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1 DEVELOPMENT OF A DENDRITIC CELL TARGETING, SYNTHETIC BIOMATERIAL MICROPARTICLE VACCINE FOR THE PREVENTION OF TYPE 1 DIABETES By JAMAL S. LEWIS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERISTY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012
2 2012 Jamal Lewis
3 T o Mom, Dad, Ghilly B, Shaq and the loving memory of my Granny J. In time of te st, family is best. ~ Burmese Proverb
4 ACKNOWLEDGEMENT S This work would not have been possible without the help, support and encouragement of several people. Firstly, I would especially like to express my sincerest gratitude to my graduate advisor Dr. Ben jamin Keselowsky. Under his mentoring, guidance, positive and inspiring advice, I have matured as a research scientist and engineer. To my supervisory committee Dr. Brandi Ormerod, Dr. Chris Batich, Dr. Michael Clare Salzler, and Dr. Todd Brusko your d iverse scientific backgrounds afforded me the exceptional opportunity of expert advice. Thank you for your guidance and constructive suggestions. I like to express my deepest appreciation to my colleagues in the laboratory for their understanding and supp ort during my studies. Thank you Abhinav Acharya, Toral Zaveri, Jerome Karpiak, Matt Carstens, Natalia Dolgova, Charles Crooks, Chris Roche, Cindy Ying and my adjacent laboratory members/ associates: Young Mee Yoon, Clive Wasserfall, Dr. Chang Qing Xia, Be n Looney and Marda Jorgensen. To my friends Shomari, Barrington, Michael Paul, Kinda, Keira, John Janelle thank you for your friendship and all the fun we had during my studies. I would also like to acknowledge the support and unremitting encourageme nt I received from Aunty Doreen, Uncle Kenny, Dr. Verian Thomas, Dr. Neil James, the late Dr. Colin Benjamin, Mr. Nurse, Uncle Ab, Aunty Bonnie, Uncle Donald, Aunty Bev and Aunty Barbara. Thank you for your warm and sincere affection. My family has always been a driving force for me and I would like to express my deepest gratitude to my parents, Dawn Erica and Leslie Oswald Lewis, my sister, Ghislaine and my brother Shaquille. Their support, caring, unconditional love, friendship
5 and encouragement in variou s ways throughout the years have inspired me. Their interest in my research was phenomenal and provided me with the stimulus to complete the project. I deeply regret that my dearest Grandma, Monthee Alicia Homer Jacobs, did not complete this journey with me. Her infectious curiosity in my research was motivating. She was my always my source of comfort. Her memory will live forever. To Tamara, I wish to express my deepest appreciation for our endless tte tte, your friendship, love, encouragement, and s upport. You are a rare jewel.
6 TABLE OF CONTENTS page ACKNOWLEDGEMENTS ................................ ................................ ............................... 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 11 ABSTRACT ................................ ................................ ................................ ................... 13 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 19 Type 1 Diabetes (Significance) ................................ ................................ ............... 20 PLGA as a Vaccine Carri er ................................ ................................ ..................... 22 Microparticle Injection Route of Administration ................................ ....................... 22 Tolerogenic Dendritic Cells ................................ ................................ ..................... 23 Tolerance inducing Agents ................................ ................................ ..................... 26 Agents with Intracellular Targets ................................ ................................ ...... 26 Agents with Extracellular Targets ................................ ................................ ..... 27 Non activating Dendritic Cell Targeting Agents ................................ ...................... 28 2 MICROPARTICLE SURFACE MODIFICATIONS TARGETING DENDRITIC CELLS FOR NON ACTIVATING APPLICATIONS ................................ ................. 34 Introductory Remarks ................................ ................................ .............................. 34 Experimental Procedure ................................ ................................ ......................... 37 Experimental Animals ................................ ................................ ....................... 37 Preparation of Fluorescent Microparticles ................................ ........................ 37 Cross linking of Ligands to Microparticles ................................ ........................ 38 Quantification of Cross linked Ligands ................................ ............................. 39 S Potential of Ligand modified Microparticles ................. 40 Dendritic Cell and Macrophage Cell Culture ................................ ..................... 41 Internalization of Microparticles ................................ ................................ ........ 42 Dendritic Cell Activation and Cytokine Analysis ................................ ............... 42 Cell Viability ................................ ................................ ................................ ...... 43 Single Cell and Mixed Culture Preferential Phagocytosis Studies .................... 43 Antigen Presentation by Dendritic Cells After Phagocytosis of Surface modified Microparticles ................................ ................................ ................. 44 Uptake and Translocation of Microparticles to Lymph Nodes ........................... 45 Significance Testing ................................ ................................ ......................... 46 Results ................................ ................................ ................................ .................... 46 Impact of Study ................................ ................................ ................................ ....... 53
7 3 MICROSPHERE TECHNOLOGY OFFERS COMBINATORIAL AND LOCAL DRUG DELIVERY TO DENDRITIC CELLS FOR ENHANCEMENT OF SUPPRESSIVE FUNCTION ................................ ................................ ................... 71 Introductory Remarks ................................ ................................ .............................. 71 Experimental Procedure ................................ ................................ ......................... 76 Microparticle Preparation ................................ ................................ .................. 76 Microparticle Characterization Sizing, Loading, Release Kinetics ................. 77 Dendritic Cell Culture and Microparticle incubation ................................ .......... 77 Dendritic Cell Phenotype Matura tion and Tolerogenic Markers ..................... 79 CD4+ T cell and Treg Isolation ................................ ................................ ......... 80 Mixed Lymphocyte Coupling (MLC) T cell Suppression and Treg Generation ................................ ................................ ................................ .... 80 Significance Testing ................................ ................................ ......................... 80 Results ................................ ................................ ................................ .................... 81 Impact of Study ................................ ................................ ................................ ....... 89 4 A VITAMIN D3 GM CSF AND TGF 1 ENCAPSULATING DUAL MICROPARTICLE VACCINE SYSTEM THAT PREVENTS ONSET OF TYPE 1 DIABETES IN NOD MICE ................................ ................................ ..................... 104 Introductory Remarks ................................ ................................ ............................ 104 Experimental Procedure ................................ ................................ ....................... 109 Experimental Animals ................................ ................................ ..................... 109 Microparticle Preparation ................................ ................................ ................ 109 Microparticle Characterization Sizing, Loading, Release Kinetics ............... 111 Bone Marrow derived DC Culture and MP incubation ................................ .... 112 Bone Marrow derived Dendritic Cell Phenotype Maturation and Tolerogenic Markers ................................ ................................ ................... 113 CD4+ T cell Isolation ................................ ................................ ...................... 114 Mixed Lymphocyte Coupling (MLC) T cell Suppression and Treg Induction 114 Antigen Presentation by Dendritic Cells after Microparticle Phagocytosis ...... 115 Diabete s Prevention Studies ................................ ................................ .......... 115 Midpoint Mechanistic Studies Histopathology, APC and Treg Analysis ...... 117 Statistical Analysis ................................ ................................ .......................... 118 Results ................................ ................................ ................................ .................. 118 Impact of Study ................................ ................................ ................................ ..... 128 5 CONCLUSIONS AND FUTURE DIRECTIONS ................................ .................... 155 LIST OF REFERENCES ................................ ................................ ............................. 160 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 179
8 LIST OF TABLES Ta ble page 3 1 MP size and loading characterization ................................ ................................ 96 4 1 Microparticle characterization: Encapsulation Efficiency ................................ .. 135
9 LIST OF FIGURES Figure page 1 1 Two classes of injectable MPs will be fabricated ................................ ................ 31 1 2 Molecular structure of Poly(lactic co glycolic acid) ................................ ............ 31 1 3 Dendritic cell lineage ................................ ................................ .......................... 32 1 4 Molecular structure of Rapamyci n ................................ ................................ ...... 32 1 5 Molecular structure dihydroxyvitamin D3 ................................ ............ 33 1 6 Molecular structure of all trans retinoic acid ................................ ....................... 33 2 1 Size and surface density characterization of surface modified microparticles .... 59 2 2 Internalization of surface modified MPs by dendritic cells (DCs) is demonstrated via confocal microscopy ................................ ............................... 60 2 3 Immature dendritic cell phenotype is maintained even after exposure to ligand conjugated MP modifications ................................ ................................ ... 61 2 4 Co culture of surface modified MPs with dendritic cells (DCs) has negligible effects on cell viability ................................ ................................ ......................... 64 2 5 Microp article (MP) phagoc ytosis by dendritic cells (DCs) and macrophages ................................ ............................ 65 2 6 MP surface modification ................................ ................................ ..................... 66 2 7 Functional antigen presentation improved by surface modified MPs ................. 67 2 8 Phagocytosis and trafficking of micropar ticles (MPs) by dendritic cells (DCs) ........... 68 3 1 MP size distribution was determined using dynam ic light scattering techniques ................................ ................................ ................................ ......... 96 3 2 MP release kinetics In vitro relea se profiles of Rapamycin, Retinoic acid, TGF IL 10 in a 2% Tween 20 PBS solution ................................ ............ 97 3 3 Co culture of dual MP systems result in reduced levels of surface expression of MHC II and costimulatory markers (CD80, CD86) relative to iDCs ................ 98 4 1 Microparticle characterization: Size Distribution ................................ ............... 135
10 4 2 Microparticle character ization: Release Profile ................................ ................. 136 4 3 Co culture of the Vit D3/ TGF levels of surface expression of MHC II and costimulatory markers (CD80, CD86) relative to iDCs ................................ ................................ ...................... 137 4 4 Co culture of the Vit D3/ TGF expression of Indoleamine 2,3 dioxygenase ................................ ................... 138 4 5 Co culture of Vit D3/ TGF CD4+ T cells ................................ ................................ ................................ ..... 139 4 6 Co culture of Vit D3/ TGF treated DCs w ith CD4+ T cells result s in expansio n and induction of Tregs and increased secretion of the suppressive cytokine, TGF ................................ ................................ .............................. 141 4 7 Antigen encapsulated in MP can be processed and loaded onto MHC II complexes by DCs and fu nctionally presented to T cells ................................ .. 1 43 4 8 Study I : Vit D3/ TGF CSF/ Ins B MPs injections into NOD mice at 4 weeks of age prevents diabetes onset ................................ ............................. 144 4 9 Study I : Vit D3/ TGF CSF/ Ins B MPs injections into NOD mice at 4 weeks of age influences immune cell phenotype ................................ .............. 145 4 10 Study II : (A) Vit D3/ TGF CSF/ hInsulin MP injection into NOD mice at 8 wee ks of age delays diabetes onset ................................ .......................... 147 4 11 Study II : Vit D3/ TGF CSF/ hInsulin MPs injections into NOD m ice at 8 weeks of age influences immune cell phenotype ................................ ........... 148 4 12 Study III : (A) Vit D3/ TGF CSF/ hInsulin MP in jection into NOD mice at 8 weeks of age prevents diabetes onset ................................ ...................... 151 4 13 Study III : Vit D3/ TGF CSF/ hInsulin MPs injections into NOD mice at 8 weeks of age influences immune cell phenotype ................................ ........... 152
11 LIST OF ABBREVIATION S ANOVA An alysis of variance APCs Antigen presenting cells BrdU Bromodeoxyuridine CD Cluster of differentiation o C Degrees Celsius DC Dendritic cell DMEM EDC 1 ethyl 3 (3 dimethylaminopropyl) carbodiimide ELISA Enzyme linked immunoso rbent assay FBS Fetal bovine serum GM CSF Granulocyte macrophage colony stimulating factor ICAM Intercellular adhesion molecule IDO Indoleamine 2,3 dioxygenase IFN Interferon IL Interleukin Ilt Immunoglobulin like transcript LPS Lipopolysaccharide Macrophage MHC Major histocompatibility complex MLR Mixed lymphocyte reaction MP Microparticle MTOR Mammalian targets of rapamycin NF Nuclear factor NHS N hydroxysuccinmidyl ester
12 NOD Non obese diabetic PBS Phosphate buffered saline PEG Polyethylene gly col PLGA Poly (d lactic co glycolic acid) RA Retinoic acid RAPA Rapamycin TGF Transforming growth factor T1D Type 1 diabetes TNF Tumor necrosis factor VDR Vitamin D receptor
13 Abstract of Dissertation Presented to the Graduate School of the University of F lorida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DEVELOPMENT OF A DENDRITIC CELL TARGETING, SYNTHETIC BIOMATERIAL MICROPARTICLE VACCINE FOR THE PREVENTION OF TYPE 1 DIABETES By Jamal Sana Lewis A ugust 2012 Chair: Benjamin G. Keselowsky Major: Biomedical Engineering Type 1 Diabetes (T1D) in both humans and non obese diabetic (NOD) mice results from a breakdown of self tolerance that is characterized by T cell cell destruction. Recently this failure in homeostatic regulation has been attributed to a deficiency in the number and/ or function of dendritic cells (DCs) and regulatory T cells (Tregs). DCs are professional antigen presenting cells (APCs) with the capacity to instigate either the inflammatory or anti inflammatory arm s of the adaptive immune system. DCs influence peripheral immune tolerance via a number of modalities including the induction of effector T cell anergy and deletion, immune deviation expansion and induction of regu latory T cells. With respect to Tregs, numerous studies have shown that this subpopulation of CD25+, FoxP3 expresssing T cells have functional defects and reduced frequency with progression of diabetes in NOD mice. These findings have inspired a novel, per sonalized approach to the treatment of autoimmune diseases including T1D, known as dendritic cell based immunotherapy. Although DC based immunotherapy has been developed to the point where it is currently being tested in humans, problems such as the plasti city and complexity of DC
14 maturation, ex vivo stability, shelf life, and high costs restrict the widespread application of this therapeutic approach. One design strategy focuses on in vivo targeting of DCs with injectable, synthetic particulate systems th at can deliver vaccine components including immunomodulatory agents. We are developing a multifunctional, synthetic microparticle encapsulated vaccine that can be easily administered with simultaneous and continuous delivery using controlled release materi als (poly lactide co glycolide). Poly (lactic co glycolic acid) (PLGA) particulate systems have gained widespread attention as a viable option for delivery of vaccines in the last quarter century. Qualities such as control of size, loading and release kin etics, immunogenicity, antigen processing and presentation, and surface functionalization superimpose to make PLGA microparticulate systems ideal for vaccine delivery to dendritic cells (DCs). Recent, efforts at controlling the DC polarity for mitigation o f autoimmune disorders, such as type 1 diabetes (T1D) have intensified Herein, we describe the development of a dendritic cell targeting, PLGA microparticle vaccine for the abrogation of type 1 diabetes and applicable to other autoimmune diseases. The fir st objective was to enhance passive targeting of dendritic cells by microparticles. A number of studies have shown that active targeting of DCs using surface modified particulate systems improves DC uptake and trafficking, and further escalates downstream immuno stimulatory responses for cancer vaccine applications. However, given the immuno stimulant pathways activated by the receptors targeted in these studies, other candidates which are non inflammatory must be considered for auto immune applications. W e investigate d the targeting of DCs using PLGA
15 m icroparticles (MPs) in a non stimulatory manner and assess efficacy in vitro and in vivo We modified MPs by surface immobilizing DC receptor targeting molecules antibodies (anti CD11c, anti DEC 205) or pept ides (P D2, RGD), where anti CD11c antibody, P D2 and RGD peptides target integrins and anti DEC 205 antibody targets the c type lectin receptor DEC 205. Our results demonstrate the modified MPs are neither toxic nor activating, and DC uptake of MPs in vit ro is improved by the anti DEC 205 antibody, the anti CD11c antibody and the P D2 peptide modifications. The P D2 peptide MP modification significantly improved DC antigen presentation in vitro both at immediate and delayed time points. Notably, MP functio nalization with P D2 peptide and anti CD11c antibody increased the rate and extent of MP translocation in vivo by DCs and macrophages ( ) with the P D2 peptide modified MPs demonstrating the highest translocation. This work informs the design of non activating polymeric microparticulate applications such as vaccines for autoimmune diseases. We then evaluated the efficacy of different M P formulations to confer tolerogenicity to DCs in vitro The goal of this MP approach, inspired by DC based immunotherapy, is to modulate DC phenotype in situ through targeted, local delivery of immunosuppressive agents. C ognizant that most immunomodulator y effects on immune cells occur when a plurality of agents are acting in concert we designed a unique dual MP system to deliver immunomodulatory agents simultaneously and precisely to their respective receptors. Specifically, we fabricated two classes of M Ps: (i) phagocytosable MPs that could deposit the i r payload intracellularly and (ii) MPs that could not be readily taken up and release agents into the extracellular milieu. Phagocytosable MPs were loaded with pharmaceutical agents, either of rapamycin (RA PA), or retinoic acid (RA).
