVE 1 (Figure 4 2 C ) . Cytokine Productions by Adjuvant Stimulations In Vitro Study I In order t o examine the effect of adjuvant stimulations on cytokine production, IL 10 and IFN levels in the collected supernatants were analyzed via ELISA. As a result, significant amounts of IL 10 were detected in the supernatants obtained from C pG alone (p<0.001) or CpG/Hb:Hp (p<0.001) stimulated samples compared to the supernatant collected fr om unstimulated (cell culture medium alone) sample s (Figure 4 3A and B) . Interestingly , GM CSF along with CpG (24 hours p<0.01; 48 hours p< 0.001 ) or CpG/ Hb:Hp (p<0.0001) did not significantly boost IL 10 production ( Figure 4 3 A and B ) . However, combination of GM CSF and CpG (p<0.0001) or CpG/Hb:Hp (24 hours p<0.01; 48 hours p<0.0001) notably induced IFN secretion compared to st imulation with CpG alone or CpG/ Hb:Hp ( Figure 4 3 C and D ) . Insulitis Scoring Study I A week after their third injection, 2 or 3 mice of each treatme nt group were randomly select ed and sacrificed. The pancreas was isolated from e ach mouse and stained with H&E to assess the degree of islet infiltration . Although mice given 3 doses of hydrogel (GM CSF/CpG/Hb:Hp) + i nsulin MPs ( g roup E ) exhibited a relatively
71 healthier condition of islets compared to control groups A and B ( Figure 4 4 ), this trend was not statistically significant. Flow Cytome try Analysis of Treg Population Study I To determine if the vaccine treatment had an impact on increasing Treg population s , the spleens from the surviv ing mice at the study end point (28 weeks o f age) were isolated and purified (n=1 5) . The proportion of Treg population w as determined by flow cytometry as a percentage of CD4+CD25+FoxP3 + ( Figure 4 5 ) cells . Although there was no statistical significance , the best survival group , that given hydroge l (GM CSF/CpG/Hb:Hp) + insulin MPs , exhibited a relatively higher percentage of Treg population compared to the control group. However, the group of mice injected with hydrogel (GM CSF/CpG/Hb:Hp) + empty MPs appeared to have a similar percentage of Treg po pulation. In Vivo Survival Curves of NOD Mice Study II Vaccine formulations and injection schedules for Study II were modified based on the results of Study I. The modified vaccine formulations were as follows (n=10/group) : Group A Hydrogel ( e mpty) + E mpty MPs; Group B Hydrogel (CpG/Hb:Hp) + Insulin MPs; Group C Hydrogel (CpG) + Insulin MPs; Group D Hydrogel (CpG/Hb:Hp) + Insulin MPs; Group E Soluble forms of CpG/Hb:Hp/denatured insulin; Group F No treatment control. Initially, a total of nine injections of hydrogel (CpG/Hb:Hp) + i nsulin MP were planned for group D at 8, 10, 12, 15, 18, 21, 24, 27, and 30 weeks of age . However, lethal anaphylaxis occurred on the day of the fifth injection and resulted in 2 out of 9 mice d ying . Due to this unexp ected reaction, the treatment plan was revised from 9 to 5 injections. All the other treatment groups were given 3 injections at 8, 10 , and 12 weeks of age. As a result, the group given 5 doses of hydrogel (GM -
72 CSF/CpG/Hb:Hp) + i nsulin MPs exhibit s the best survival proportion at the end of the study ( Figure 4 6; o verall p=0.0019; vs no treatment control group F p=0.0049). The second best survival proportion was 60%, which resulted from 3 injections of hydrogel (CpG) + i nsulin MP ( Figure 4 6; vs group F p=0. 0022). Additionally, mice given 3 injections of hydrogel (CpG/Hb: Hp) + insulin MP exhibited 55.55% of survival proportion ( Figure 4 6 ; vs group F p=0.0223) , w hereas mice treated with the same vaccine agents but in soluble form had poorly maintained euglyce mia; only 11.11% of the mice were still surviv ing by the study end point (Figure 4 6 ; vs group F p=0.6570 ) . Width of Granuloma Study II In Study I, we already confirmed that multiple injections of autoantigen encapsulated MPs caused granuloma formation at the site of injection. This trend was also observed in treatment group s B, C, and D in Study II ( n=5 10; Figure 4 7) . The s ize of the granuloma gradually decreased following the final injection , which was given at 12 weeks of age. Interestingly, a fourt h injection performed at 15 weeks of age boosted the width of the granuloma again. The increased size of granuloma started to diminish two weeks later . However, a fifth injection given at 18 weeks of age caused a marginal increase of the size. Then, the gr anuloma seemed to be resolved until 25 weeks of age. During this study, t hree mice of group D had ruptured abscess es, and this symptom seemed to be responsible for increasing the width of the granuloma again at 25 weeks of age . Although m ice of group B and C showed fluctuations in terms of granuloma size change , dramatic change was not observed until the study end point . Insulitis Scoring Study II At 13 weeks of age, 2 or 3 mice of each treatment group were randomly selected and sacrificed for a mid point mechanistic study. Isolated pancreata were used for
73 insulitis scoring. Despite euglyce mia, signs of islet infilt ration were detected in all mice (Figure 4 8) . However, mice injected with either hydrogel (CpG/Hb:Hp) + insulin MP or hydrogel (CpG) + insulin MP showed significantly lower insulitis scores compa r ed to the control group (p<0.0001) . Soluble adjuvant and antigen treated group s also exhibited less severe insulitis compared to no treatment control group (p <0.01 ) . In Vivo Survival Curves of NOD Mice Study III To determine if the prevention of diabetes resulted mainly from multiple CpG treatments along with insulin MPs, we decided to exclude Hb:Hp from vaccine formulations in Study III and maintain injection schedules performed at 8, 10, 12, 15, and 1 8 weeks of age for all treatment groups. In addition, the number of animals was increased to 23 mice per group in order to obtain statistical power and significance for the vaccine treatments . The vaccine formulations of Study III were a s follows: Group A Hydrogel (e mpty) + Empty MPs; Group B Hydrogel (CpG) + Insulin MPs; Group C Hydrogel (CpG) + Insulin MPs; Group D Soluble forms of CpG/ denatured insulin; Group E No treatment control. Disappointingly , the fifth injection of hydrogel (CpG) + insul in MPs caused anaphylaxis a nd led to loss of 3 out of 9 mice. Due to the animal loss , we decided not to give the fifth injection to the remaining mice. Therefore, a total number of 13 mice was designated as a separate group and the mice were given a total of 4 injections (Group B) . In addition, group A did not receive th eir fifth injection. Curiously , a fifth injection of soluble CpG along with denatured insulin did not cause lethal anaphylaxis. The blood glucose levels of the mice were monitored once a wee k until 28 weeks of age. Overall , statistical significance could not be achieve d in this study ( Figure 4 9; p=0.1630). Group D , which was treated with soluble CpG along with denatured insulin exhibited an unexpectedly high survival proportion ( Figure 4 9; vs
74 control group E p= 0.0442). Although five injections of hydrogel (CpG) + insulin MPs yielded a 66.67% survival proportion , this was not statistically significant ( Figure 4 9; vs group E p=0.0817). Interestingly, 4 doses of the same formulations resulted in the worst survival proportion ( Figure 4 9; vs group E p=0.4496). Granuloma Size Change Study III As we have demonstrated previously, m ultiple treatments with denatured insulin incorporated MPs induced granuloma formation at the site of injection . To obtain more precise width of the granuloma, the mean value of skin thickness of the control group was deducted from measured granuloma size (n=4 23) . The granuloma was first detected at 11 weeks of age (Figure 4 10) . The width of granuloma steadily decreas ed until 15 weeks of age, but was boosted again a week after the fourth injection. Multiple treatments with soluble vaccine or blank biomaterial s did not induce the granuloma formation. Flow Cytometry Analysis of T Cell Subpopulations Study III Due to the loss of 3 animals from lethal anaphylaxis, we decided not to perform a midpoint mechanistic study. However, mice that survived until 28 weeks of age were sacrificed and the organs were dissected including the spleens and skin draining brachial LNs in o rder to investigate the immunomodulatory effect of each treatment. Three different sets of antibody panels were prepared to detect specific T cell subpopulations, particularly Treg s , memory T cell s , and insulin B(9 23) reactive T cell s. Isolated cells were stained with each set of antibody cocktails. Surprisingly, mice given 5 doses of hydrogel (CpG) + insulin MPs exhibited a significantly higher percentage of CD4+CD25+Foxp3+ Treg population s in splenocytes compared to all other groups ( Figure 4 11A; vs gro up A or B p<0.001; vs group D p<0.0001 ; vs group E p<0.01) . In
75 addition, this group of mice appeared to have a significantly higher percentage of CD4+CD44+CD62+ central memory T cell population s in splenocytes ( Figure 4 11B; vs group A p<0.01; vs group B, D or E p<0.0001) . However, no statistic al significance was detected in splenic CD4+CD44+CD62L effector memory T cell s (Figure 4 11C) or CD4+ I A g7 i nsulin B (9 23) tetramer+ population s (Figure 4 11D) in all groups . T cell subpopul ations in the brachial LNs yielded contrasting results. The p ercentage of Treg population s w ere significantly higher in the no treatment control group compared to other treatment groups ( Figure 4 12A; vs group A p<0.01; vs group B p<0.05; vs group D p<0.0001) . Whereas mice given 5 injections of hydrogel (CpG) + insulin MPs appeared to have significantly higher Treg population s in the LNs , only when compared to the mice of group D ( Figure 4 12A; p<0.01). There was no statistical difference in the central memory T cell population s in all groups (Figure 4 12B) . M ice treated with 5 doses of hydrogel (CpG) + insulin MPs exhibited higher percentage of effector memory T cell population compared to the soluble vaccine treated group ( Figure 4 12C; p<0.05). Curiously, i nsulin B(9 23) reacti ve CD4+ T cell population s w ere significantly less in the mice of group C ( Figure 4 12D; vs group D or E p<0.05). Discussion Antigen specific immunomodulation is considered a gold standard therapeutic approach towards autoimmune diseases including T1D  . This type of therapy has the goal of inducing autoantigen specific tolerance by increasing the Treg population , enhancing autoreactive T cell deletion , or skewing cytokine profiles toward anti inflammatory and regulatory type s [118, 119, 135] . To achieve this goal, APCs must encounter an antigen and it must then be presented by the APCs to T cells  . S oluble vaccine antigen has been criticized because the soluble form of antigen tends to be
76 cleared by the immune system without having a c hance to encounter targeted APCs [74, 115] . To overcome the aforementioned drawback, we utilized two biomaterials , PLGA MP and hydrogel, which are a phagocytosable size d antigen delivery vehicle and an injectable c ontrolled release material, respectively [73, 80] . For the first in vivo testing for prevention of T1D in NOD mice, we selected 3 different agents including GM CSF, CpG, and Hb:Hp. While CpG and Hb:Hp were used as vaccine adjuvants, GM CSF was mainly expected to act as a chemoattractant at the injection site. We first injected the vaccine formulations in NOD mice at 8 weeks of ag e . T wo additional injections were given at 10 and 12 weeks of age. The principal outcom e of this Study I was that treatments with hydrogel (CpG/Hb:Hp/ GM CSF ) and insulin MPs significantly prevented the development of diabetes. In addition , administrations of hydrogel (CpG/ GM CSF ) and insulin MP s exhibited comparable survival proportion s . Int erestingly, the use of Hb:Hp alone as an adjuvant instead of CpG or a combination of both adjuvants revealed the worst survival proportion at the end of study. Th ese result s led us to test in vitro adjuvant stimulations of NOD splenocytes. We initially exp ected that treatment with Hb:Hp could induce production of anti inflammatory cytokine IL 10. However, significant amount s of IL 10 were only detected in the presence of CpG. In addition, the addition of GM CSF did not boost IL 10 production but induce d sig nificant level of pro inflammatory cytokine IFN . Although IFN has an effect on inducing indoleamine 2,3 dioxygenase (IDO) , which is expresse d by tolerogenic DCs and lead s to suppress ion of T cell responses [150, 151] , this cytokine also has unappreciated role s in the pathogenesis of T1D  . A p ossible pro infla mmatory effect of GM CSF as well as an effort to simplify vaccine formulations in order to minimize unforeseen
77 adverse effects led us to exclude the GM CSF from vaccine formulations of Study II and III. In Study II, although two mice of group D died due to anaphylaxis on the day of the fifth injection, this group of mice exhibited the best survival proportion s with statistical significance at the end of the study. Three injections of hydrogel (CpG) + insulin MP or hydrogel (CpG/Hb:Hp) + insulin MP also appe ared to significantly protect the mice from development of diabetes. However , these results did not fully prove our hypo thesis that exclusion of GM CSF would enhance therapeutic outcome s . Also, we could determine that the use of biomaterials to delive r our vaccine formulations was an improved and effective means for prevention of diabetes compared to the vaccine delivery via soluble form. Based on the survival proportion results of both Study I and II , we assumed that CpG is crucial as an adjuvant, but Hb: Hp alone might not work as an anti inflammatory adjuvant in our settings . In addition, we inferred that Hb:Hp might be responsible for inducing fatal anaphylaxis in Study II since these two proteins are purified human derived proteins . Lu et al . demonstrat ed that four or six injections of purified human alpha 1 antitrypsin (hAAT) protein or human albumin induced fatal anaphylaxis in NOD mice  . Interestingly, hAAT delivery via recombinant adeno associated virus in NOD mice did not cause anaphylaxis  . The authors pointed out that multiple treatments with purified human derived protein might be res ponsible for anaphylactic death  . Based upon previously published literature and our observation s , we decided to exclude Hb:Hp from vacci ne formulations in Study III .