16 While un phagocytosable MPs encapsulated either transforming growth factor beta1 (TGF 1), or interleukin 10 (IL 10). We demonstrate d that we can prepare MPs to desired size and encapsulating immunomodulatory agents via single and double emulsion, solvent evaporation techniques. The release kinetics of these MPs were all characterized, in general, they last for 4 weeks or greater. Additionally, we observed that in vitro MP formulations with encapsulated agents generally effected greater modulation of DC stimulatory molecules than their soluble eq uivalent counterparts. Further, combination of s ingle factor MPs led to cumulative dampening of DC activation markers but this is dependent on the combination of encapsulated factors. Similarly, we investigated the impact of our dual MP systems on inhibitory molecules. Results show that single dose agents at the concentrations and in the delivery format used in this study also inhibit expression of suppressive surface molecules on DCs. This wa s further intensified by simultaneous delivery of cytokines by un phagocytosable MPs However, downregulatio n of DC inhibitory molecules did not affect the ability of MP treated DCs to suppress proliferation of allogenic T cells To begin to decipher the observed severe immunosuppression by dual MP treated DCs, we tested for generation of regulatory FoxP3+ T cel ls under the same conditions. Our results demonstrate that, with the exception of the RA/ TGF 1 MPs group, the CD25+ FoxP3 expressing subpopulation of T cells is relatively constant for all treatments. This indicates the strong levels of suppression we ob serve for our MP formulations are most likely due to DC hypo stimulatory state and T cell anergy, in addition to other uninvestigated mechanisms like T cell apoptosis.
17 One dual MP system which showed great potential for in vivo tolerance induction based on in vitro testing was the Vit D3 MP in combination with the TGF 1 MP. We compiled a dossier on the in vitro immune effects of this dual MP system then tested its efficacy as a type 1 diabetes prevention therapy. Moreover, w e fabricated two classes phagocytosable) The phagocytosable MPs were loaded with Vit D3 and insulin. The un phagocytosable MPs encapsulated TGF B1 and GM CSF. We confirmed loading and release kinetics of these drug loaded using conventional particle degradation and drug detection methods. The release profiles show an initial burst release within the first week followed by a more gradual release for the next three weeks T he effects of the Vit D3/ TGF MP system on the expression of stimulatory molecules (MHC II, CD80, CD86) on DCs as well as the relevant control treatments were studied In comparison to iDCs, all of these activating markers are down regulated signif icantly by incubation with the combination of VitD3/ TGF Further, Vit D3/ TGF treated DCs considerably inhibit proliferation of allogenic T cells. These MPs were injected into a cohort of 8 wk old, female NOD mice to investigate their effica cy at diabetes prevention. Injection of the combination of GM CSF MP s + Vit D3 MP s + TGF B1 MP s + hI nsulin MP s resulted in a survival rate of 60% non diabetic mice, compared to the blank MPs at 10% non diabetic after 28 wks Kaplan Meier analysis at this p oint reveals the survival proportions for this group is significant compared to the control with a p value of 0.045. These studies demonstrate that our engineered microparticle vaccine formulations can: (a) target DCs in vivo for uptake and trafficking; (b ) modulate DC phenotype and further promote allogenic T cell hypor esponsiveness to exposed DCs; (c ) prevent the
18 onset of diabetes in NOD mice when treated at an age that is therapeutically relevant. In the future, we will investigate diabetes prevention wi th the targeted MPs, as we expect improved uptake of MPs in vivo to further promote diabetes prevention.
19 CHAPTER 1 INTRODUCTION The debate on the cause of autoimmune diseases, particularly diabetes, has shifted recently from auto reactive cell escape of clonal deletion to reduction in number and function of regulatory T cells (Tregs) which suppress low levels of physiologic auto reactive cells  This new opinion has inspired the development of moderately successful T1D therapies that seek to generate g reater numbers of functional regulatory T cells in patients afflicted with insulin dependent diabetes  Of particular interest to our group is Dendritic Cell natural adjuvants to recruit the adaptive arm of the immune system in foreign role in the maintenance of peripheral tolerance  To this extent, scientists have developed vaccines in which DCs are either (i) arrested in a state of immaturity (T cell anergy) or (ii) are engineered (genetically, chemically, etc) to produce immunosuppressive molecules (Treg induction), which ultimately curtail excessive immune responses  However, problems such as the plasticity and complexity of DC maturation, donor specificity, ex vivo stability, shelf life, and cost abrogate the benefits of this therapeutic approach  An intelligent design strategy might focus on the development of a synthetic particl e encapsulated vaccine, or vaccine particle that can be easily administered with simultaneous delivery of both prime & boost doses using time release materials (e.g., poly lactide co glycolide). This flexible approach greatly simplifies issues related to m anufacturing, storage, and shipping as biomaterial encapsulation provides vaccine stability and improved shelf life. Additionally, vaccine particles can be engineered to be
20 modular and multifunctional, targeting specific antigen presenting cells, and provi ding both intracellular and extracellular delivery of immuno modulatory agents. In this body of work, we rationally designed and engineered a DC targeting, two component synthetic microparticle system with encapsulated antigen and immuno modulatory agents that direct tolerogenic immune cell phenotypes and promote antigen specific protection from T1D development in non obese diabetic mice (Figure 1 1 ) Ultimately, the goal is to develop a novel synthetic microparticle vaccine that is easily administered and capable of prevention and reversal of the onset of type 1 diabetes. This work is unique and represents the archetype of this microparticle vaccine system. Type 1 Diabetes (Significance) Type 1 Diabetes (T1D) is an autoimmune disease characterized by T cell mediated destruction of insulin cells . The aggressive infiltration and inflammation of the pancreas by auto reactive cells leads to an inability to secrete insulin, lack of homeostatic control of glucose levels and ultimately patient dependence on exogenous insulin sources for survival . Recently, lack of glycemic control has been linked to micro and ma crovascular complications, hypertension and dyslipidemia . In the US only, more than 1 million persons are afflicted by this disease which frequent onsets in early adolescence and reduces life expectancy by 10 15 years [8,9]. Various epidemiological s tudies have confirmed the incidence of T1D is increasing at an average rate of 3% per year, especially in the 14 and under age group . The socioeconomic impact of T1D, in terms of personal, reduced productivity and related health care cost, is astronom ical. In 2002, it was estimated that 132 billion was spent on health care costs related to diabetes [6,8]. Although only a fraction of this sum is attributed to T1D, it is still quite economically burdensome. For instance, children faced
21 with a lifetime of glucose monitoring and insulin injections have twice the health care costs than children without T1D [8,10]. Traditionally, life long insulin replacement via various delivery methods (subcutaneous, oral, nasal) has been used to manage glucose levels in T 1D patients [11 13]. However, the failure of insulin replacement to continuously sustain euglycemia in conjunction with associated physiologic complications (e.g. hypoglycemia, obesity) and excessive cost of care has led to the development of innovative ne w therapies for T1D within the last 30 years [7,14] Newer approaches to re establish homeostatic conditions in patients afflicted by T1D include organ and insulin producing cell transplantation and generation of immunological tolerance to auto reactive d iabetogenic epitopes [2,15]. Currently, pancreas transplantation is recognized as the term insulin independence. However, the associated risks of surgical procedures, long term immunosuppressive drug treatment and donor availability limit the success of pancreas replacement therapy. Similar problems plague insulin producin g cell transplantation therapy [15,16]. A more attractive strategy to regenerative medicine is the induc tion of tolerance to islet auto reactive antigens in T1D patients. Of particular interest to our group is DC based immunotherapy. In this personalized medical procedure, blood derived monocytes are manipulated ex vivo to produce antigen specific tolerogen ic DCs which are then re However, this procedure has a number of drawbacks including inconsistency with DC maturation, donor specificity, ex vivo stability, shelf life, and cost restrict these approaches. This work atte mpts to
22 address these inadequacies by developing a robust biomimetic material and b iomolecular approach to prevent T1D onset PLGA as a Vaccine Carrier Poly (L lactide co glycolide ; PLGA) is a polymer composed of two hydroxy acid monomers lactic and glyco lic acids. It has been sanctioned by the F ood and D rug A dministration for use in humans in various medical applications, including single injection vaccine formulations, because of its biodegradability, biocompatibility, mechanical properties, release kine tics, ease of fabrication and customizability [18,19] PLGA can be formulated into nanoparticles, microparticles of multiple geometries by fabrication techniques including double emulsion, coacervation and spray drying methods [19 23] PLGA polymers can be converted into insoluble microsphere s of defined size (1 7m) that are ideal for phagocytosis by macrophages and DCs [24,25]. Upon phagocytosis, PLGA is hydrolyzed and loaded antigen is processed by both MHC pathways and cross presented stimulating both T helper and CTL cells [25 28]. The degradation rate and release kinetics can be tuned by altering polymer molecular weight, peptide loading and terminal end group ( CHO or COOH ) [20,29,30]. The biodegradability and biocompatibility properties of PLGA are attributed to the enzymatic and non enzymatic hydrolytic cleavage of PLGA upon contact with biological fluids that produces lactic and glycolic acids which can be entered into the Krebs cycle and catabolized into CO 2 and H 2 O [31,32] Microparticle Injection Route of Administration Poly (lactide co glycolide) microparticula te systems are under serious consideration for in situ delivery of vaccine components including DNA, antigen and immuno modulatory agents [21,24,29,33]. At a size range between 1 and 7 MPs
23 cannot freely drain into the lymphatic system following injecti on, but are instead cleared from the site by circulating APCs including dendritic cells [34,35]. Above this size range, MPs are un phagocytosable but can release their contents extracellularly to invading APCs as well as surrounding cells . In light o f this, it is important to consider the route of administration for MP injections as this parameter will have decided effects on drug delivery efficiency and safety. Incidentally, only a few studies have explored the trafficking of phagocytosable PLGA MPs in vivo Newman et al. investigated how the type of phagocytic cell involved in PLGA MP uptake varies with MP injection site. This report demonstrated that MPs delivered intraperitoneally (i.p.) in mice are predominantly intercepted by macrophages, whereas intrademal (i.d.) injection results in more MP associated DCs in draining lymph nodes (LNs) . Similarly, Lewis et al demonstrated that phagocytosable PLGA MPs injected subcutaneously (s.c.) are more likely to be trafficked to draining LNs by DCs than macrophages over a 72 h period post injection . Moreover, the injection site also affects the destination of trafficked MPs. For instance, in preliminary MP accumulation studies by Phillips et al ., polymeric microspheres injected s.c. were shown to dr ain to the pancreatic LN based on the proximity of the injection site to that LN . These observations were instructive in considering the site of injection of MPs for the live animal studies in this work. Tolerogenic Dendritic Cells Dendritic cells (DCs) were first discovered in 1973 by Steinman and Collette. They have traditionally been defined as the ubiquitous immune cell type exhibiting stellate morphology that system [39,40] Thei presenting cells (APCs) has been well documented. A plethora of reports have outlined the key qualities of DCs as
24 including: (i) an ability to uptake and transport antigen (Ag) from peripheral tissues to T cell zones is marked by upregulation of major histocompatibility complex II (MHC II), co stimulatory surface molecules (e.g. CD80, CD83, CD86) and loss of adhesion molecules and uptake assoc iated molecules (CD25, CD54); (iii) Ag processing and presentation on both MHC I and MHC II complexes (cross presentation); (iv) T cell interaction and stimulation. Moreover, attention has been paid to the role of DCs as strong, n atural immunological adjuv ants [41,42]. More recently, it has been recognized that DCs also play a critical role in central tolerance and the maintenance of peripheral tolerance [43,44]. The race is now on to utilize the tolerogenic potential of DCs as therapeutic agents for autoimmune diseases and transplant rejection. The mechanisms by wh autoimmune protection have yet to be clearly defined. However, evidence has emerged that gives some insight and support for proposed mechanisms such as deletion of self reactive T cells mediated by thymic DCs (central toler ance), and T cell anergy induction and generation of regulatory T cells (Treg cells) for peripheral tolerance [39,42] Classic immunobiology describes the basis of T cell anergy to be the lack of a second required signal in the interaction between APCs an d nave T cells. The delivery of signal 1 (MHC I or II peptide complex to TCR ) in the absence of signal 2, usually costimulatory molecules (CD80, 86) or soluble cytokines (IL 12, TNF (signal 3) results in an antigen specific non responsive T cell [45, 46]. In addition to anergy, peripheral tolerance is maintained by induction of antigen specific CD4+ CD25+ FoxP3+ suppressor T cells (Tregs) [47,48] Depletion of this
25 subset of T cells has been shown to accelerate and induce autoimmunity in various anim al models [49 51]. Researchers postulated that Tregs are thymic CD4+ T cells with selection and developed into CD 25+ T cells. Suppression of effector T cells is accomplished by t he ability of Tregs to impair antigen presentation by mature DCs. However, the crucial step in Treg mediated immuno suppression is generation of Tregs and this is initiated by dendritic cells of definite phenotypes  A number of biological and pharmac eutical agents, including thymic stromal lymphopoietin (TSLP), IL 10 and transforming growth factor beta1 (TGF capacity to instigate CD25+ T cell proliferation and differentiation from CD25 T cells [52,53] The maturation state of these DCs is also important and it has been demonstrated that Treg Phenotypical characteristics of tolerogenic DCs include: increase expression of surface markers programm ed death ligand (PDL ) 1 and PDL2, immunoglobulin like transcript (ILT) 3 and ILT4; and reduced surface MHC II and co stimulatory molecules (CD80/ 86). Functionally, tolerogenic DCs show increased production of anti inflammatory cytokines (e.g. IL 10 ) and in doleamine 2, 3 dioxygenase (IDO ) [54,55] Another important concept to consider is the contribution of apoptotic cells to are taken up by phagocytes via a repertoire CR4). Ligation of apoptotic cells to these receptors initiate immunosuppressive signaling cascades in DCs and pro mote tolerogenic DC phenot ypes [49 51].
26 T olerogenic DC s, therefore, offer a number of immunosuppressive modalities with potential for autoimmune protection. The different modalities include: (i) obstruction of cell surface costimulatory molecules either by inhibition of expressio n or direct interaction (e.g. CTLA 4); (ii) suppression of proliferation of Ag specific T cells (e.g. I n doleamine 2, 3 deoxygenase); (iii) deletion of Ag specific T cells (e.g. FasL, TRAIL). Administration of either biological agents like cytokines (IL 10, TGF intestinal peptide (VIP), calcitonin gene related peptide]), and vitamin D3 or pharmacological drugs as aspirin, corticosteroids, cyclosporine, rapamyc in, and deoxyspergualin may be therapeutically beneficial. These factors have been shown to induce DCs c apable of generating CD25+, Fox P3+ T cells (Tregs) and further halt or reverse autoimmunity in animal models . We elaborate further on some tolerance inducing factors of interest below. Tolerance inducing Agents There have been a number of biological and pharmacological agents, acting on DCs, which have been described to hold therapeutic benefit. Researchers have demonstrated that a dministration of eit her biological agents like cytokines (IL 10, TGF ), and vitamin s (A, D3 ) or pharmacological drugs such as rapamycin are effective for DC immunotherapy applications  Agents with Intracellular Targets Rapamycin (a.k.a., sirolimus) is a drug known to a ffect DC suppression in multiple modalities. Rapamycin is an immunophilin ligand which obstructs downstream signaling from m ammalian targets of rapamycin (m TOR), a ubiquitous protein involved in signal transduction pathways for cellula r activation and pro liferation [4,56]. Rapamycin treated
27 DCs have been shown to inhibit allogenic T cell responses. The observed effect is believed to be due to reduced IL 1 2 and further IFN the active metabolite of vitamin D3 has been shown to have immunosuppressive effects on DCs. Piemonti et al  can: (1) pre vent differentiation of bone marrow derived monocytes into DCs; (2) inhibit maturation of LPS and TNF conditioned immature DCs (inhibition of increased MHCII and co stimulatory molecule expr ession) [59,60]. The downstream effect of this inhibition was r educed T cell proliferation and hyporesponsiveness in a n antigen non specific manner. Griffin et al. and function by binding to an intracellular receptor protein known as vitamin D receptor (VDR) which binds to vitamin D receptor element (VDRE). This couple can block NF fixation and transcription of genes involved in DC maturation including IL 12p40 . Vitamin A treated DCs have b een shown to induce FoxP 3+ regulatory T cells from nave CD 4+ T cells in the presence of TGF occurs is yet to be resolved but studies have shown that retinoic acid (RA), a derivative of vitamin A increases the convers ion of nave T cells to Tregs by DCs exposed to TGF Agents with Extracellular Targets Transforming growth factor beta 1 (TGF pleiotropic cytokine s. TGF I, co stimulatory molecules (CD 40, 80, 86) and inflammatory cytokines (IL 1, 6, 8, and TNF d production of indoleamine 2,3 dioxygenase ( IDO ) has also been observed  Further, associa tion of TGF molecule with the transforming growth factor beta receptor ( TGF ) results in activation of PI3K Akt and non canonical NF
28 pathways which culminates in IDO expression. Induction of Ag specific Tregs as well as Ag specific deletion of effe ctor T cells (apoptosis) has been observed in T cells exposed to TGF treated DCs [62,65]. Interleukin 10 is an anti inflammatory cytokine secreted by Th2 cells, B cells, dendritic cells, macrophages, and keratinocytes. I nterleukin 10 treated DCs show r educed expression of MHC II, co stimulatory molecules (CD 80, 86), intracellular adhesion molecules (ICAM 1) and inflammatory cytokines (IL 1, 6, 8, TNF mechanism by which this occurs has yet to be described. The downstream effect of IL 10 treated DCs on T cells is antigen specific T cell anergy.  Granulocyte macrophage colony stimulating factor (GM CSF) is a critical mediator in the differentiation and development of myeloid DCs that is effective in low doses; resulting in two types of toleroge nic DCs: High and low CD45RO+ with low expression of MHC II, co stimulatory molecules (CD 80, 86), intracellular adhesion molecules (ICAM 1) and inflammatory cytokines (IL 1, 6, 8, TNF L1. These DCs have been shown to induce Tregs wi th increase secretion of IL 10 [67 69]. Non activating Dendritic Cell Targeting Agents and process information, and deliver the message to T cells for a guided response of the adap tive immune system  To facilitate this function DCs are equip ped with a plethora surface receptors (including LOX C type lectins and complement receptors) for recognition, uptake and DC priming via intracellular signaling  Scientists have begun to exploit this feature of DCs by incorp orating targeting moieties, such as antibodies against surface receptors, with active metabolites and drug delivery systems . However, most of these studies
29 integrated DC targeting moieties that enhanced DC adjuvant activity for immunogenic or cancer applications. For non activating applications, DC receptors that do not trigger immuno stimulatory pathways, or that are tolerance inducing, are a ppropriate. In the CD11c and DEC 205 antibodies and P D2 peptide, we ha ve identified t hree candidates with the potential of not only directing efficient DC specific phagocytosis but also enhanc ing antigen presentation via the CD11c/CD 18 and DEC 205 receptors [72 74]. The CD11c/ CD18 protein by leukocy tes particularly myeloid DCs . Since this molecule is abundantly present on the surfaces of DCs, it provides a rational choice of receptor for DC targeting Targeting of DCs via the CD11c antibodies has been shown to enhance humoral responses in mice [ 75]. In addition to the use of antibodies, DC specific targeting through the CD11c surface receptor may also be effected through the use of receptor binding peptides. P D2 (VTLTYEFAAGPRG) is a synthetic peptide derived from domain 2 of the extracellular po rtion of intracellular adhesion molecule 4 (ICAM 4) . ICAM 4 as well as CD11a/C D18, CD11b/CD18 and CD11c/CD18 . CD11c is prominent on the surface of DCs and Castro et al  confirmed its efficacy as an immuno target for delivery of antigen to DCs for directe d CD4 and CD8 T cell responses. While Gahmberg et al  have demonstrated the avidity of the ICAM 4 derived peptide P D2 for CD11c/CD18 receptor It is therefore expected that P D2 may also be an effective tool for targeted delivery of antigen and tolerogenic factor loaded microparticles through this receptor Similarly DEC 205 (CD205), a hallmark for DCs, has been demonstrated to be an efficient immu no target for improving both immunog enic and tolerogenic responses
30 . Studies have shown that antigen conjugated anti DEC 205 delivery to immature DCs results in antigen specific T cell anergy (reversible) as well as Treg induction. These antigen DEC205 antibody conjugates have also been shown to prevent and reverse the onset of Type 1 diabetes in mouse models . Another prospective receptor for non activating, DC targeting is Although not unique to DC s receptor was believed to be involved in recognition, uptake and i nitiation of intracellular signaling along a tolerogenic pathway . Later studies resolved its function to that of recognition and engulfment of ACs. It was also revealed that its interaction with the thrombospondin receptor (CD36) and anionic sites on ACs may be responsible for anti inflammatory signaling. The affinity of RGD integrin has been well documented [81 85]. It follows that surface modification of microparticles with RGD ligands could potentially improv e microparticle uptake as well as prime DC phenotype along a tolerance inducing pathway.
31 Figure 1 1. Two classes of injectable MPs will be fabricated. A .) Pha gocytosable: Surface modified Ag delivering particles, loaded with pharmacological agents targeting intracellular receptors. PLGA particles provide delivery of antigen to endosomes for processing and MHC II loading as well as endosomal escape for cross p resentation on MHC I molecules and cytosolic deliv ery of pharmacological agents. B .) Un Phagocytosable: Biological factors targeting cell surface receptors are incorporated into large, un phagocytosable particles. These factors act as DC chemoattractants as well as condition DC phenotype. Figure 1 2. Mo l ec ular structure of Poly(lactic co glycolic acid) X represents the number of repeating lactic acid units and y represents the number of repeating glycolic acid units A .) Phagocytosable Particles: Targeting DCs, Delivering Antigen and Immunosuppressive Drug: Surface Modifications: P D2, Dec 205 Ab, CD11c Ab Size: 1 2 m Encapsulated: Rapamycin, Vitamin D3 Retinoic Acid Encapsulated: Rapamycin Insulin Peptide Dual Microparticle System for Dendritic Cell Recruitment, Conditioning & Targeted Ag/Drug Delivery B .) Un Phagocytosable Particles to Locally Deliver DC C hemokine & Pro Tolerogenic Biological Agent : Encapsulated Factor: TGF 1 IL 10 and GM CSF Size: 35 m
32 Figure 1 3 Dendritic c ell lineage. This schematic illustrates the lymphoid related and myeloid related dendrtitic cell developemtn pathways. Adapted from Shortman et al Stem Cells; 1997. Figure 1 4 Molecular structure of Rapamycin
33 Figure 1 5 Molecular structure dihydroxyvitamin D3 Figure 1 6. Molecular structure of all trans retinoic acid
34 CHAPTER 2 MICROPARTICLE SURFAC E MODIFICATIONS TARG ETING DENDRITIC CELL S FOR NON ACTIVATING APPLICATI ONS Introduct ory Remarks Poly (lactic co glycolic acid) (PLGA) particulate systems have gained widespread attention as viable option s for vaccine delivery in the last quarter century . PLGA particles at the micron and sub micron sizes are contemporary immuno therapy tools being used to deliver antigen [86,87] adjuvant  DNA [89 91] and pharmacological drugs [92,93] PLGA as a biomaterial has been extensively characterized and has been shown to demonstrate qualities such as biocompatibility, biodegradability [29,94]. Additionally, PLGA particulate systems offer control of size and shape of the delivery system, hydrophobicity, loading and release kinetics of a wide range of biomolecules, modulation of immunogenicity, antigen processing and presentation. Furthermore, PLGA particulate matter provide capability for surface func tionalization . These qualities combined make PLGA microparticulate systems ideal for vaccine delivery to antigen presenting cells (APCs) including dendritic cells (DCs). First discovered in 1973 by the Steinman group, it is now well understood that DCs are directly involved in initiation and modulation of T cell and B cell immunity . Dendritic c ells are the most efficient antigen presenting cells due to their exceptional ability to uptake, process and present antigen [41,96,97]. More recently, i t has been recognized that DCs play a critical role in central tolerance and maintenance of peripheral tolerance. The implication is that through DCs, the direction and magnitude of immune response can be manipulated. Therefore, DCs present a therapeutic t arget for modulation of autoimmune diseases and transplant rejection .