78 We modified and simplified the va ccine formulations in Study III in order to prevent anaphylaxis, yet improve therapeutic outcome s . Disappointingly, the fifth injection of hydrogel (CpG) + insulin MPs induced fatal anaphylaxis and led to loss of 3 out of 9 mice. However, the fifth injecti on of soluble vaccine formulations did not induce any adverse effect. In the Study III , we could not detect significantly improved therapeutic effect s in terms of diabetes prevention in the group injected either 4 or 5 times with hydrogel (CpG) + insulin M Ps . Interestingly, only the soluble vaccine treated group was significantly protected from the development of diabetes. This result was the opposite of the data which we obtained from Study II , although t here was a difference in injection frequency of 3 in jections in Study II and 5 injections in Study III . Thus, two additional injections exhibited clear evidence for improved efficacy . This may explain why most antigen specific approaches with soluble agents have multiple injection schedules in order to elic it higher efficacy [155 157] . In spite of th e disappointing results, it was important to investigate the potential disease prevention mechanism . To examine the mechanism, we analyzed T cell subpopulations in the sp leen and brachial lymph nodes of the surviv ing mice . Given the significant increase in the percentage of Treg population s in group C, we assumed that delivery of vaccine formulations via biomaterials rather than as soluble forms might be a more effective m eans of enhancing Treg population s in vivo . The a dvantage of the biomaterial based vaccine was confirmed again in the increased percentage of the splenic central memory T cell population s . Memory T cells can be classified into central memory T cells and e ffector memory T cells. Central memory T cells are very sensitive to antigenic stimulation and can be activated by
79 antigen presentation via mature DCs in secondary lymphoid organs. The central memory T cells secrete high levels of IL 2 yet little IFN in response to antigen . While e ffector memory T cells can migrate to inflamed nonlymphoid peripheral tissues and interact with nonprofessional APCs , they mainly produce IFN in order to clear the source of inflammation [158 160] . Given th is , antigen delivery via PLGA MPs might be a favorable way to generate c entral memory T cell population. I nsulin reactive CD4+ T cell s that evade peripheral tolerance mechanisms can , following an encounter with the antigen , be ac tivated and differentiat e into pathogenic Th1 cells that can be involve d in T1D pathogenicity [161, 162] . Although there were no differences in the T cell population s in the splenocytes of all groups, we detected a significantly lower percentage of i nsulin B(9 23) reactive CD4+ T cell population s in brachial LNs of group C and D , which were treated with 5 injections of the full com bination of vaccine formulation v ia biomaterials or soluble form , respectively, compar ed to a no treatment control group. This result implies that multiple treatments with denatured insulin had possible influences on deletion or inact ivation of the insulin reactive CD4+ T cell population in the LNs . Remarkably, we confirmed that our biomat erial based vaccine formulations contain ing denatured insulin caused granuloma formation with infiltration s of T, B and macrophage populations and features of a tertiary lymphoid organ . The size of granuloma decreased after the third injection, but the add itional two injections boosted formation of the granuloma again. Collectively, th ese result s suggest that insulin antigen recall response had occurred by repeated administration of the antigen.
80 In summary, a series of five injections of hydrogel incorporat ed with CpG, Hb:Hp, and insulin MPs appeared to have the best therapeutic efficacy relative to the control groups . However, this result was not statistically different than the group only receiving a series of three injections of the same formulation or th ree injections of CpG and insulin MPs . In addition, the fifth injection of biomaterial based vaccine containing denatured insulin caused lethal anaphylaxis in roughly 20 30% of mice. Although mice surviv ing after the fifth injection exhibited nearly 70 80% of protection from the development of diabetes, risk factors associated with anaphylaxis should be identified and resolved in order to mov e towards clinical translation.
81 Figure 4 1. Study I: Kaplan Meier plots of diabetes incidence. Five groups o f female NOD mice (n=10/group) were given a total of 5 subcutaneous injections of the biomaterial based vaccine formulations. Combination of adjuvants along with insulin MPs treatments led to prevention of diabetes (Overall, p=0.0199).
82 Figure 4 2. St udy I: Histology of granuloma. Subcutaneous injections of matrix containing insulin MPs induced granuloma formation at the injection site. A & B) H&E staining of the granuloma section exhibited infiltration of nucleated cells. Immunohistochemical staining revealed some of the nucleated cells were D) T cells , E) B cells, or F) macrophages. Additionally, the surrounding area of granuloma exhibited positive staining for C) a lymphatic endothelial marker LYVE 1.
83 Figure 4 3 . Study I: Effect of adjuvant s timulations on cytokine production in vitro . Stimulation of CpG alone or CpG/Hb:Hp induced significantly higher levels of IL 10 production from NOD mice splenocytes (8 weeks old, n=3). Whereas Hb:Hp alone did not enhance IL 10 production (A; 24 hours, and B; 48 hours). Addition of GM CSF did not boost IL 10 secretion (A; 24 hours, and B; 48 hours), but did induce IFN production (C; 24 hours, and D; 48 hours). (ns not significant, * p<0.05, ** p<0.01, *** p<0.001, and ****<0.0001)
84 Figure 4 4. Study I: Insulitis scoring of pancreatic islets at 13 weeks of age. A week after the last injection, 2 or 3 mice was rand omly selected for midpoint mechanistic study. Despite euglycemia, signs of islet infiltration were noted in all mice. Although mice of group E, treated with hydrogel (GM CSF/CpG/Hb:Hp) and insulin MPs, exhibited relatively healthier islets, this trend was not statistically significant .
85 Figure 4 5. Study I: Treg frequency in the spleen of surviv ing mice. Although the mice were protected from the development of diabetes for up to 28 weeks of age, there were no signs of increasing Treg populations in the spleens. Group A (n=1) Hydrogel (GM CSF) + Empty MPs; Group B (n=2) Hydrogel (GM CSF/CpG/Hb:Hp) + Empty MPs; Group C (n=4) Hydrogel (GM CSF/CpG) + Insulin MPs; Group E (n=5) Hydrogel (GM CSF/CpG/Hb:Hp) + Insulin MPs.
86 Figure 4 6. Study II: Kaplan Meier survival curves of NOD mice. Multiple subcutaneous injections of adjuvants along with insulin MPs starting at 8 weeks of age resulted in the prevention of diabetes (n=10/group). The group with a series of five injections of hydrogel (CpG/Hb:Hp ) along with insulin MPs did exhibit disease prevention relative to control groups including no treatment and soluble factors, but were not statistically different than the group only receiving a series of three injections of the same formulation (p=0.4495 ).