35 The versatility of DCs to guide immune responses is attributed to their lineage and, maturation state . Immature dendritic cells (iDCs) circulate throughout the body and are abl They are equipped with a wide array of endocytic and phagocytic surface receptors that recognize a host of molecules including proteins, lipids, sugars, glycoproteins, glycolipi ds and oligonucleotides [98,99]. Notably, the receptor type engaged during phagocytosis by DCs directs subsequent change in maturation . Researchers have sought to exploit these traits by incorporating targeting molecules such as pathogen associated mo lecular patterns, and antibodies against surface receptors in conjunction with proteins, polymeric particles and other drug carriers [71,100,101]. These approaches are intended to augment drug uptake by DCs as well as bolster adjuvant activity for increase d immunogenicity [100,102 104]. However, there are numerous applications in which targeting factors to DCs in a non stimulating context is perceived to be desirable such as microparticle (MP) based vaccines correcting T1D . For non stimulatory applicat ions, DC receptors that do not trigger immuno stimulatory pathways, or that are tolerance inducing, are appropriate. The endocytic receptor, DEC 205 (CD205) represents one such potent ial candidate for non activating DC targeting. DEC 205 is an integral mem brane protein highly expressed on the surface of DCs found in lymphoid areas critical for immunity and tolerance . It is a member of the C type lectin family which binds carbohydrates and mediates endocytosis . Considerable effort has gone towards t argeting DCs via DEC 205 antibodies and single chain fragment variables (scFv ) [71,79,102]. Bonifaz et al showed that proteins targeted to this receptor improve antigen presentation by 100
36 fold . Further, DEC 205 targeting has been linked with DC abil ity to induce tolerance in vitro as well as in animal models [105,106]. Therefore, iDCs can possibly be primed along a tolerogenic pathway through targeting of the DEC 205 receptor. The implications of this cannot be overstated if the goal is the developme nt of a DC targeting MP vaccine for autoimmune diseases. Another surface receptor abundantly present on DCs, which provides a rational choice for DC targeting, is the CD11c surface molecule. The CD11c/CD18 protein is part of the family of 2 integrins expr essed by DCs [41,107]. Targeting of DCs via the CD11c antibodies has been shown to en hance humoral responses in mice [41,75,107]. In addition to the use of antibodies, DC specific targeting through the CD11c surface receptor may also be effected through th e use of receptor binding peptides. The P D2 peptide is derived from the Ig like domain 2 of intercellular adhesion molecule 4 (ICAM 4 ) . All four members of the 2 integrin family have a strong binding affinity for ICAM 4 which has been shown to be involved in erythrophagocytosis a process thought to be involved in self recognition and immune homeostasis [77,108]. While we are not aware of any work which directl y provides evidence that P D2 peptide could enhance DC phagocytosis, blocking studies by Ihanus et al highlight the high affinity that P D2 peptide has for CD11c, and motivate investigation for use in MP targeting . The use of targeting peptides has t he benefit that, unlike whole antibodies, they causing Fc receptors and are less expensive to produce than antibodies . The goal of coupling DC targeting ligands to the surface of MPs is to provide increased payload of drug /biological/antigen delivered, thereby improving response and reducing the number of administrations required. Our approach is unique in that the DC
37 targeting ligands investigated here may function in a non activating manner, which may prove useful for app lications such as immunotherapies to correct autoimmune diseases or promote transplant tolerance. Experimental Procedure Experimental Animals Female, C57Bl/6 mice aged 6 weeks old were purchased from t he University of Florida Animal Care Services (ACS) (Ga inesville, FL). All animals were housed in specific free environment conditions in University of Florida ACS facilities and used in accordance with detailed experimental protocols approved by The University of Florida Institutional Animal Care and Use Comm ittee (IACUC). Preparation of Fluorescent Microparticles A 50:50 polymer composition of poly (d lactide co glycolide) (PLGA; av erage molecular weight ~ 44,000 g/mol) in methylene chloride (Lactel, AL, USA) was used to generate MPs. Poly vinyl alcohol (PVA; molecular weight ~ 100,000 g/mol) was purchased from Fisher Scientific (NJ, USA) and was used as an emulsion stabilizer. Distilled water (DiH 2 O) was used as the aqueous phase to form the emulsions while methylene chloride (Fisher Scientific, NJ, USA) was used as the organic solvent to dissolve PLGA polymer. Microparticles were formed using a standard oil water solvent evaporation technique  Briefly, 100 mg of PLGA polymer was dissolved in methylene chloride at 5% w/v ratio. Fluorescent dye (either rh odamine 6g [Sigma Aldrich, MO,USA], 1,1' dioctadecyl 3,3,3',3' tetramethylindodicarbocyanine, 4 chlorobenzenesulfonate salt [DiD; Invitrogen, Karlsruhe, Germany] or Anthracenecarboxylic acid [ANC; Fluka, Buchs, Switzerland]) was directly loaded into 2 mL o f the 5% PLGA in methylene chloride solution and
38 emulsified at 35,000 rpm for 180 s using a tissue miser homogenizer (Fisher Scientific, NJ, USA) to form a primary emulsion. The primary emulsion was added to 2 mL of 5% PVA solution in DiH 2 O and the homogen izing was continued at 19,500 rpm for 60 s. This was added to 30 mL of 0.5% PVA solution. The particles thus formed were agitated using a magnetic stirrer (Fisher Scientific, NJ, USA) for 24 h to evaporate residual methylene chloride. The remaining solutio n was centrifuged at 10,000 x g for 10 min to collect MPs which were subsequently washed three times with DiH 2 O. The water was aspirated from the centrifuged MPs, which were then flash frozen in liquid nitrogen and kept under vacuum in dry ice overnight. T he MPs were stored at 20 o C until used. Reagents whose vendors were not specified were purchased from Sigma Aldrich. Cross linking of Ligands to Microparticles Ligands were tethered to the surface of PLGA MPs by conjugation of unbound available amine gro ups in ligands to free carboxyl terminals of the PLGA polymer using carbodiimide chemistry. The free carboxyl groups on PLGA MPs were activated with a 1 (3 dimethylaminopropyl) 3 ethylcarbodiimide hydrochloride (EDC; Acros Organics, Belgium) and N hydroxys ulfosuccinimide (NHS; Pierce Biotechnology, IL, USA) solution for 15 min while agitating at 50 rpm. The ligand (e.g. Anti mouse CD11c mAb [clone was introduced to the suspension which was shaken vigorously for 16 h. After in cubation, the particles were centrifuged at 10,000 rpm for 10 min and washed twice with phosphate buffered saline (PBS) (Hyclone, UT, USA) and re suspended for use. The peptides, P D2 [amino acid sequence: GGVTLTYQFAAGPRDK] and RGD [amino acid sequence: GG GRGDSPCGGDK] were produced by the University of Florida ICBR peptide synthesis core facility. For quantification purposes, a fluorescent tag (either 5 Carboxytetramethylrhodamine [ 5
39 TAMRA ] or 7 amino 4 methylcoumarin [ MCA ] ) was added to the amino terminal of these peptides. The concentration of the ligands used during the conjugation step was based on 10:1 mole ratio of the ligand to the amount of PLGA polymer present. As a control group for some experiments, polyethylene glycol moieties were adsorbed ont o the surface of MPs by incubating a known mass of PLGA MPs in a 10% Pluronic F127 (BASF) solution for 16 h under gentle agitation. This group was classified as the PEG MP group. Quantification of Cross linked Ligands The method used to quantify conjugate d ligand levels was dependent on ligand type. For peptides, each PLGA MP batch (1 mg dry mass) conjugated with fluorescently tagged polypeptide was suspended in 100 L of 6 mg/ml bovine pancreatic trypsin (MP Biomedicals LLC, Solon, OH) to cleave the fluor escent tag portion of the polypeptide derivative from the surface of the PLGA MPs. Standards containing TAMRA RGD and trypsin in solution were incubated simultaneously. The suspensions were centrifuged to pellet MPs and 75 L of supernatant were transferre d to black, flat bottom, half area, polystyrene 96 well plates (Corning Inc., Corning, NY) and fluorescence quantified using a SpectraMax M5 plate reader (Molecular Devices, LLC). Antibodies tethered onto the surface of PLGA MPs were quantified using a dot blot procedure. Antibody conjugated MPs incubated with 6 mg/mL trypsin overnight. Samples were then boiled i n 2% SDS 15 min, cooled and spotted onto a polyvinylidene difluoride ( PVDF ) membrane (BioRad). Standards consisting of serial dilutions of the tryp sinized antibody in 2% SDS were also spotted onto the PVDF membrane. The spotted membranes were allowed to dry for 3 h at room temperature and blocked
40 blocking solution (5% milk casein, 0.2% Tween 20, 0.02% sodium azide in P BS). Blocking solution was then decanted off and 20 mL of the antibody solution (1:10,000 dilutions of alkaline phosphatase tagged antibody raised against the tethered antibody) was incubated with the membrane at room temperature for 1 h with gentle shakin g. The antibody solution was then removed and three 20 ml washes with TBS tween (20 mM Tris pH 7.6, 0.8% NaCl, 0.1% Tween 20 in deionized water) were performed to remove unbound antibody. The membrane was then washed with 20 mL of PBS to remove residual TB S tween solution. The bottom of a light shielded container was covered with 1 mL of an alkaline phosphatase colorimetric was laid face down onto the substrate. The membrane was incubated at room temperature in the substrate for 25 min then rinsed with DI water. A Molecular Dynamics Storm 850 Imager was used to determine image fluorescence ( 480/520 nm excitation/emission) that was analyzed with Axio Vision software. Densitometric means were taken by recording average pixel brightness in the spot and were background corrected. Antibody levels were compared to protein antibody standards for quantification. Potential of Ligand modified Microparticles The MP s ize distribution was measured by a Microtrac Nanotrac Dynamic Light Scattering Particle Analyzer (Microtrac, Montgomery, PA). The zeta potential of ligand conjugated MPs was determined using a Brookhaven ZetaPlus zeta potential analyzer (Brookhaven Instrum ents Corp., NY, USA). For each experimental coating and control, three samples were analyzed at room temperature in distilled water.
41 Dendritic Cell and Macrophage Cell Culture 12 week old, female, C57BL6/j and Non Obese Diabetic (NOD) mice in accordance with guidelines approved by University of Florida using a modified 10 day protocol. For DC culture, mice were euthanized by CO 2 asphyxiation followed by cervical dislocation and tibias and femurs were harvested for isolating marrow cells. The marrow cells were obtained by flushing the shaft of the bones with a 25g needle using RPMI medium (MP Biomedicals, OH USA) containing 1% fetal bovine serum (Lonza, Walkersville, MD) and 1% penicillin streptomycin (Hyclone) and mixed to make a homogenous suspension. The suspension was then strained using 70 m cell strainers (Becton Dickinson, NJ, USA) and cells were col lected at 1300 rpm for 7 min. The red blood cells (RBCs) were removed by lysing with ACK lysis buffer (Lonza, Walkersville, MD) followed by centrifugation at 1500 rpm for 5 min to recover leukocytes. Leukocytes were then re suspended in DMEM/F 12 with L glutamine (Cellgro, Herndon, VA), 10% fetal bovine serum, 1% sodium pyruvate (Lonza, Walkersville, MD), 1% non essential amino acids (Lonza, Walkersville, MD), 1% penicillin streptomycin (Hyclone) and 20 ng/ml GM CSF (R&D systems, MN, USA; DC media) and plate d on tissue culture flasks for 2 d in order to remove adherent cells. At 2 d the floating cells were transferred to low attachment plates and cultured in fresh DC media for expansion of DC precursor cells. At 7 d, cells were transferred to tissue culture plates t o allow for DC adhesion and proliferation. At 10 d, they were lifted from tissue culture plates and used. CSF (Millipore, MA, USA) .
42 Internalization of Microparticles Phagocytosis microscopy (Olympus DSU IX81 Spinning Disc, MA, USA). Twenty thousand cells per well were cultured in LabTek (Nunc, Roskilde, Denmark) eight well chamber glass slides one day prior to being fe d surface modified rhodamine loaded MPs at a 10:1 ratio (MPs cells). After incubation at 37 o C for 1 h, un phagocytosed particles were removed by washing three times with 1X PBS and cells were fixed using 4% paraformaldehyde at room temperature for 10 min. The morphology of the cell was elucidated by staining with Oregon Green 488 phalloidin (Invitrogen, Karlsruhe, Germany) while the nucleus was highlighted by Hoechst 33342 nucleic acid stain (Invitrogen, Karlsruhe, Germany). Dendritic Cell Activation and Cytokine Analysis DC maturation was quantified by measuring cell surface marker levels using flow cytometry. Briefly, DCs were lifted by incubating with 5 mM Na2EDTA solution in 1 M PBS solution at 37 o C for 20 min. Dendritic cells were then washed with 1% fetal bovine serum in PBS and incubated with antibodies raised against CD16 BD Pharmingen, CA, USA) for 40 min at 4 o C against CD80 (clone 16 C II (clone BD Pharmingen) for 40 min at 4 o C. Species specific isotypes were used as controls. Data acquisition was perfor med using (FACScalibur, Becton Dickinson, NJ, USA) flow cytometry and the geo metric fluorescent intensities determined. More than 10,000 events were acquired for each sample and data analysis was performed using FCS Express version 3 (De Novo Software, Los Angeles, CA).
43 Cell culture supernatants were collected after 24 h of cell cu lture with various surface modified MPs, centrifuged to remove any cell debris and stored at 20 o C until analysis. The IL 12 cytokine subunit, IL 12p40, and IL 10 cytokine production was analyzed using sandwich enzyme linked immunosorbant assay (ELISA) ki ts (Becton Dickinson, NJ, USA) according to manufacturer's directions. Cell Viability To examine the potential for cytotoxicity of the surface modified PLGA MPs, a lactate dehydrogenase kit (Roche,Mannheim, Germany) was used per manufacturer instructions. In brief, 1 x 10 6 cells were plated in 6 well tissue culture plates. After being exposed to the various surface modified PLGA MPs for 24 h, the conditioned media were collected and centrifuged to remove debris and the supernatants were stored at 20 o C unti l further measurement. Dendritic cells were lysed by adding 10% Triton X 100 Dehydroge nase activity in supernatants and controls were determined by incubation with the chromogenic substrate at 37 o C for 1 h, and the optical density value was detected at 490 nm by a microplate reader (MRX II, Dynex technologies, VA, USA). The relative cytotox icity of each treatment was calculated by finding the difference between Single Cell and Mixed Culture Preferential Phagocytosis Studies Mi Following a washing to remove free DiD, cells (1 x 10 5 cells in 0.2 ml) were co cultured
44 wit h various surface modified rhodamine loaded MPs for 1 h while being gently agitated at 50 rpm at 37 o C in a 2 mM dextrose solution. Microparticles outnumbered cells by a 10:1 ratio. Flow cytometric analysis was then carried out to determine the mean fluores cent intensity of cells in the rhodamine channel as a measure of phagocytic activity. Controls including cell only and MP only suspensions were also assessed by flow cytometry. A similar method was used to study preferential phagocytosis in a mixed culture of labeled with either calcein (Invitrogen) DCs or DiD Experimental and control ANC loaded MPs were added to cells in a 10:1 ratio. These suspe nsions were a gitated at 50 rpm for 1 h at 37 o C and flow cytometric analysis to determine the mean fluorescent intensity of cells in the ANC channel after incubation as a measure of MP uptake levels. The appropriate controls including cell only and MP only suspensions were also assessed by flow cytometry. In order to block Fc receptors from binding the Fc portion of surface immobilized antibodies, Fc Block (anti CD16/32, clone 2.4G2) was added to the suspensions that contained MPs modified with antibodies. A ll experiments were repeated using three separate preparations with three replicates each time for each ligand modified MP group. Antigen Presentation by Dendritic Cells After Phagocytosis of Surface modified Microparticles NOD DCs (2.5 x 10 4 / well) were c o incubated with surface modified, 1040 55 mimetope loaded MPs, as well as the relevant control treatments, in a 96 well tissue culture plate for 1 h in 2 mM dextrose at 37 o C. MPs outnumbered NOD DCs by 100:1.
45 After thoroughly washing away all un phagocyt osed and unbound MPs, NOD BDC2.5 CD4+ T cells (1.25 x 10 5 / well) were added to each well and incubated at 37 o C for 3 d. Bromodeoxyuridine (BrdU) (kit from Beckton Dickinson) was added to the culture for the last 4 h. T cells were then immunofluorescently stained for BrdU according to proliferation for the different treatments. The proliferation index which is indactive of functional antigen presentation is the ratio of the perce ntage of BrdU positive cells for each treatment to that of the Unmodified, 1040 55 loaded MP group. In order to determine how prolonged antigen presentation was impacted by MP surface modification, MP treated DCs after washing were left in DC culture media for 4 d. Subsequent to this period, NOD BDC2.5 CD4+ T cells were added for a 3 day mixed lymphocyte coupling (MLC) followed by BrdU inoculation and staining for quantification of proliferation. Uptake and Translocation of Microparticles to Lymph Nodes Thi s experiment was designed to determine the targeting efficacy of our ligand modified MPs in an in vivo environment. Female, 6 week old C57Bl/6 mice were divided into 4 experimental groups (based on MP surface ligand type) and three control groups r each group). Each animal from the experimental groups received a subcutaneous injection into the footpad of both hind limbs, containing 0.5 mg of ligand conjugated DiD loaded MPs suspended in 5 PBS. The animals in the control groups were : unmodified DiD loaded MPs, 50 loaded MPs (which are too large to be phagocytosed), and u nmodified unloaded MPs. The 50 DiD loaded MP was included as a control to locally release DiD staining cells at the
46 injection site without being taken u p in order to determine if this effect contributed to the number of DiD positive cells translocated to the LN. At 24, 48 and 72 h post injection, mice were sacrificed by CO 2 asphyxiation and cervical dislocation, and the draining popliteal lymph nodes fr om each hind limb harvested and prepared into single cell suspensions. Cells were then prepped for flow cytometry by incubation at 4 o C for 0.5 h in a cocktail of antibodies specific for DCs (anti F4/80, clone A3 1) and T cells (anti CD3, clone 17A2). All antibodies were from BD Biosciences Pharmingen (CA, USA) except anti F4/80 that was from AbD Serotec (Oxf ord, UK). Appropriate isotypes were used for each antibody species as negative controls. Following staining, multi color flow cytometry was used to determine the fraction of each cell type stained with the DiD dye as a measure of the extent particle uptake and Significance Testing Statistical analyses were performed using general linear nested model ANOVA. test was subsequently performed to make pa ir wise comparisons. Differences were considered significant when p San Jose, CA). Results We characterized surface modified MPs, determining size distribution, ligand surface loading and zeta potenti als. PLGA MPs were prepared via a double emulsion solvent evaporation technique. Fabricated MPs were determined to have an average
47 representative plot shown in Figure 2 1A Conjugation of ligands to the MP surface did not alter the size distribution of the particles (data not shown). Ligands were tethered to the surface of PLGA MPs. Carboxyl groups present at the MP surface were activated by EDC/ NHS and coupled to primary amine groups on the decorating molecules (except PEG, which was surface adsorbed). Surface modification of MPs by PEG, DEC 205 and CD11c anti bodies, P D2 and RGD peptides was characterized by zeta potential analysis. Unmodified PLGA MPs demonstrated a neg ative zeta potential (~ 46 mV). Surface conjugation with CD11c antibody further reduced the zeta potential to approximately 59 mV. Conversely coating with PEG masked the negative surface charge to 20 mV. These observations are consistent with similar pu blished studies . Conjugation of Anti DEC 205, P D2 and RGD peptides only slightly increase the surface charge (~ 37 to 42 mV). We verified antibody conjugation by enzymatic cleaving of the proteins. Cleaved antibodies were spotted onto PDVF membra nes and quantified by dot blot analysis. Peptide conjugation was also validated by enzymatic cleavage of fluorescently tagged peptides from the particle surface followed by detection using a plate reader. The surface loading of the CD11c Ab, DEC 205 Ab, P D2 peptide and RGD peptide were revealed to be substantial at 109 ng/mg, 88 ng/mg, 4359 ng/mg and 19 ng/mg PLGA respectively as tabulated in Figure 2 1B It was also determined, that the surface density of all ligands was substantially increased by crosslin king. The surface density of the CD11c Ab, DEC 205 Ab, P D2 peptide and RGD peptide, in the absence of the cross linker, was determined to be 77 ng/mg, 60 ng/mg, 3373 ng/mg and 11 ng/mg PLGA respectively.
48 Confocal laser scanning microscopy was used to con firm the internalization of actin while a nucleic acid stain highlighted the nucleus and rhodamine loaded MPs were used to show particle engulfment. Figure 2 2 is a represe ntative confocal microscopic image of the DC cytoskeleton (green) surrounding the rhodamine loaded MP (orange) in both the x y and x z planes, confirming MP internalization. To assess whether our MP surface modifications were relevant for non activating a pplications, we examined their effect on DC maturation. Specifically, we investigated whether or not the various ligand grafted MPs increased expression of the DC maturation markers, CD83, CD86 and MHC II on bone marrow derived iDCs. The levels of express ion of CD83, CD86 and MHCII on DCs co cultured with any of the surface grafted MPs were found to be not statistically (p value > 0.05) different to that of iDCs and representative dot plots are shown ( Figure 2 3 ). In contrast, DCs challenged with LPS as a positive control, show significantly higher expression levels of CD83, CD86 and MHCII. These results suggest these ligand grafted MP modifications do not activate DCs. Furthermore, we demonstrated that there is no DC toxicity associated with MP co incubati on (p value > 0.05) ( Figure 2 4 ). In order to investigate the uptake of MPs surface modified with ligands by DCs and labeled MPs in suspension, serum free. Fc Block (anti CD16/32, clone 2.4G2) was included to the antibody modified MP groups to block recognition by Fc receptors in order to drive uptake by the targeted receptors for. Uptake levels were based on the intensity of MP fluorescence associated with cells.
49 Phagocytosis of MPs by DCs was altered based on the surface modification of the MP ( Figure 2 5A ). All surface modifications, with the exception of the PEG and RGD modified MPs, demonstrated the capacity to significantly increase MP uptake by DCs compared to the unmodified MP. The antibody groups, anti CD11 c and anti DEC 205 both significantly improved DC uptake by ~50% compared to unmodified MP. The P D2 peptide enhanced DC phagocytosis the most, doubling the number of MPs phagocytosed over the unmodified MPs. modified MP s was also investigated. MPs surface conjugated with the P D2 peptide substantially increased phagocytosis by ~ 40% over uptake levels for unmodified MPs. All other surface modifications yielded similar levels of MP uptake as the unmodified. Because in vi vo can compete for MP uptake, we were interested in cells have equal acces s to MPs. We found that under these conditions, DCs take up uptake is influenced by ligand surface modification. Similar trends, based on the type of surface modification, seen for uptake in both single cell suspension uptake systems were observed for uptake in the mixed culture competitive uptake system. Anti CD11c and anti DEC 205 antibodies and P D2 peptide significantly improved uptake for both take of unmodified MPs. Moreover, differences in uptake specificity were observed, suggesting that compared to other treatments, the anti DEC 205 antibody MP modification provided both high levels of uptake and
50 specificity of DC targeting. In contrast, whi le MPs surface modified with anti CD11c antibody and P D2 demonstrated high levels of uptake, DC selectivity was low in this in vitro model In order to determine efficacy of DC antigen presentation following phagocytosis of antigen loaded MPs, NOD derived DCs cells were allowed to take up ligand grafted MPs encapsulating the peptide antigen, 1040 55 mimetope, then subsequently incubated with NOD BDC2.5 T cells. The NOD BDC2.5 mouse is engineered with T cell receptors that are specifically engaged by the 104 0 55 mimetope. Upon binding the 1040 55 peptide (e.g. when presented on APCs), T cells are stimulated to proliferate. In our experimental setup, MPs loaded with 1040 55 peptide were incubated with DCs for 1 h, unbound MPs removed, and then DCs were either immediately co cultured with NOD BDC 2.5 T cells, or were cultured for 4 d before the addition of NOD B DC 2.5 T cells. By comparing the 4 d delayed experiment to the no delay test, the ability for MP loading to provide prolonged antigen presentation by DCs to T cells was investigated ( Figure 2 7 ). Regarding the early antigen presentation of the no delay experiment, the P D2 modified MPs was the only group that significantly improved T cell response above the level observed for unmodified, 1040 55 peptide loaded MPs ( Figure 2 7A ). The positive control, a soluble bolus with the equivalent amount of peptide encapsulated in the MPs was included. This control provoked the largest T cell response, over 2 fold higher than the P D2 modification, while unloaded MPs showed minimal T cell proliferation. Investigating prolonged antigen presentation by the inclusion of a 4 d delay, MP surface modification was found to impact the response. The incubation delay resulted in
51 a dramatic decline in the response of T cells to the soluble antigenic peptide control. Most MP modifications maintained a low magnitude T cell response similar to that seen without the incubation delay. Only the P D2 peptide conjugated MP loaded with antigen significantly increased proliferation ( Figur e 2 7B ). The unloaded, unmodified MP group was included as a control to demonstrate that T cell proliferation was indeed in response to the encapsulation of 1040 55 peptide in other MP modifications. For applications targeting phagocytes in vivo it is cri tical to investigate the effect of MP surface modifications using an in vivo model. We therefore used a mouse footpad injection model to determine the rate and extent of surface modified MP translocation from the injection site to proximal lymph nodes, via loaded MPs were surface modified and injected into the footpad of mice. Subsequently, proximal draining lymph nodes were excised and analyzed for cell MP co localization. The percent of MP + cells harvested from draining lymph nodes was used as a measure staining positive for either CD11c or F4/80, respectively. At 24 h after injection, no modification showed distinction in improving trafficking to the draining lymph node ( Figure 2 8A ). However, by 48 h, lymph node harvested DCs recovered from mice injected with anti CD11c and P D2 modified MPs displayed a marked increase in the percent of MP+ DCs to 4.8 % MP + and 6.2% MP + respectively compared to 2% MP + for significance only for the CD11c antibody modification, which increased the number of cells with MPs to 2% MP + compared to the 0.5% MP + seen for unmodified MPs. Afte r 72 h the P D2 modified particles substantially improved trafficking to draining lymph nodes
52 for DCs (4.4% MP + + ) cells compared to the controls (1.8% MP + and 0.8% MP + respectively). Unexpectedly, the isotype antibody for CD11c also impr oved the number of MP + trafficked DCs at 72 h implicating the role of DCs Fc receptors at this time point. This illustrates the influence the choice of antibody species and IgG type have when using whole antibodies for in vivo targeting. In addition to elu cidating the extent of MP trafficking, this time series study also contextualizes the impact of MP surface modification on the kinetics of MP trafficking. These data strongly suggest that the rate of uptake and translocation of MPs is greatly superior when MPs are surface modified, primarily with the P D2 peptide and to a lesser extent the CD11c antibody, for DCs ( Figure 2 8B Figure 2 8C ) over a 3 day period. The CD11c antibody which peaks at 48 h but then drops to control levels at 72 h. The P D2 MP also shows improved uptake and trafficking for DCs which peaks at 48 h but then drops to control D2 MP maintains increasing levels throughout the experimental time period. In general, these in vivo results correlate well with the in vitro results, except that the modification with the anti DEC 205 antibody did not translate to uptake and trafficking levels as high as indicated in vitro This in vivo study also illustrates differences in the kinetics of uptake and number of MP + CD11c + cells occurs at 48 h. For F4/80 + cells, there is no peak for the duration of the study for an y of the modifications with the exception of P D2 grafted MPs in the study time period. These findings corroborate reports of slower lymph node .
53 Impact of Study PLGA particulate systems have been identified as a valuabl e immuno therapeutic tool. Particles at the micron and sub micron level allow for targeted delivery of a plethora of immuno modulating agents to critical elements of the immune system [29,86]. Targeted delivery of factors to DCs is of particular interest a s DCs play a pivotal role in the activation and maintenance of suppressive networks within the immune system . Efforts at controlling DC polarity for mitigation of autoimmune disorders, such as type 1 diabetes (T1D) have intensified in the past decade . efficient at antigen presentation and therefore may be considered to be less potent at manipulation of the immune system [41,113]. Notably, researchers hav e begun to incorporate PLGA MPs as a cell immuno modulatory system into DC immunotherapy [25,93]. Phillips and associates  demonstrated prevention of T1D in NOD mice by polymeric microspheres loaded with anti sense oligonucleotides that passively targe ted DCs by virtue of being micron range particulate, and which were demonstrated to manipulate the immuno regulatory function of DCs. Others have shown that active targeting of DCs using surface modified particulate systems improves DC uptake and trafficki ng, and escalating downstream immuno stimulatory responses, primarily for cancer vaccine applications . Active targeting of MPs is accomplished by surface ligation of ligands that specifically bind molecules on the surface of DCs. Prior work has demon strated efficacious targeting of MPs to DC receptors such as toll v 3 and v 5 integrins . However, binding these receptors can result in DC activation. For example, it is well established that toll like receptors and CD40 recep tor are stimulatory
54 in nature . Additionally, we have recently shown that DC adhesion to various v integrins are also activating [116 118]. Similarly, others have shown that targeting MPs v integrins results in DC activation . Given the stimulatory pathways induced by these receptors, other candidates which are either non inflammatory or pro tolerogenic are of interest for non activating applications. Ligands th at target DC surface receptors such as the integrin CD11c/CD18 and the DEC 205 receptor have been identified as potential candidates for this goal. We were therefore interested in the use of antibodies against CD11c and DEC 205 as well as the CD11c binding peptide, P D2 peptide as ligands in this class. Both CD11c and DEC 205 antibodies have been previously coupled to antigen loaded liposomes for targeting of DCs to enhance protection from tumor in mice challenged with malignant melanoma cells [102,119]. Bo nifaz et al  and Birkholz et al  conjugated DEC 205 monoclonal antibody and anti DEC 205 single chain fragment variable directly to antigenic proteins for targeting to DCs to improve antigen specific immune stimulation. More recently, Fahmy and associates reported grafting of anti DEC 205 to PLGA nanoparticles and its effect on DC uptake and function. Their study demonstrated that surface grafted DEC 205 nanoparticles induce IL 10 expression but are only modestly effective in improving nanoparti cle internalization by DCs . As the popularity of particulate systems as contemporary medical drug delivery devices increases, the need for particle optimization arises (including active targeting, release kinetics, protein repulsion, etc). This study begins to address these parameters, principally for applications requiring targeting to phagocytes while maintaining a non activated state. Another consideration is that MP modification
55 with targeting peptides instead of antibodies is a particularly attra ctive candidate as it will not target Fc receptors and has a lower production cost . To promote phagocytosis, we fabricated our particles with a range of 0.5 2.5 m in size with an average diameter of 1.15 m and confocal microscopy confirmed that the MPs were readily taken up by DCs. Below this micron size range, particles can be taken up by pinocytosis which is not limited to antigen presenting cells. In this regard, nanoparticles are therefore not as selective a s microparticles for phagocytes [12 4,125]. Well established EDC/NHS chemistry was used to ligate ligands to the surfaces of PLGA MPs. Verification of conjugation was accomplished by peptide and protein quantitative methods, and corroborated by zeta potential measurement. These results show that a substantial amount of each molecule is immobilized on the surface of the MP, at levels comparable to those previously reported [115,126]. Differences in uptake observed between the different surface modifications of MPs can be attributed to factors affecting the activity of the immobilized ligand including the quantity of immobilized ligand, the specific receptor targeted, the site of ligand binding to the receptor (i.e., does binding activate/de activate the receptor) and the effective binding affin ity of the immobilized ligand [113,115,127]. These factors can explain why modifications in our hands may act differently from prior reports, and their potential influence over our results is discussed further below. In vitro testing to determine the stimu latory capacity of MP modifications showed there is no significant change in the expression of DC activation markers investigated with any of the surface modified MPs. Further, neither the modified or unmodified MPs showed any toxic effects. These outcomes are important considerations for designing
56 DC targeting MPs for applications such as vaccines for amelioration of autoimmune disorders. We also determined that secretion of cytokines, IL 12 and IL 10, does not vary significantly for DCs exposed to any of the MP modifications (data not shown). With regard to DEC 205 MPs, this result is seemingly contrary to work by the Fahmy group which described an increase in DC secretion of IL 10 upon DEC 205 nanoparticle exposure . This disparity may be explained by the fact that their reported surface density was 50 fold greater than the density measured on our DEC 205 MP, the size of their particle was much smaller at 200 250 nm, and the DEC 205 presentation on their particle was different, consisting of palmit ate avidin molecules incorporated into the surface of the particle binding a biotinylated DEC 205 antibody. was initially evaluated in vitro The CD11c and DEC 205 antibody modi fied and P D2 peptide surface functionalized MPs provided significant enhancement of DC phagocytosis while CD11c and P quan tification methods revealed that the surface density of the RGD peptide was 19 ng/ mg which equates to approximately 11,500 fmol/cm 2 Petrie et al  reported that an RGD surface density of approximately 2,000 fmol/cm 2 on solid substrates promoted muri ne adhesion. Therefore, it was expected our RGD modified MPs should encourage MP phagocytosis. Given that our RGD modification did not improve uptake may suggest that peptide presentation in our formulation was sub optimal. Alternatively, increasing MP pha gocytosis may simply require higher threshold levels of RGD surface density
57 than levels required only for adhesion, as Corre and associates  reported that microspheres functionalized with 20 phagocytosis. Whilst ligand quantity and presentation are limiting factors, differences in so influence [129,130]. Notably, these receptor levels and activation states can certain ly differ between in vitro and in vivo scenarios. Particulate based antigen delivered to APCs can serve as an intracellular antigen depot providing prolonged antigen presentation due to sustained release [86,131]. We investigated MP surface modification t o increase intracellular stores of antigen thereby increasing prolonged presentation of antigen. We determined that at an initial time point, ligand directed MP uptake influences antigen presentation significantly for the P D2 peptide ligand and to a lesse r extent and significance level (p <0.09) for the DEC 205 antibody modified MP group. Further, we demonstrated that microparticulate systems allow for prolonged, continuous presentation of antigen, and that ligand surface modification, particularly with P D2 peptide, increases the level of this protracted presentation. This is important, because taking into account time considerations after subcutaneous injection for APC antigen interception, uptake and translocation, antigen administered in soluble form ca n be diluted and degraded before effective functional interaction by APCs c an occur in T cell rich regions . This result indicates controlled release packaging of antigen in targeted PLGA MPs can overcome this issue. In vitro studies provided a well co ntrolled platform to identify the best candidates for DC uptake. Furthermore, these results are important for applications intending to enhance in vitro / ex vivo loading of APCs using polymeric microspheres [98,131].