87 Figure 4 7. Study II: Size change of granuloma. A week after the second injection, granuloma was only detected in groups that included insulin MPs. The s ize of the granuloma was measured each week using a digital caliper. The g ranuloma almost resol ved two weeks after the third injection, but the fourth injection boosted its formation. (n=5 10)
88 Figure 4 8. Study II: Insulitis scoring of islets from a midpoint mechanistic study. A week after the third injection, 2 or 3 mice per group w ere random ly selected for assessing degrees of insulitis. Despite euglycemia, signs of islet infiltration were observed in all mice. However, insulin MPs along with CpG/Hb:Hp ( group B and D) or CpG alone ( group C) injected groups exhibited significantly lower insuli tis scores compared to no treatment control group ( group F). (ns not significant, ** p<0.01, and **** p<0.0001)
89 Figure 4 9. Study III: Diabetes incidence following the vaccine injections. The group with a series of five injections of soluble CpG an d denatured insulin did prevent the disease relative to no treatment control group (p=0.0442). However, treatments with the same formulation via biomaterials did not successfully protect the mice from the development of diabetes compared to no treatment co ntrol group (5 injections p=0.0817; 4 injections p=0.4496). (n=6 23, Overall p=0.1630)
90 Figure 4 10. Study III: Formation, regression, and regrowth of granuloma. Multiple subcutaneous injections of matrix containing insulin MPs caused granuloma format ion beginning at a week after the second injection. Peak size of granuloma was roughly 5.5mm. Width of granuloma started to wane a week after the third injection, but the fourth injection boosted its size again. (n=4 23)
91 Figure 4 11. Study III: Fre quency of T cell subpopulations in the spleens of surviv ing mice. At the study endpoint, T cell subpopulations in the splenocytes of surviv ing mice were examined (n=4 14). The group injected with 5 doses of hydrogel containing CpG and insulin MPs had signi ficantly more Treg and central memory T cell population s compared to control groups including no treatment, soluble factors, and blank biomaterials (A and B). However, there were no statistical differences in effector memory and insulin B(9 23) reactive CD 4 T cell population s (C and D). (* p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001)
92 Figure 4 12. Study III: Frequency of T cell subpopulations in the brachial lymph nodes (LNs) of surviv ing mice. The group injected with 5 doses of hydrogel contain ing CpG and insulin MPs had significantly more A) Treg and C) effector memory T cell population s compared to soluble form injected group. However, t here was no statistical difference in B) central memory T cell population s . Interestingly, five injections o f the biomaterial based vaccine or soluble factors yielded significantly less D) insulin B(9 23) reactive CD4 T cell population compared to no treatment control group. (n=4 14, * p<0.05, ** p<0.01, and **** p<0.0001)
93 CHAPTER 5 IMP AIR MENT OF NOD MICE SPL ENOCYTE MOBILIZATION IN RESPONSE TO GM CSF AND G CSF Background The NOD mouse is the most widely accepted animal model in T1D research. This mouse strain has multiple defects in immunological function, which collectively lead to spontaneous autoimmune diab etes  . GM CSF is a cytokine that acts as a hematopoietic growth factor and immune modulator , particularly a crucial mediator of DC devel opment . This cytokine is also able to recruit circulating monocytes, neutrophils, and lymphocytes [95 97] . G CSF is widely us ed as a mobilizing agent of hematopoietic stem cells in clinics  . This cytokine induces production of neutrophils, release of the cells from the bone mar row, and activity of mature neutrophils [164 166] . Previous studies have noted that t reatment with GM CSF or G CSF resulted in diabetes prevention in NOD mice w ith evidence of increased tolerogenic DC s or regulatory T cell populations [97 99, 167, 168] . Interestingly, we found that mobilization defects in response to GM CSF or G CSF exist ed in NOD mice splenocytes but a normal response in BM cells was observed in an in vitro migration assay. To determine whether this p eripheral migration defect in response to the cytokines is NOD mouse specific, we performed the same set of experiments with immune cells of C57 and NOR mice , widely used Th 1 dominant and diabetogenic H 2 g7 MHC haplotype contained diabetes protective mous e strain , respectively [169, 170] . These two control mouse strains exhibited normal migration patterns in response to GM CSF or G CSF indepen dent of age or source of cells. This chapter presents and discusses the new observations of peripheral migration defe cts in NOD mice splenocytes i n response to GM CSF and G CSF. The
94 migration patterns will be compared to those of the control mouse strains. Follow ing this observation, migrated cells of each mouse strain will be examined by flow cytometry analysis in order to determine what types of NOD immune cells do not mobilize by the cytokines compared to the other two strains . Subsequent to the results, differences in gene expression levels in response to GM CSF or G CSF stimulation will be presented and discussed in order to identify the underlying defect at the genetic level. Materials and Methods In Vitro Migration Assay Three million BM cells and splenocytes isolated from each mouse strain including C57, NOD , and NOR (n=3/strain/age) in 300 L RPMI1640 cell culture medium were placed in upper chamber of a sterile 8 m po lycarbonate cell culture insert (Millipore, Billerica, MA, USA). The individual insert was placed in each well of a 12 well cell culture plate ( Corning Inc., Tewksbury, MA, USA ). Each b elow chamber was contained cell culture medium alone, GM CSF (200ng/mL), or G CSF (50ng/mL) in 500 L of the cell culture medium. The cell culture plate was placed in 37 o C CO 2 incubator for 24 hour s. Following this incubation , migrated cells were collected and then counted number of live cells to obtain percentage of migrated cells . In Vitro Stimulation and RNA Isolation The f irst set of experiment s prepared 3x10 6 BM cells and splenocytes from 8 and 12 week old C57, NOD , and NOR (n=3/strain/age) mice . The s econd set of expe riment s prepared CD11c+ DCs from the BM and spleen of 8 week old C57, NOD, and NOR (n=10/strain) mice. The purification of CD11c+ cells was performed using mouse CD11c MicroBeads and autoMACS Pro Separator (Miltenyi Biotec, San Diego, CA, USA). Due
95 to limi tations of the cell population , stimulated cell numbers were as follows: BM DCs (1 .5 x10 5 cells/stimulations); splenic DCs (2x10 6 cells/stimulations). While the first set of experiment s was tested in triplicate, the second set of experiment s was performed i n duplicate. The isolated cells were placed in each well of a 12 well cell culture plate ( Corning Inc., Tewksbury, MA, USA ) and stimulated with nothing, GM CSF (200ng/mL) , or G CSF (50ng/mL) for 8 hours. Following th is stimulation , the cells were collected and then centrifuged at 300xg for 10 minutes. Supernatants were decanted carefully and the sample contained tubes were transferred to a fume hood (PCR Workstations; Jeio Tech, Seoul, South Korea). All RNA related work was performed in the hood to prevent potential contamination. RNeasy Mini Kit (Q IAGEN, Inc., Valencia, CA, USA) was used for RNA isolations. Concentration of isolated RNA was determined using NanoVue Spectrophotometer (GE Healthcare Life Sciences, Pittsburgh, PA, USA) . Isolated RNA was stored at 80 o C until used. Gene Expression Analysis Isolated RNA samples were convert ed to cDNA for qPCR (Quantitative polymerase chain reaction) analysis. For the reverse transcription process, 250ng of RNA was transferred to a 0.1mL PCR tube (Eppendorf, Haupp auge, NY, USA). Then, 2.5 L Oligo dT (500 g/mL; Promega, Madison, WI, USA) was added to each tube. The reaction volume was brought up to 24.5 L by adding DEPC treated water (Life Technologies, Grand Island, NY, USA). The sample tubes were then transferred to Mastercycler thermal c ycler (Eppendorf, Hauppauge, NY, USA) and incubated at 70 o C for 5 minutes. Following this incubation, the samples were chilled on ice immediately. Master mix for cDNA synthesis was composed of 25mM MgCl 2 , dNTP, RNAsin, 5x