58 However, applications utilizing in vivo targeting of MPs require in vivo experimental systems. Based on the low uptake levels in vitro we eliminated the RGD modified group and compared in vivo uptake and translocation to draining lymph node after subcutaneous injection of our best modification s. The P D2 peptide conjugated MPs in particular, and to a lesser extent, the anti CD11c conjugated MPs were effective in increasing trafficking of MPs to the draining lymph node by both DCs (CD11c+) and in vitro result, the anti DEC 205 conjugated MPs failed to alter MP trafficking by DCs. It is expected that the DCs present at the murine subcutaneous injection site possess a different repertoire of receptors (expression levels and activation states) than those of the bone marrow derived cells used in our in vitro experiments. For instance, Jefford et al demonstrated that peripheral blood DCs generated in vivo differed from in vitro monocyte derived DCs in phenotype, migratory capacity and T cell stimulation . Furthermore, DC subpopulations in vivo show phenotypic diversity [133,134]. While Steinman and colleagues  reported that antigen coupled DEC 205 antibody injected intravenously successfully targeted lymphoid DCs, it is possible that the DCs present at the subcutaneous injection site have a lower expression of DEC 205 . Consequently, subcutaneously injected MPs modi fied with the anti DEC 205 antibody for non activating DC targeting may prove ineffective.
59 Figure 2 1. Size and surface density characterization of surface modified microparticles. (A) Microparticle size was determined to be 1 DLS. (B) PLGA MPs surface modified with various ligands and PEG were characterized by zeta potential measurement and fluorescent quantification techniques to confirm surface loading. Zeta potential data shown represent the mean standard error for five readings of each sample. The ligand surface loading data shown represent mean standard deviation for three replicates of each sample. (A) (B) Microparticle Diameter (nm) % Volume
60 Figure 2 2. Internalization of surface modified MPs by dendritic cells (DCs) is demonstrated via confocal microscopy. DCs were incubated rhodamine loaded surface modified MPs (orange) for 1 h, fixed and stained for the actin cytoskeleton (green) and the nucleus (blue). This image shows the x y optical section as well as x z project ion, showing engulfment of the MPs
61 Figure 2 3. Immature dendritic cell phenotype is maintained even after exposure to ligand conjugated MP modifications. Dendritic cells were incubated with various surface modified MPs for 24h at 37 o C. MPs outnumbered cells by a 10:1 ratio. Cells were then extensively washed to remove unbound MPs and stained with fluorescently tagged anti CD80, CD86, MHC II and CD11c antibodies. Flow cytometric assessment revealed the levels of expression for these molecules on DCs. Im mature DCs (iDCs) and +LPS groups are included as negative and positive controls, respectively. Representative plots from three separate experiments are shown
62 Surface Ligand/ Condition CD80 v. CD86 CD80 v. MHC II iDC (neg. control) +LPS (pos. control) CD11c Ab MP Dec 205 Ab MP
63 P D2 MP RGD MP PEG MP Unmodified MP
64 Figure 2 4. Co culture of surface modified MPs with dendritic cells (DCs) has negligible effects on cell viability. Dendritic cells were incubated with variou s surface modified MPs for 24 h at 37 o C. Microparticles outnumbered cells by a 10:1 ratio. Cell supernatants were collected after 24 h for a cytotoxicity assay. The percentage viability for each specimen was calculated based on live/dead cell controls. No differences between groups were found by ANOVA (p > 0.1) Viability (%) Microparticle Type
65 Figure 2 5. Microparticle (MP) phagocytosis by (A) dendritic cells (DCs) and (B) with surface modified rhodamine loaded MPs for 1 h with agitation at 37 o C. Microparticles outnumbered cells by a 10 :1 ratio. Flow cytometric analysis was carried out to determine the mean fluorescent intensity of cells (in the rhodamine channel) after incubation as a measure of MP uptake. Pair wise significant difference from cell uptake of unmodified PLGA microparticl es is denoted by the symbol (p value < 0.05). (A) (B)
66 Figure 2 6. cultured with various surface modified AMC loaded MPs for 1 h at 37 o C while being gently agitated. Microparticles o utnumbered cells by a 10:1 ratio. Flow cytometric analysis was carried out to determine the mean fluorescent of MP uptake level. Pair wise significant difference of MP uptake betwe en Microparticle Uptake (RFU) Microparticle Type
67 Figure 2 7. Functional antigen presentation improved by surface modified MPs. (A) P D2 peptide surface modified PLGA MPs loaded with antigenic peptide leads to in creased antigen presentation and (B) improved prolonged antigen presentation. Non obese diabetic mouse DCs were cultured with either MPs loaded with 1040 55 mimitope or soluble peptide (as a control, at an equal mass to that loaded in the MPs) at a 100:1 M P to DC ratio for 1 h, followed by washing to remove unbound MPs. Subsequently, freshly isolated BDC2.5 CD4+ T cells were added to culture wells (either immediately (early) or after 4 d (prolonged)) and co cultured a 3 day mixed lymphocyte reaction. T cell proliferation was then measured using a BrdU proliferation assay as a measure of functional antigen presentation. Data shown represent the mean proliferation indices standard error (n = 3). Pair wise significant difference from the unmodified PLGA MP g roup (by ANOVA and Tukey Significance Test) is denoted by the symbol (p value < 0.05). (B) (A)
68 Figure 2 8. Phagocytosis and trafficking of microparticles (MPs) by dendritic cells (DCs) Female, 6 week old C57Bl/6 mice were given footpad injections containing 0.5 mg of ligand conjugated DiD MPs al ong with control MPs. At different time points (24 h, 48 h, 72 h) the draining lymph nodes were recovered, followed by flow cytometry analysis. The percentage of each cell type MP+ (DiD ch annel) was determined and analyzed by ANOVA. Microparticle uptake wise comparisons using the Tukey Test showing sign ificant difference in comparison to unmodified MPs (p < 0.05).
69 (B) (A)
71 CHAPTER 3 MICROSPHERE TECHNOLO GY OFFERS COMBINATOR IAL AND LOCAL DRUG DELIVERY TO DENDRITI C CELLS FOR ENHANCEMENT OF SUPPR ESSIVE FUNCTION Introduct ory Remarks Dendritic Cells (DCs) are specialized phagocytic cells whose functions are influential in the control the innate and adaptive immune responses . It is well established that DCs are the most potent antige n presentation cells (APCs) and inducers of T cell activity . Circulating through most tissues, DCs possess qualities that allow them to (a) rapidly recognize foreign from self entities, (b) efficiently intercept and process antigen, and (c) display an tigen in the context of the major histocompatibility complex (MHC) to the T cell receptor on T cells [41,95]. Accordingly, attention has been paid to the role of DCs as strong, natural immunological adjuvants . However, it is now recognized that DCs a re also instrumental in the initiation of suppressive networks required for maintenance of peripheral tolerance in the body . Tolerogenic dendritic cells (tDCs), therefore, are emerging as immunotherapeutic candidates for the induction of protective im munity . Of particular interest is the application of DC immunotherapy for the abrogation of autoimmune diseases and immune mediated transplant rejection. This personalized medical procedure normally entails the use of either in vitro or ex vivo deri ved DCs manipulated with tolerance inducing factors (e.g. oligonucleotides, corticosteroids, cytokines) to produce tDCs that are re [137,138]. Tolerogenic dendritic cells initiate a number of modalities that can lead to hyp oresponsiveness by effector immune cells. These include anergic pathways, regulatory T cell generation (Treg), as well as effector T cell deletion .
72 A number of pharmacological and biological agents have been used to generate tolerance inducin g DCs. Studies have shown that administration of either biological agents like the cytokines, interleukin 10 (IL 10) and transforming growth factor beta 1 (TGF trans retinoic acid (RA) are the rapeutically effective for this application [139 142]. Interleukin 10 is a pleiotrophic immunomodulatory cytokine expressed and secreted by helper T cells and antigen presenting cells (APCs ) . It has been shown that IL 10 binding to the extracellular subunit of the IL 10 receptor, initiates inhibitory signal cascades in immune cells. Interleukin 10 induces T cell hyporesponsiveness by inhibiting T cell proliferation and cytokine production . Additionally, IL 10, in the presence of TGF cell activity . Its effect on DCs has also been well documented. IL 10 treated DCs show reduced cytokine production (including IL 1, IL 10 itself, IL 12, TNF), and reduced MHCII and co stimulatory molecule (e.g. CD80, 86) which correlated with the ability of these DCs to inhibit activation when couple with allogenic T cells [145,146]. Interestingly, mixed DC/ T cell populations treated wi th either RAPA or vitamin D3 resulted in IL 10 producing T cell populations rich in Tr 1 and Th2 phenotypes. It is uncertain the role played by either of the cell types in this observation [147,148]. A nother multi modal cytokine with down regulatory effect s on immune cells is TGF array of lymphoid cells, particularly DCs and T cells . Interestingly, these cells are not only sources for TGF his immunosuppressive cytokine. Transforming growth factor beta 1 binds to either of three classes of
73 membrane bound or soluble, cytoplasmic transforming growth factor beta receptor. This receptor complex signals through a serine/ threonine kinase domain t o initiate translocation of SMAD molecules to the nucleus. In T cells, the results of this signaling network have been well characterized and include proliferative and functional effects that are immunosuppressive [150,151]. For example, TGF STAT pathway in T cells thereby impeding the expression of the IL 2 and IL 12 receptors which are both integral to inflammatory responses by this cell type . The scope of impact of TGF but it has been demonstrated TGF modulatory effects are inhibitory in nature and lead to a tolerogenic DC phenotype that is capable of inducing antigen specific CD25+ FoxP3+ T cells (induced Tregs) from CD+4 nave T cell population [52,53]. Not ably, TGF 1 DCs produce indoleamine 2,3 di oxgenase (IDO), an enzyme involved in tryptophan catabolism and responsible for the generation of kynurenines thought to be key factors in the spread . The pharmacologic drug, rapam ycin (RAPA) is a macrolide antibiotic derived from the filamentous bacter ium, Streptomyces hygroscopicus . Rapamycin exerts potent immunosuppressive action on immune cells including T cells and DCs. Rapamycin complexes with the intracellular FKBP12 pr otein and functions to selectively disrupt the mammalian target of rapamycin (MTOR) associated biochemical pathways . The scope of action is broad and includes modulation of cell proliferation, differentiation, migration and survival [152 154]. In DCs rapamycin has been implicated for inhibition of cytokine mediated signal transduction. It has been demonstrated that exposure of DCs to RAPA results in reduced expression of MHCII and co stimulatory molecules, LPS
74 resistance as well as inability to stimu late allogenic T cells in vitro . In vivo it has been suggested that RAPA treated DCs are capable of generating CD4+CD25+FoxP3 regulatory T cells in addition to inducing apoptosis of effector T cells resulting in transplant acceptance in various mous e models [140,155]. All trans retinoic acid (RA) is the other immunosuppressive agent of interest. All trans retinoic acid has been shown to modulate both innate and adaptive immune systems. Stephensen et al  demonstrated that Vitamin A can alter the Th1/ Th2 profile in vitro via the retinoid X receptor. Moreover, Elias and colleagues established that the Th17 differentiation is inhibited and FoxP3 Treg development enhanced through RA binding of retinoic acid receptor alpha in nave CD4 T cells . In APCs, RA treatment results in diminished production of inflammatory cytokines such as IFN [141,142]. Further, supplementation of vitamin A and its metabolites in mice have been reported to mitigate autoimmune diseases including type 1 diabetes and en cephalomyelitis [158 160]. It is interesting to note that co administration of these agents produced synergistic effects on DC populations that boost tolerance inducing activity. For example, RA acts a co factor to TGF ociated lymphatic tissue, enhancing their ability to generate FoxP3+ Tregs . Therefore, it is rationale to suggest that co delivery of these factors could have significant therapeutic benefit for autoimmune diseases and transplant rejection applicatio ns. The question do we achieve feasible, concerted delivery of such factors to specific DC subsets under in vivo delivery of combinations of these immunosuppressive factors can be achieved through
75 polymeric microparticulate delivery systems. Combined microparticulate treatment may offer reduction of doses required to produce therapeutic effects and further, harness immunoregulatory effects not realized by equivalent single factor doses. Tar geted, local, continuous delivery of these factors can be accomplished through poly (lactic co glycolic acid) (PLGA) microparticles. Poly (lactic co glycolic acid) particulate systems have been widely established as viable delivery systems for a plethora o f biomolecules, and pharmacological agents [29,33,93,162,163]. Its key qualities include biocompatibility, biodegradability and controlled release kinetics. Additionally, PLGA microparticles can be fabricated to specific micron size range which is importan t for phagocytic cell targeting [29,33]. Particles that are below the micron size range can be taken up by pinocytosis which is not limited to APCs. In contrast, particles above the phagocytosable range (1 their contents to the extracellular envi ronment, whilst microparticles in that size range are readily phagocytosed by APCs and able to deliver their encapsulated agent intracellularly . In this study we exploit this feature, by separately encapsulating RAPA and RA in phagocytosable microparti cles (~1 10 in un phagocytosable incubated bone marrow derived murine DCs with microparticle couples consisting of one of either type of MP and assessed their ability to modulate DC immune function. The goal of our approach is to not only boost immunosuppressive effects observed by single factor administration but also deliver factors at lower safe dosages, which may prove to be practical for treatment of autoimmune diseases and organ transplant reje ction.
76 Experimental Procedure Microparticle Preparation A 50:50 polymer composition of poly(d lactide co glycolide) (PLGA) (MW ~ 44,000 g/mol) in methylene chloride (MC) (Purac) was used to generate MPs. Poly vinyl alcohol (PVA) (MW ~ 100,000 g/mol) w as purchased from Fisher Scientific (NJ, USA) and was used as an emulsion stabilizer. Distilled water (DiH2O) was used as the aqueous phase to form the emulsions while methylene chloride (Fisher Scientific, NJ, USA) was used as the organic solvent to disso lve the PLGA polymer. Microparticles were formed using a standard oil water solvent evaporation technique. To make phagocytosable MPs, 100 mg of PLGA polymer was dissolved in methylene chloride at 5% w/v ratio. Either Rapamycin (RAPA) (LC Laboratories) or All trans Retinoic acid (RA) (Acros Organics) in DMSO was loaded into 2 ml of 5% PLGA solution. This solution was added to 2 mL of 5% PVA solution in DiH2O and homogenized at 35,000 rpm for 180 s using a tissue miser homogenizer (Fisher Scientific, NJ, US A) to form a primary emulsion. This was added to 30 mL of 1% PVA solution. The particles thus formed were agitated using a magnetic stirrer (Fisher Scientific, NJ, USA) for 24 h to evaporate residual methylene chloride. The remaining solution was centrifug ed at 10,000 x g for 10 min to collect MPs which were subsequently washed three times with DiH2O. The water was aspirated from the centrifuged MPs, which were then flash frozen in liquid nitrogen and kept under vacuum in dry ice overnight. The MPs were sto red at 20 o C until used. Un phagocytosable MPs (TGF and IL 10 loaded) (BD Pharmingen) were fabricated using by a double emulsion solvent evaporation technique similar to that described above but with the
77 addition of a second emulsification step and using a vortex ter (Fisher Scientific) instead of a homogenizer. Microparticle Characterization Sizing, Loading, Release Kinetics The size distribution of the MP was measured by the Microtrac Nanotrac Dynamic Light Scattering Particle Analyser (Microtrac, Montgomery, P A). Particle sizes are reported as the average diameter standard deviation (S.D.). The loading efficiency of the phagocytosable MPs was measured by dissolving 100 mg of MPs into 2 mL MC and re precipitating the PLGA with a known volume of methanol (Acro s Organics). The suspension was centrifuged and the supernatant removed to a new tube. Following evaporation, residue remaining in the tube is concentrated in a known, small quantity of DMSO and the solution concentration measured by spectrophotometer. For the second particle type, loading efficiency was measured using a solvent evaporation technique followed by spectrophotometric analysis. The in vitro release kinetics from all MPs was determined as described. Briefly, a known mass of MPs were resuspended in a known volume of phosphate buffer saline (PBS) (Hyclone, UT, USA) containing 2% tween 20 (Acros Organics). These samples were vortexed and placed in a shaking incubator at 37 o C. At regular intervals MPs were pelleted, the supernatant collected and MPs resuspended in an equal, fresh volume of release media. Drug content of the supernatant was determined through spectrophotometric detection. Dendritic Cell Culture and Microparticle incubation 12 week old, female, C57BL6/j mice in accordance with guidelines approved by University of Florida using a modified 10 day protocol. For DC culture, mice are euthanized by CO2 asphyxiation followed by
78 cervical dislocation and tibias and femurs are harvested for isolating marrow cells. The marrow cells were obtained by flushing the shaft of the bones with a 25g needle using RPMI medium (MP Biomedicals, OH, USA) containing 1% fetal bovine serum (Lonza, Walkersvill e, MD) and 1% penicillin streptomycin (Hyclone) and mixed to make a homogenous suspension. The suspension was then strained using 70 m cell strainers (Becton Dickinson, NJ, USA) and cells were collected at 1300 rpm for 7 min. The red blood cells (RBCs) we re removed by lysing with ACK lysis buffer (Lonza, Walkersville, MD) followed by centrifugation at 1500 rpm for 5 min to recover leucocytes. Leucocytes were then re suspended in DMEM/F 12 with L glutamine (Cellgro, Herndon, VA), 10% fetal bovine s erum, 1% sodium pyruvate (Lonza, Walkersville, MD), 1% non essential amino acids (Lonza, Walkersville, MD), 1% penicillin streptomycin (Hyclone) and 20 ng/ mL GM CSF (R&D systems, MN, USA) (DC media) and plate on tissue culture flasks for 2 days in or der to remove adherent cells. At 2 d the floating cells were transferred to low attachment plates and cultured in fresh DC media for expansion of DC precursor cells. At 7 d, cells were transferred to tissue culture plates to allow for DC adhesion and proli feration. At 10 d, they were lifted from tissue culture plates and used for various studies. For these studies, MPs were incubated at 37 o C for a period of 48 h prior to analysis or addition of T cells. Phagocytosable MPs (RAPA MP, RA MP) were added at a 1 0:1 MP to cell ratio, while un phagocytosable MPs (TGF 10 MP) were incubated at a mass that encapsulated the effective concentration of that respective drug for the incubation media volume. Unloaded MPs and the soluble equivalent of released drug s doses were included as controls.
79 Dendritic Cell Phenotype Maturation and Tolerogenic Markers Dendritic cell maturation and tolerogenic molecule expression were quantified by measuring cell surface marker and intracellular cytokine levels by flow cytome try. Following MP incubation, DCs were lifted by incubating with a 5 mM Na2EDTA in PBS solution at 37 o C for 20 min. Dendritic cells were then washed with 1% fetal bovine o C to eptors on DCs. Cells were washed and then stained with antibodies against CD80 (clone 16 A/I E (clone (Millipore) for 30 min at 4 o C For intracellular molecules, cells were fixed, permeabilized and stained with antibodies against IDO (clone 10.1, IgG3) (eBiosciences), IL 10 (JES5 16E3, IgG2b) IL 12 (clone C15.6, IgG1) and IFN isotypes were used for each antibody species as negative controls. Data acquisition was performed using (FACScalibur, Becton Dickinson, NJ, USA) flow cytometry and the geometric fluorescent intensities as well as percent of positively stained cells determined. More than 10, 000 events were acquired for each sample and data analysis was performed using FCS Express version 3 (De Novo Software, Los Angeles, CA). Cell culture supernatants were collected after 48 h of MP incubation, centrifuged to remove any cell debris and stored at 20 o C until analysis. The IFN 10 cytokine production were analyzed using sandwich enzyme linked immunosorbant assay (ELISA) kits (Becton Dickinson, NJ, USA) according to manufacturer's directions.
80 CD4+ T cell and Treg Isolation Mouse CD4+ T cells were purified from splenocyte suspensions by negative Mixed Lymphocyte Coup ling (MLC) T cell Suppression and Treg Generation For suppression studies, C57Bl6 DCs (2.5 x 10 4 / well) were co incubated with the different combinations of MPs, as well as the relevant control treatments, in a 96 well tissue culture plate for 72 h at 37 o C in culture media consisting of RPMI 1640 with 10% FBS and 1% penicillin streptomycin (Hyclone). After thoroughly washing away all un phagoctyosed and unbound MPs, BALB/c CD4+ T cells (1.25 x 10 5 / well) were added to each well and incubated at 37 o C for 3 d. Bromodeoxyuridine (BrdU) (kit from Beckton Dickinson) was pulsed into the culture media for the last 4 h. T cells were then cytometry was then used to quantify T ce ll proliferation for the different treatments. A similar method was employed for Treg generation studies with purified CD4+ BALB/c T cells. However, following DC T cell co culture for 72 h, T cells were immunofluorescently stained using anti CD4 PECY7 (clone RM4 5), anti CD25 APC (clone 7D4) and anti Foxp3 PE(FJK 16s) antibodies (BD Pharmingen). Cells were analyzed using the FCS express V3 software (De Novo Software, Los Angeles, CA) Significance Testing Statistical analyses were performed using genera l linear model ANOVA, followed by posthoc assessment using Tukey test to make pair wise comparisons. Differences were considered significant when equal or less than p = 0.05 using Systat (Version 12, Systat Software, Inc., San Jose, CA).
81 Results We success fully fabricated MPs using a single emulsion, solvent evaporation technique for phagocytosable MPs and a double emulsion, solvent extraction method for the un phagocytosable MPs. We characterized these MPs determining their size distributions, loading effi ciencies and in vitro release kinetics. The size range of phagocytosable MP was determined by dynamic light scattering, calculated by volume, MPs and IL 10 MPs were 1. More details on the physical characteristics of the fabricated MPs are reported in Table 1 including the encapsulation efficiencies and the amoun t drug encapsulated in the MPs on a per mass basis. The entrapment efficiencies for RAPA MPs, RA MPs, TGF MPs and IL 10 MPs were 72.2 3.2%, 62.3 6.2%, 61.0 4.9% and 51.9 5.1% respectively. Differences in encapsulation efficiencies reflect not on ly differences in microparticle preparation techniques between phagocytosable and un phagocytosable MPs but also differences in initial loading concentrations and initial loading solution composition. The in vitro release kinetics for all MPs was studied at pH 7.4 in a 2% Tween20 PBS solution. Figure 3 2 shows the release profiles of RAPA MPs (Panel A), RA MPs (Panel B), TGF 10 MPs (Panel D). After 30 d, the cumulative release of RAPA and RA reached 95% and 100% respectively. Whereas, cumulative release for the un phagocytosable M Ps was slower over the course of 30 d only reaching 61% for TGF 10 MPs. The release profiles follow Fickian transport laws in that there is an initial burst release within the first week
82 followed by a more gradual release for the next three weeks. There are differences observed between the release profiles of phagocytosable and non phagocytosable MPs. However, given that the methods used to load the agents as well as the size of the agents are very dissimilar, these differences are ant icipated. Further, there is an unexpected discrepancy between the release profiles of TGF 10 MPs with the later being a more linear release profile, even though they were fabricated similarly and differ only slightly in molecular weights. A p ossible explanation for this difference can be found if we scrutinize the loading solutions of each MP. TGF dissolved in an aqueous solution of PBS, citric acid and BSA whereas IL 10 loading solution consisted of only PBS. The additional acid in the TGF accelerated the initial rate of degradation of the polymer observed. To begin to assess the immunosuppressive ability of our dual MP systems, we studied their effect on DC maturation. More specifically, we investigated the impact du al MP incubation had on the expression of the maturation markers CD80, CD86 and MHC II. Dual MPs were co cultured with DCs for 48 h and the same conditions were applied for the relative controls including untreated DCs (iDCs), unloaded MPs, the soluble e quivalent doses and single MP systems. Figure 3 3A displays the effects of combined RAPA MP and TGF incubation on DC maturation as well as associated controls. Our results show that MHC II remained unchanged statistically for all groups. CD80 expression decreases significantly for the RAPA MP, TGF CD86 surface levels decreased dramatically for RAPA MP, Sol RAPA, TGF RAPA/ TGF 3 3E in which the comprehensive
83 maturation indices are displayed further highlights these c hanges in maturation state for the RAPA/ TGF average of the combined individual molecule maturation indices. For the RAPA/ TGF dual MP system, this value is significantly than that of the iDC, Unloaded MPs and TGF well as the Sol RAPA group is considerably lower than the iDC and Unloaded MPs groups. These results indicate that dual RAPA/ TGF on can have a substantial tolerogenizing effect on DCs via reduction of stimulatory molecules. Overall, the expression of these molecules on DCs treated with RAPA/ TGF significantly lower than all control groups except the TGF We exa mined the effects of RAPA/ IL 10 MPs on the maturation state of DCs. MHC II expression levels varies only slightly amongst various groups. Whereas there are significant reductions in CD80 surface levels for the RAPA MP, and RAPA / IL 10 MPs groups and CD86 surface quantities for the RAPA MP, Sol RAPA and RAPA/ IL 10 MPs groups (Figure 3 3B). Again, we used the CMI as an overall measure of maturation state for the RAPA/ IL 10 MPs and control treatments. Figure 3 3F illustrates that combined treatment signifi cantly reduces the CMI compared to all groups except the RAPA MP and Sol RAPA groups. From this we can deduce that dual delivery of RAPA MPs and IL 10 MPs can appreciably modulate expression of activation molecules on the surfaces of DCs. However, this red uction in stimulatory molecule expression could also be achieved by administration of RAPA MPs or an equivalent dose after 48 h, in static in vitro culture conditions.