96 Reaction Buffer, and Reverse Trans cri ptase (Promega, Madison, WI, USA). The total volume of 25.5 L master mix wa s added to each sample tube , resulting in a final reaction volume of 50 L. The reaction temperature and time were as follows: 25 o C for 5 minutes; 42 o C for 1 minute; 70 o C for 15 minutes . Expression of Itgam , Socs3 , Csf3r , and Csf2ra relative to Gapdh was analyzed by quantitative PCR. The primer and corresponding melting temperature (Tm) for each gene were as follows: Itgam (Tm: 55 o C) 5 TGC TGC GAA GAT CCT AGT TG 3 and 5 CCC CAA TTA CGT AGC GAA TG 3 Socs3 (Tm: 58 o C ) 5 AAG GCC GGA GA T TTC GCT 3 5 AAC TTG CTG TGG GTG ACC AT 3 Csf3r ( Tm: 52 o C) 5 GCT GAG GCC TAC CAT GAA 3 5 GCG TTG GCT TCC AGA AC 3 Csf2ra ( Tm: 52 o C) 5 CTT TCG TTG ACG AAG CTC AG 3 5 GAG TCT CGC CCT TCG GTT 3 ; Gapdh 5 GTG GTT CAC ACC C AT CAC AA 3 5 AGC TTG TCA TCA ACG GGA AG 3 (Integrated DNA Technologies, Inc., Coralville, IA, USA) . PCR conditions (total 34 cycles) were as follows: 95 o C for 3 minutes; 95 o C for 30 seconds; the corresponding Tm for 30 seconds; 72 o C for 30 secon ds. Each cDNA template (5ng/reaction) was plated in duplicates. SsoAdvanced SYBR Green supermix from Bio Rad (Hercules, CA, USA) was used for qPCR reaction . R esults Migration Patterns of Bone Marrow Cells i n Response to GM CSF or G CSF Whole BM cells isola ted from all three strains, C57, NOD, and NOR were tested for whether the cells could be mobilize d by GM CSF or G CSF (Figure 5 1) . The BM cells from all three strains isolated at 8 weeks of age were substantially mobilized by GM CSF or G CSF ( Figure 5 2A; C57 by both cytokines p<0.05 ; NOD and NOR
97 p<0.001 by GM CSF, p<0.01 by G CSF). However, 50ng/mL of G CSF did not induce age (Figure 5 2B) . Whereas BM cells obtained from 12 weeks old NOR mice exhibited normal migration in response to G CSF ( Figure 5 2B; p<0.05). In addition, BM cells from all three strains purified at 12 weeks of age significantly mobilized by GM CSF ( Figure 5 2B; C57 and NOD p<0.05; NOR p<0.01). Migratio n Patterns of Splenocytes i n Response to GM CSF or G CSF We also tested mobilization patterns of splenocytes isolated from C57, NOD, and NOR mice at 8 and 12 weeks of age in the same in vitro settings as we already demonstrated with BM cells. Splenocytes o f 8 week old C57 and NOR mice exhibited normal migration in response to GM CSF ( Figure 5 3A; C57 p<0.01; NOR p<0.05). Although splenocytes obtained from 12 week old C57 mice appeared to increase mobilization in response to GM CSF or G CSF, the proportion w as not statistically significant (Figure 5 3B) . However, NOR splenocytes isolated at the same age substantially migrated in response to GM CSF or G CSF ( Figure 5 3B; p<0.01 by GM CSF, p<0.05 by G CSF). Surprisingly , NOD splenocytes obtained at 8 (Figure 5 3A) and 12 (Figure 5 3B) weeks of age did not mobilize by either GM CSF or G CSF . The percent migration was comparable to the negative control condition (cell culture medium alone) . Flow Cytometry A nalysis of Migrated Splenocytes To further assess mobiliza tion patterns by GM CSF or G CSF, migrated cells were collected and their phenotypes were examined by flow cytometry analysis . The collected cells were stained with anti CD11c, MHC Class II, CD19, and CD11b antibodies. Fold change of migrated immune c ells including DCs, macrophages, and B
98 cells was obtained by comparing differences between the number of specific immune cell populations at the beginning in each mouse strain and the number of migrated cell populations by GM CSF or G CSF . As a result, 8 week o ld NOD DCs and B cells appeared to have migration impairments in response to GM CSF compared to those of the other strains ( Figure 5 4A; DCs p<0.0001 vs C57, p<0.001 vs NOR; B cells p<0.05 vs C57 or NOR ). The migration defects appeared to be worse in the i mmune cell populations of 12 weeks old NOD splenocytes (Figure 5 4B) . All type s of immune cells including macrophages, DCs, and B cells from the NOD mice did not migrate in response to G CSF (p<0.0001). Additionally, the DCs were defective in mo bilization by GM CSF (p<0.05). Differences in Gene Expression Levels in Bone Marrow Cells and Splenocytes To identify the underlying defect, differences in gene expression levels in response to GM CSF or G CSF were determined by RT qPCR analysis. Whole BM cells and s plenocytes were stimulated with either GM CSF or G CSF for 8 hours. Following this stimulation, RNA was isolated and then the RNA was reverse transcribed into cDNA. The cDNA was used for qPCR for Itgam , Socs3 , Csf3r , and Csf2ra . Firstly, level of Itgam gen e expression did not dramatically change by GM CSF or G CSF stimulation in BM cells of all three strains (Figure 5 5A) . BM cells isolated from 8 weeks old NOR mice tended to express the gene in lower levels compared to C57 and NOD mice (Figure 5 5A) . NOD s plenocytes isolated at 8 weeks old appeared to increase of the Itgam gene expression by GM CSF stimulation ( Figure 5 5B; p<0.05), but this trend was no longer observed in splenocytes obtained at 12 weeks of age (Figure 5 5D) . Additionally , Itgam gene expre ssion level upon either GM CSF or G CSF
99 stimulation did not much change in splenocytes obtained from 12 week old C57 and NOR mice (Figure 5 5D) . However, d ifferences in Socs3 gene expression levels among the mouse strains were very notable (Figure 5 6) . GM CSF or G CSF stimulation significantly induced Socs3 gene expression in BM cells isolated from 8 week old C57 and NOD mice (Figure 5 6A; C57 p<0.001 by GM CSF, p<0.01 by G CSF; NOD p<0.05 by GM CSF, p<0.01 by G CSF) . Interestingly, GM CSF stimulation sign ificantly increased Socs3 gene expression in 8 week old NOD splenocytes (Figure 5 6B; p<0.05) . L evel of Socs3 gene expression was also significantly induced in 8 week old C57 splen o cytes (Figure 5 6B; p<0.05) , but it was lower than the level detected in NO D splen o cytes. Curiously, Socs3 gene expression was hardly noticeable in 8 week old NOR mice BM cells and splenocytes (Fi g u r e 5 6A and B) . T his gene expression pattern appeared to be reversed in 12 week old immune cells. Socs3 gene expression was significa ntly increased by GM CSF or G CSF stimulation in NOR mice BM cells (Figure 5 6C; p<0.01 by GM CSF, p<0.05 by G CSF) . In addition, NOR mice splenocytes expressed significant level of Socs3 gene by GM CSF or G CSF stimulation co mpared to NOD mice splenocytes (Figure 5 6D; p<0.05) . Csf3 r gene expression level did not dramatically change in response to GM CSF or G CSF stimulation in all mouse strains and all ages (Figure 5 7) . BM cells and spl e n ocytes isolated from 8 week old NOD mice expressed higher level of Csf3r gene compared to other strains (Figure 5 7A and B; BM compared to NOR p<0.05; SPL compared to C57 p<0.01, or NOR p<0.0001) , but, conversely , NOR mice BM cells and splenocytes expressed very low level of Csf3 r gene, regardless of the cytokine
100 stimulat ions. Stimulation with GM CSF or G CSF did not make notable change in Csf3 r gene expression in 12 week old BM cells and splenocytes of all mouse strains (Figure 5 7C and D) . However, NOD and NOR mice BM cells appeared to express significant level of the ge ne by GM CSF stimulation compared to C57 mice BM cells (Figure 5 7C; p<0.01) . Additionally, G CSF stimulation resulted in a significant level of Csf3 r gene expression in NOD mice BM cells when compared with C57 mice BM cells (Figure 5 7C; p<0.05) . Csf2ra g ene expression in response to GM CSF or G CSF stimulation was almost unrecognizable in 8 week old NOR mice BM cells and splenocytes (Figure 5 8A and B) . C57 mice BM cells isolated at 8 weeks of age exhibited down regulation of Csf2ra gene expression in res ponse to GM CSF or G CSF stimulation, but this result was not statistically significant (Figure 5 8A) . However, GM CSF stimulation on C57 mice splenocytes did not down regulate this gene expression (Figure 5 8B) . Both BM cells and splenocytes of 8 week old NOD mice appeared to express the gene in similar levels regardless of the cytokine stimulations (Figure 5 8A and B) . I nterestingly, NOD mice splenocytes obtained at 12 weeks of age expressed very low level of Csf2ra gene in all conditions (Figure 5 8D) . D ifferences in Gene Expression Levels in BM DCs and Splenic DCs To evaluate differences of the gene expression levels in purified cell population, CD11c+ DCs were isolated from the BM and spleen of C57, NOD, and NOR mice at 8 weeks of age. Due to low yield of CD11c+ DCs , in vitro stimulation experiment was performed in duplicate. Therefore, statistical significance could not be obtained in this part of the experiment. Three genes including Itgam , Csf3r , and Csf2ra expressed comparably among all three strains regardless of the cytokine stimulation s or source of
101 the DCs (Figure 5 9A, B, and C) . Interestingly, no Socs3 gene expression was detected in BM DCs from all three strains and splenic DCs obtained from C57 . Splenic DCs isolated from NOR mice expressed ext remely low level of Socs3 gene, whereas increasing Socs3 gene expression was observed in NOD splenic DCs by GM CSF or G CSF stimulation (Figure 5 9D) . Discussion Impairment in chemotactic mobilization of immune cells including neutrophil s, lymphocytes , and DCs has been investig a ted and some of the impaired immune cell populations appeared to be correlated with inflammatory diseases [171 175] . Our results demonstrated that a migration defect existed in NOD mice splen ocytes in response to GM CSF or G CSF but normal migration was observed in the BM cells. In fact , control strains C57 and NOR exhibited mobilization by the cytokines regardless of the source of the cells. Both cytokines have been widely investigated for th eir ability prevent or reverse diabetes in NOD mice . Disease prevention or reverse mechanism seemed to be associated with peripheral tolerance induction via increasing tolerogenic DCs or Treg populations [97 99, 167 , 168, 176] , therefore detection of peripheral migration defect in NOD mice in response to the cytokines was quite surprising . Our in vitro migration assay revealed that most NOD splenic immune cell populations including DCs, B cells, and macrophages appe ared to be responsible for the lower percent migration compared to the cell po pulations of the control strains. Development of T1D is closely associated with presence of multiple genetic susceptibility loci. Nearly 40 loci have been linked to T1D in human [177, 178] and g enome wide linkage studies have revealed mo re than 20 insulin dependent diabetes (Idd) loci in the NOD mouse strain  . To examine underlying defects in terms of
102 down regulation or up regulation of selective genes , we compared differences in gene expression levels upon GM CSF or G CSF stim ulation among the mouse strains C57, NOD, and NOR. T he RT qPCR analysis revealed that Socs3 gene expression in response to the cytokines was notable in 8 week old NOD mice splenocytes compared to the sple nocytes of C57 and NOR mice. In addition, the gene e xpression in BM cells of C57 and NOD mice at 8 weeks of age was also r emarkably increased by GM CSF or G CSF stimulation . However, this trend was no longer observed in 12 week old NOD mice splenocytes. Surprisingly, Socs3 gene expression in both BM cells a nd splenocytes of 8 week old NOR mice was hardly detectable in all conditions . Additionally , NOR mice splenocytes isolated at 12 weeks of age notably expressed Socs3 gene by GM CSF or G CSF stimulation compared to 12 week old NOD mice splenocytes. Interest ingly, differences in Itgam , Csf2ra , and Csf3r gene expression patterns were quite notable among the mouse strains . Both BM cells and splenocytes, which were obtained from 8 week old NOR mice expressed extremely low level s of Csf2ra and Csf3r gene compared to those of C57 or NOD mice. Although Itgam gene expression was detectable in BM cells and sp l enoctyes of 8 week old NOR mice, the gene expression level was relatively low. However, all three strains exhibited comparable level of Itgam gene expression in both BM cells and splenocytes isolated at 1 2 weeks of age. NOR mouse, a recombinant inbred diabetes resistant strain, has 88% of NOD derived and 12% of C57BLKS/J derived genetic backgrounds . The C57BLKS/J strain was introduced by inbreeding and contained C 57BL/6J and DBA/2J genetic backgrounds [170, 180 182] . Particularly, t he C57BLKS/J derived alleles at Idd4, 5, 9,
103 11, and 13 have been correlated with the diabetes protection in NOR strain  . However, NOR strain inherit ed Itgam , Socs3 , and Csf2ra gene allele s , whi ch are located on chromosome s 7, 11 , and 19, respectively , from NOD strain according to and < http://t1dbase.org> . Interestingly , the Csf3r gene is within Idd11 locus on chromosome 4. P revious reports have indicated that the Idd 9/ 11 locus in the NOR strain contributes to diabetes protection with evidence of inducing anergy of autoreactive B cells and suppressing activation of diabetogenic CD4 T cells [183, 184] . R emarkable differences of the Socs3 , Csf2ra , and Csf3r gene expression patterns, which were observed between NOD and NOR mice , were quite intriguing in terms of providing potential evidence of diabetes correlation with these genes . Ferraro et al . noted that streptozotocin induc ed diabetic mice had impairment in mobiliz ation of hematopoietic stem and progenitor cells from the bone marrow after G CSF treatment , and the authors pointed out that the poor mobilization in response to G CSF stimulation may result from abnormality in mo dulation of CXCL12 expression , which is known to be critical for chemotaxis, cell s urvival, and cell proliferation [185, 186] . Thus, several previous studies have addressed that impact of genes on regulation of dia betes pathogenesis. Although the question still remained whether our results could be correlated with progression of T1D, specifically in NOD mice, the silver lining of this study is that identification of peripheral migration impairments and differences i n gene expressions in NOD mice in response to GM CSF and G CSF would be valuable information for better understanding of immunological defects and disease pathogenesis .
104 Figure 5 1. Schematic illustration of in vitro cell migration assay. To test chemot actic migration, we set up in vitro cell migration assay utilizing 8 m pore size polycarbonate membrane cell culture inserts. Briefly, 3x10 6 bone marrow cells or splenocytes were suspended in 300 L of cell culture medium and placed in the upper chamber of the insert. Cells were incubated for 24 hours at 37 o C in the presence of GM CSF (200ng/mL) or G CSF (50ng/mL) in the below chamber of the insert. Following this incubation, migrated cells were collected, counted, and then used for further analysis.
105 F igure 5 2. Percentage of bone marrow (BM) cell migration at 8 and 12 weeks of age. BM cells were mobilized by GM CSF or G CSF regardless of strains or ages. (n=3/strain/age, ns not significant, * p<0.05, ** p<0.01, and *** p<0.001)
106 Figure 5 3. Perce ntage of splenoc yte migration at 8 and 12 weeks of age. Splenocytes isolated from C57 and NOR mice exhibited normal migration. However, NOD splenocytes did not migrate in response to GM CSF or G CSF. (n=3/strain/age, ns not significant, * p<0.05, ** p<0.01 , and *** p<0.001)
107 Figure 5 4. Fol d change of migrated splenocyte populations. A) DCs and B cells from NOD mice did not migrate much in response to GM CSF at 8 weeks o f age compared to other strains. B) Macrophages, DCs, and B cells from 12 weeks old NOD mice were signif icantly less mobilized by G CSF. (n=3/strain/age, * p<0.05, *** p<0.001, **** P<0.0001)
108 Figure 5 5. Itgam gene expression at 8 and 12 weeks of age. GM CSF or G CSF stimulation did not significantly induce Itgam gene expression i n BM cells regardless of strain or age. Notably, A) NOR BM cells isolated at 8 weeks of age exhibited significantly lower Itgam gene expression compared to other strains. B) GM CSF stimulation significantly induced this gene expression in 8 weeks old NOD s plenocytes. However, C & D) BM cells and splenocytes isolated at 12 weeks of age expressed comparable levels of Itgam gene in all three strains regardles s of the cytokine stimulations . (n=3/strain/age, * p<0.05, ** p<0.01, and *** p<0.001)
109 Figure 5 6 . Socs3 gene expressi on at 8 and 12 weeks of age. A) GM CSF or G CSF stimulation remarkably induced Socs3 gene expression in BM cells from 8 week old C57 and NOD mice . Notably, B) stimulation of GM CSF did induce this gene expression in splenocytes from 8 week old C57 and NOD mice. Interestingly, Socs3 gene expression was hardly detectable in 8 week old NOR mice A) BM cells and B) splenocytes . However, C) BM cells obtained from 12 week old NOR mice significantly induced Socs3 gene expression by both of the cytokines . While NOD mice D) splenocytes isolated at 12 weeks of age expressed very low level of Socs3 gene regardless of the cytokine stimulations. (n=3/strain/age, * p<0.05, ** p<0.01, and *** p<0.001)
110 Figure 5 7. Csf3r gene expression at 8 and 12 weeks of age. Csf3r gene expression level s did not change much by the cytokine stimulations regardless of strains, ages, or source of cells. However, A) BM cells and B) splenocytes obtained from 8 week old NOD mice notably expressed higher level Csf3r g ene compared to those of NOR mice . Additionally, the gene expression in C) BM cells isolated from 12 week old NOD mice in response to GM CSF or G CSF was higher than C57 BM cells. However, there was no statistical difference in D) splenocytes. (n=3/strain/ age, * p<0.05, ** p<0.01, and **** p<0.0001)
111 Figure 5 8. Csf2ra gene expression at 8 and 12 weeks of age. The level s of Csf2ra gene expression did not change much by GM CSF or G CSF stimulation regardless of strains, ages, or source of cells. Intere stingly, A) BM cells and B) splenocytes from 8 week old NOR mice expressed extremely low level s of the gene compare d to other strains . Conversely, Csf2ra gene expression was relatively low in NOD mice C) BM cells D) splenocytes isolated at 12 weeks of age compared to other strains, but this was not statisticall y different. (n=3/strain/age, * p<0.05, and ** p<0.01)
112 Figure 5 9. The levels of gene expression in BM DCs and splenic DCs. GM CSF or G CSF stimulation did not induce much of A) Itgam , B) Csf3r , a nd C) Csf2ra expression in BM DCs or splenic DCs of all strains. However, splenic DCs from NOD mice exhibited relatively higher D) Socs3 gene expression by GM CSF stimulation compared to those of other strains. This set of experiments was performed in dupl icate.
114 CHAPTER 6 CONCLUSIONS AND FUTURE DIRECTIONS The overall goal o injectable matrix material incorporating a combination of adjuvants and autoantigen, which is capable of inducing the antigen specific tolerogenic immune responses , therefore affording a safe and effective manner for the prevention of type 1 diabetes (T1D). Exogenous insulin administration , most widely accepted treatment for T1D allows the patients to manage their blood glucose level and pursue a normal lifestyle. However, insulin therapy cannot perfectly control blood glucose level s and prevent the patients from T1D related complications , such as nephropathy, retinopathy, and cardiovascular disease [187, 188] . In 19 65 , Gepts reported remarkable findings based on a sufficient number of pancreas samples that inflammatory infiltrates of the isl ets of Langerhans were present in 68% of acute dia betes patients  . Continuous efforts by many to characterize the disease pathogenesis revealed that the presence of autoreactive T and B cells, and aberrant innate immune cells resul ted from genetic or environmental factors that contribute d to the destruction of insulin producing cells in the pancreatic islets of Langerhans, which ultimately lead to overt diabetes [187, 190, 191] . Ample evid ence of autoimmune mediated pathogenesis led to the investigat ion of immune based therapy for T1D . The first immunotherapy to cure T1D was attempted almost three decades ago usi ng an immunosuppressant drug, cy closporin e [187, 188] . However, this type of systemic im muno suppressive regimen carries its own risk including increased susceptibility for infection, or cancer [60, 134 136] . Due to this limitation, a ntigen specific immunomodulation has gained widespread attention as a more selective
115 strateg y in 1990s  . However, there were many obstacles in the delivery of the antigen to immune cells, as well as the education of these cells [60, 192] . High dosages of the antigen were required to educate the immune cells  and this requirement can increase financial burden s and the risk of side effects. Since selection of the proper physical form of a vaccine is one of the critical considerations to achieve the best therapeutic outcome, we selected phagocytosable PLGA MPs as an antigen delivery vehicle. W e also utilized self assembli ng peptide hydrogel to achieve localized vaccine delivery through controlled release of vaccine formulations. The first part of this dissertation characterized the biomaterials both in vitro and in vivo . Combination of hydrogel and MPs appe ared to readily organize 3D scaffold. Size distributions of MPs encapsulating denatured insulin exhibited an average diameter of 5. 01 m , which is a phagocytosable size. CpG or GM CSF incorporating in the h ydrogel was released almost thoroughly within 48 hours in vitro and the release profile appeared to be associated with the size of the incorporating agent. We also determined that the gel released GM CSF did work as a chemoattractant of whole BM cells obtained from 8 week old NOD mice. Next, we confirmed biodistribution a nd accumulation of hydrogel PLGA MPs matrix in vivo . We subcutaneously injected hydrogel contain ing insulin MPs and CpG into dorsal neck area of 8 week old NOD mice. Unexpectedly, fluorescence signal released fr om the hydrogel was detected in the ventral s ide of the body as early as 3 hours post injection, but it was not surprising due to the fact that live animals have proteolytic enzymes, which could degrade the hydrogel  . Considerable amounts of hydrogel and MPs still remained at the injection site at 75 hours post injection. Notably , we confirmed that both MPs and the fluorescence si gnal
116 released from the hy drogel were accumulated in the cervical, axillary, and brachial LNs, spleen, and pancreas. Although additional supplementary data w ould be necessary, we believe that th e se biomaterials were transported and trafficked in the organs via APCs. Taken together, t his body of work highlighted features and roles of the MPs hydrogel matrix as a n efficient vaccine delivery system . In our first attempt to test the efficacy of the vaccine formulations, we loaded several different agents including GM CSF, CpG, and Hb:Hp as well as insulin MPs into hydrogel. The first injection occurred at 8 weeks of age followed by two additional injections at 10 and 12 weeks of age. We demonstrated that a group administered with hydrogel (GM CSF/CpG/Hb:Hp) + insulin MPs exhibited the be st survival proportion at the end of the study. Additionally, the group with the second best survival was that administered GM CSF, a pro inflammatory adjuvant CpG , and insulin MPs. Whereas treatments with an anti inflammatory adjuvant Hb:Hp along with GM CSF and insulin MPs apparently yielded the worst survival proportion. Th ese in vivo result s could possibly relate with a mechanism that acute inflammation induced by pro inflammatory mediator is subsequently resolved by activation of endogenous anti inflam matory signals [193, 194] . Although we need to further examine the mecha nism in our setting, we believe that the use of CpG may be linked to induction of acute inflammation and subsequent resolution of inflammation , followed by enhancement of the resolution process via post activation of HO 1 pathway induced by administration of Hb:Hp. Next , in vitro tests with ad j uvant(s) and chemoattractant revealed that combination of GM CSF, CpG and/or Hb:Hp induced IFN production from 8 week old NOD mice splenocytes. Although IFN may be able to act as an IDO inducer [150, 151] , we
117 decided to eliminate GM CSF from our next vaccine formulations in order to prevent potential pro inflammatory effect of GM CSF . Additional significan ce of this study was detection and histological characterization of granuloma. Interestingly, this lesion was only discovered in mice injected with insulin MPs regardless of types of adjuvant. Although Mooney group demonstrated that an implantable macropor ous PLG matrix incorporating GM CSF could recruit and house DCs at the implanted site in mice  , our gran uloma exhibited more organized tertiary lymphoid organ like structure with infiltration of T, B , and mac rophage populations . After the first in vivo study, we modified our vaccine formulations and schedules. We excluded GM CSF and changed injection schedules from 3 to 9 time s in order to boost vaccine efficacy. Unfortunately, two mice died after the fifth injection of hy drogel (CpG/Hb:Hp) + insulin MP. The death appeared to be caused by anaphylactic shock. Due to the unexpected loss of animals, we decided not to give addition al injections to the surviving mice. Interestingly, almost 80% of the surviv ing mice maintained euglycemia until 28 weeks of age. However, this survival proportion was not significantly higher than the survival rate obtained from a group administered 3 tim es with the same vaccine formulation. Additionally, a group of mice given 3 injections with hydrogel (CpG) + insulin MPs exhibited comparable survival proportion. In this set of study, we traced size change of the granuloma each week from 11 to 28 weeks of age and observed that the fourth injection boosted the size of the granuloma. In our third trial , we only used CpG as a vaccine adjuvant. Disappointingly, the fifth injection of our biomaterial based vaccine formulation caused lethal anaphylaxis again . Ho wever, the fifth injection with a soluble form of the vaccine did not cause any
118 adverse effect. Elimination of Hb:Hp could not yield improved efficacy or prevent anaphylaxis. We assumed that absence of Hb:Hp in the vaccine formulation might be less efficac ious in terms of eliciting tolerogenic immune response and prevention of diabetes based on the survival proportion data of Study II and III . Instead of Hb:Hp , we will choose the alternative HO 1 inducer, hemin for the future study. S ynthetic h emin (trade n ame Panhematin) is already an approved drug for acute porphyria patients  , therefore the use of hemin would facilitate clinical translation in the future. In spite of the discouraged results, we could determine the possible mechanism behind the prevention of diabetes by this vaccine . Notably, splenocytes isolated from hydrogel (CpG) + insulin MPs administered group (5 injections) appeared to have a higher Treg population than the other groups. Another interesting result was t hat significantly lower insulin B(9 23) reactive T cell population in the brachial LNs of the hydrogel (CpG) + insulin MPs treated mice was observed . We already confirmed through in vivo imaging that the subcutaneous injected vaccine formulation was accumu lated in the brachial LNs, therefore the pathogenic T cell population in the LNs could be substantially influenced by the vaccine agents. Given these results, our biomaterial based vaccine appear ed to work as expected . However, foremost, we should resolve the anaphylaxis issue in order for our vaccine to be clinically translatable . P retreatment with a combination of antihistamine and platelet activating factor antagonist will be considered to prevent this issue for the future study. In depth study about gra nuloma formation caused by multiple subcutaneous autoantigen injections will be informative in both preclinical and clinical aspects. First, confirmation of IDO expressing DCs, Tregs, and insulin B(9 23) reactive T cell populations by flow cytometry analys is will be a good
119 approach for better understanding the granuloma. Additionally , adoptive transfer of immune cells obtained from granuloma to non obese diabetic severe combined immunodeficiency (NOD SCID) mice would be necessary in order to examine whether diabetes can be prevented or accelerated in the donor mice. Ultimately, clinical testing of granuloma formation and examination on humans by injecting hydrogel loaded with clinical grade CpG and GMP grade PLGA MPs encapsulated with denatured insulin autoa ntigen would possibly provide crucial information for the use of the granuloma as a biomarker for T1D. Next, the detection of peripheral migration defect in response to GM CSF or G CSF existing in NOD mice splenocytes has led us to study in depth b oth cell ular and genetic levels along with control strains, C57 and NOR. In vitro migration assays showed that mobilization defect in response to these cytokines existed in NOD mice splenocytes but a normal response in BM cells was observed . C ontrol mice had a mig ration in response to the cytokines independent of source of cells. In addition, further assess ment revealed that immune cells including DC, macrophages, and B cells from NOD splenocytes were significantly less mobilized by GM CSF or G CSF. Analysis of gen e expression levels in response to GM CSF or G CSF stimulation provided information about possible underlying defects in the immune cells of NOD mice . Particularly, comparisons of gene expression levels between NOD and NOR mice revealed significant differe nces. All tested genes including Itgam , Socs3 , Csf3r , and Csf2ra expressed lower levels in both BM cells and splenocytes isolated from 8 week old NOR mice compared with those of NOD mice. Interestingly, these gene alleles of NOR mice seemed to be inherited from NOD mice. Although NOR strains share almost
120 88% of the genome with NOD mice, few disease resistance genetic loci contribute to complete protection from diabetes in NOR mice  . Therefore, it would be worth it to further investigate the gene expression in additional specific cell populations including neutrophils and B cells from all three strains , as well as the correlation with the progression to T1D, specifically in NOD mice.
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136 BIOGRAPHICAL SKETCH Young Mee Yoon was born in 1983 in Ulsan, South Korea. In February 2001, she graduated from Joong Following high school, she enrolled at Sangmyung University in Seoul, South Korea in March 2001 . After her freshmen year, Young Mee t ravelled for the first time by herself to Melbourne, Australia and she stayed in the city for 2 months to learn English and experience a new environment. This trip broadened her horizons . In March 2003, she transferred from Sangmyung University to Kyung He e University in Seoul, South Korea as a junior year student. She majored in Biology and earned a Bachelor of Science with Honors in February 2005. Following college graduation, Young Mee started to pursue a Master of Science degree in Biomedical Science s a t Hanyang University, Seoul, South Korea. During her graduate program, she joined immunological defense mechanisms against bacterial pathogens in human intestinal epithelial cells. She presented her research posters at n umerous national researc h conferences and published her works in p eer reviewed journals. In February 2007, she earned a Master of Science degree and started to work as a commissioned research scientist at Nano Bio Research Center, Korea Institute of Scienc e and Technology (KIST) , Seoul, South Korea. While in the KIST, Young Mee investigated the use of biomaterial for efficient cell culture device s . In June 2008, Young Mee moved to Gainesville, Florida in order to pursue her doctoral degree in the Interdisc iplinary Program (IDP) in Biomedical Sciences at the University of Florida , where she was awarded an Alumni Fellowship . After three research lab rotations, she joined Dr. Mark Atkinson laboratory and i mmunology concentration of the IDP. As a doctoral stu dent , Young Mee investigated development
137 of an injectabl e biomaterials based tolerogenic vaccine to prevent t ype 1 d iabetes in the n on obese diabetic mouse model , with the eventual goal of translation to clinical application s . After graduation, Youn g Mee p lans to continue working in the field of t ype 1 d iabetes research.