84 We then combined TGF effect on MHC II, CD80 and CD86 expression levels. MHC levels are statistically unchanged for all treatments. For CD80, only application of single factor MP groups significantly l owered expression. Similarly, for CD86 only the single factor MPs in addition to the Sol RA groups reduced expression levels dramatically below that of iDC and unloaded MPs groups (Figure 3 3C). The CMI (Figure 3 3G) for these groups reflect our observatio ns for the individual molecule maturation indices. The RA/ TGF MPs group, although it reduces the CMI compared to iDC and Unloaded MPs groups, this change is not meaningful. Interestingly, single MP groups (RA MP and TGF lower the CMI significant ly below that of iDC and Unloaded MPs groups. This may suggest that combined administration of these two types of MPs has a neutralizing, instead of additive, effect of DC maturation state. Finally, we investigated how stimulatory molecules expressions on DCs varied due to treatment with combined RA MP and IL 10 MP treatment as well as associated control groups. Our results show that MHC II level is relatively unaffected compared to those seen for the iDC group. The RA MP group is the only group that signi ficantly reduces CD80 expression levels in comparison to the iDC group. This group as well as the Sol RA group also lowers CD86 surface expression considerably (Figure 3 3D). The overall effect is that stimulatory molecule expression is only reduced for RA MP and Sol RA groups. Disappointingly, the maturation profile for DCs treated with both RA MP and IL 10 MP looks similar to that of the iDC group (Figure 3 3H). Generally, we can conclude from these results that dual MP treatment to DCs either maintains a maturation profile similar to that of iDCs or significantly reduces it to a
85 state that maybe capable of tolerance induction. Unexpectedly, combination MP treatments. In some ca ses, there is a reversal of the effect seen with single MP treatments. For instance, the RA MP and TGF stimulatory molecules expression significantly below that of iDC and unloaded MPs. However, addition of these two gro ups simultaneously to DCs only maintained the expression levels. We also observed that MP treatments have a greater effect on DC maturation state that their comparative soluble dose s. This effect can result from a combination of factors including local/ di rect delivery of agent to cells by MP formulations. To determine the extent to which the MP treated DCs are resistant to stimulative conditions, we exposed DCs to lipopolysaccharide (LPS) for 24 h subsequent to 48 h MP incubation. We considered the express ion of the stimulatory molecules MHC II, CD80 and CD86, to be representative of the maturation state and therefore resistance to LPS stimulation for the assorted combination treatments and accompanying control treatments. To simplify our analysis, we cal culated the comprehensive maturation resistance index (CMRI) for each group which is an average of the individual stimulatory molecules maturation index for each group. Figure 3 4 displays the levels of the CMRI for DCs treated with the various dual MP sys tems and their associated controls. Generally, the combined MP treatment maintained the levels of expression of stimulatory molecules seen for iDCs even after 24 h LPS stimulation. The maturation resistance observed for these dual MP systems were significa ntly different from the increases observed for Sol LPS, Sol LPS + Unloaded MPs, single factor un
86 pha gocytosable MP, single factor soluble equivalent dose (in both phagocytosable and un phagocytosable MPs), and dual factors soluble dose groups. Interestingl y, single factor phagocytosable MP groups showed similar maturation resistance to activating stimuli as the combination MP groups. The common denominator between these categories of treatments is the phagocytosable single factor MP, which may suggest that ingested MPs are co ntinuously releasing agents int r a cellularly which counteracts the external maturation stimuli. Alternatively, the observed maturation resistance for dual MP treatments could be as a result of expression of inhibitory molecules such as im munoglobulin like transcript 3 (Ilt3). To further investigate the phenotype of DCs in response to combinatorial MP treatment, we evaluated the expression of this tolerance inducing molecule. Figure 3 5A demonstrates the effect of combined RAPA MP and TGF 1 MP on Ilt3 expression in DCs. Unexpectedly, RAPA/ TGF the corresponding single factor MP groups (RAPA MP and TGF these groups, treatment of DCs with the soluble dose equivalen t for RAPA MP and RAPA/ TGF that seen for iDCs. RAPA/ Il 10 MPs treatment also dramatically lowered Ilt3 surface abundance on DCs, although not to the level observed for the RAPA MP gro up (Figure 3 5B). The only other combination of MPs that significantly moderated Ilt3 expression on DCs was the RA/ TGF complement of this group also alters Ilt3 to similar expression levels indicating that may be some synergism with this combination of agents in affecting Ilt3 expression (Figure
87 3 5C). Finally, Figure 3 5D illustrates that neither RA/ IL 10 MPs group nor its associated control groups effected noteworthy changes in Ilt3 expression levels on DCs. To determine the suppressive effect of dual MP treated DCs on proliferation of effector T cells, we tested the response of allogenic CD+ T cells to DCs priorly cultured with the assorted dual MP systems and their relevant controls. Briefly, C57Bl6 bo ne marrow derived DCs were incubated for 2 d then unbound MPs and unused soluble agent washed from wells. Subsequently, splen ic CD4+ T cells isolated from BALB/c mice were added to culture wells so that they outnumbered DCs by a 6 to 1 ratio. These cells w ere co cultured for 3 d then inoculated with BrdU. BrdU incorporation was used as a measure of T cell proliferation and the percent of BrdU positive cells for each treatment compared to that for the iDC group to determine the allogenic T cell proliferation index (API). Figure 3 6A demonstrates the extent of suppression for the RAPA/ TGF important to note that Unloaded MPs have a significant effect on T cell proliferation, reducing the proliferation index from a value of 1 for the iDC group to approximately 0.7. Dual RAPA/ TGF levels observed for both the iDC and Unloaded MPs groups. Although the level of suppression o bserved for this MP pair (API ~ 0.21) is significantly higher than that of the TGF (API ~ 0.27). The RAPA/ IL 10 combination of MPs resulted in significant reduction of T cell s timulatory capacity compared to both iDCs and Unloaded MP treated DCs. However, both single factor MP treatments (RAPA MP API ~ 0.27; IL 10 MP API ~ 0.28) also effected similar levels of T cell hyporesponsiveness (Figure 3 6B). Figure 3 6C
88 illustrates the effect of the RA/ TGF treatments, on T cell proliferation. Combined RA/ TGF proliferation the most for all MP formulations. The API for this dual MP treatment is approximately 0.3 which is only slightly lower than that for the RA MP group with an API around 0.36, but significantly less than that of the TGF only combination of MPs that prompted suppression of T cell proliferation to levels significantly lower th an both of its corresponding single factor MPs was the RA/ IL 10 MPs group. The API for this group was approximately 0.19 compared to APIs of 0.36 and 0.28 for the RA MP and IL 10 MP groups respectively. Finally, it should be noted that without an allogeni c DC stimulus, T cell proliferation is minimal and this is represented by the T cell only group. After revelation of the highly suppressive nature of our dual MP treated DCs, we wanted to elucidate the mechanism behind the functional modulation by these D Cs. It is well known that tolerogenic DCs play an integral role in the induction of CD4+ CD25+ FoxP3+ regulatory T cells which are powerfully suppressive in nature.  Therefore, given that our previous experiment demonstrated impressive inhibition of all ogenic T cell proliferation, particularly for the dual MP systems, we assessed if these results were due to generation of Tregs by the various MP formulations. Briefly, MP formulations were incubated with C57Bl6 DCs for 2 d followed by washing to remove un bound MPs. Subs equently, freshly isolated BALB/c CD4+ T cells were added to culture wells and co cultured in a mixed lymphocyte coupling for 3 d. Cells were then immuno stained using antibodies against CD4, CD25 and FoxP3. Figure 3 7A shows the percent of CD25+ FoxP3+ T cells generated for the assorted MP formulations. With the exception of the
89 RA/ TGF comparable to that of the iDC and Unloaded MPs. The data suggest that coupling of these selected agent loaded MPs does not necessarily boost Treg induction. Moreover, the RA/ TGF mitogens, phorbol 12 myristate 13 acetate (PMA)/ ionomycin (Iono) treatment. Finally, the v ast difference observed between the percent of Tregs observed in the coupling of iDCs and allogenic T cells illustrates that, at least under in vitro conditions, DCs are required for induction of Tregs. Impact of Study Over the past decade, exploration of PLGA particulate systems for delivery of a wide range of drugs in therapeutic applications has increased exponentially [29,33,162,163]. In the immunology field, researchers have recognized that the versatility of polymeric particulate systems could be adop ted for DC based immunotherapy  Dendritic cells, the most potent APCs, play a critical role in initiation of adaptive immunity, as well as maintenance of suppressive networks that prevent autoimmunity [40,95]. The underlying dogma is that DCs can guide the direction and scale of adaptive immune responses [40,41,95]. DC based immunotherapy exploits this quality of DCs by attempting to preprogram the DC so as to create a functionality that provokes desired, downstream immune responses that would not be po ssible in vivo under diseased conditions. Typically, in vitro or ex vivo derived DCs are manipulated with tolerance inducing factors (e.g. oligonucleotides, corticosteroids, UV radiation) to produce tDCs that can be re [1 35,164]. In a study by Pedersen et al.  human monocyte derived DCs exposed to a combination of dihydroxyvitamin D induced tolerogenic DCs that secreted
90 IL 10 and, to which allogenic T cells were non responsive Until recently, DCs have been exclusively manipulated ex viv o or in vitro but scientists have begun to realize the advantages of in vivo modulation and are now seeking to employ innovative measures to target drugs to DC populations in vivo [5,98]. include the use of PLGA particulate system that can be tailored to be bimodal and bi functional in actively targeting DCs for delivery of drug payloads. For instance, Jhunjhunwala et al.  demonstrated that PLGA MPs encapsulating rapamycin can modulated DC phenotype and function for trans plant rejection application Our interest in the use of PLGA delivery systems for modulation of DCs lies in the realm of protective autoimmunity, particularly against type 1 diabetes. As proved by Phillips and associates, there is huge potential for thera py of type 1 diabetes through in vivo delivery of drug loaded MPs to DCs and other APCs . We were also aware that most immunomodulatory effects on immune cells occur when a plurality of agents are acting in concert [53,144,161,166]. Therefore, we hypot hesized that PLGA MPs could be used for targeted, local, and parallel delivery of multiple factors to DCs. Further, we believed that combinatorial delivery of immunosuppressive factors would result in synergistic effects and ultimately the generation of a DC with superior tolerogenic features. We tested these suppositions by fabricating PLGA MPs that delivered combinations of either RAPA or RA intracellularly, and either TGF 10 extracellularly to murine bone marrow derived DCs. The aim was to efficiently target the respective DC receptors for these agents. To accomplish this, we would have to prepare tw o classes of MPs: (i) phagocytosable MPs that could deposit their payload intracellularly and (ii) MPs that
91 could not be readily taken up and release agents into the extracellular milieu. Therefore, the first challenge was fabrication of these agent loaded MPs to the appropriate size requirements. We demonstrate that we fabricated phagocytosable MPs with an average diameter between 1.5 contrast, our un in diameter are beyond the dimensional extent to which an APC can phagocytose particulate matter . Further, prepared MPs were effectively loaded with desired immunosuppressive agents as verified using well established pharmaceutical drug and protein detection methods [35,167,168]. The release of the agents from their biodegradable microspheres followed general solute release behavior The general profile shows an initial burst release within the first week followed by steady incremental emission. Alth ough, the release kinetics of our fabricated MPs are not tightly controlled, it is important to note that release profiles can be manipulated through modification of various parameters including polymer molecular weight, lactide to glycolide ratio as well as particle size . Tolerogenic DCs are typically characterized by reduced levels of expression of stimulatory molecules (e.g. MHC II, CD40, CD80, CD86) and conservation of expression levels of inhibitory markers (e.g. Ilt2, Ilt3, Ilt4 ) . In ligh t of this knowledge, we determined the expression levels of CD80, CD86 and MHC II on DCs exposed to our selected combinations of MPs as well as their control MP and equivalent soluble dose formulations. Surprisingly, for all formulations both single and mu ltiple MPs only moderately lowered constitutive expression of MHC II on DCs. Conversely, CD80 and CD86 our results show that levels of expression were significantly moderated to differing degrees based on MP size, encapsulated agent, and combination. The
92 c omprehensive maturation index, an average calculated based on equal weighting of the stimulatory expression for each treatment relative to that of iDC, was used to elucidate and compare the maturation states of DCs of treatments i.e. combined factors MPs vs. single factor MP, or combined factors MPs vs. soluble equivalent release. There are some salient points to be highlighted from this analysis (i) all formulations including the unloaded MP group at least maintained an iDC maturation profile, (ii) MPs with encapsulated agents generally effect greater modulation than their soluble equivalent counterparts and, (iii) combination of single factor MPs does not always lead to cumulative dampening of DC activation markers. Indeed, depending on the agent combi nation, effects seen with single factor MPs can be reversed. MP delivery of agent is expected to prompt greater DC responses as local and direct delivery create higher concentrations of active agents at receptor sites and therefore increased frequency for receptor drug interactions compared to an equivalent soluble bolus . With regard to the first point, a number of authors have reported on the stimulatory nature of PLGA particulates and further, its use as an adjuvant which boosts immunity against ta rgeted antigens [170,171]. While others have described PLGA particles as being immuno inert systems that only function as vehicles . Indeed, our results for expression of positive stimulatory molecules by Unloaded MPs corroborate this finding. Additio nally, there are reports that indicate degradation of PLGA into it constituent monomers, particularly lactic acid, has the effect of downregulating stimulatory molecules on DCs following exposure [172,173]. Paradoxically, our results on LPS resistance of M P treated DCs lend plausibility to this phenomenon.
93 Under diseased conditions, DCs are exposed to external stimuli that dictate an activating phenotype . For intervention of autoimmunity in this activating environment, DCs must therefore have the capa city to resist maturation. Accordingly, we tested our dual MP systems in comparison to single MPs and soluble equivalent dosages to compare their efficacy at engendering LPS resistant DCs. As aforementioned, a most interesting observation was the significa nt diminution in maturation state of DCs treated with Unloaded MPs and soluble LPS in comparison to the Sol LPS group. We interpret these results to be indicative of PLGA degradation which leads to high lactic acid concentrations. Other authors have demons trated the effects of high lactic acid concentration on expression of positive stimulatory molecules in DCs [172,173]. Single factor phagocytosable MP groups, as well as, combination MP groups were the treatments that generally resisted LPS mediated matura tion suggesting that MPs act as long term depots of agent with gradual release intracellularly that prevents upregulation of positive stimulatory molecules on DCs. This is in keeping with observations made by our group and other groups . Another promi nent feature of tDCs is their ability to maintain the levels of expression of inhibitory molecules (e.g. Ilt2, 3, 4) while downregulating stimulatory markers such as CD40, 80, 86 . We investigated the expression of the inhibitory molecule, Ilt3, with the view of further characterizing the tolerogenicity of MP treated DCs compared to iDCs and the other associated controls. Ilt3 is a member of the immunoglobulin super family and signal through immunotyrosine based inhibitory motifs (ITIM) which typically leads to calcium dependent downregulation of NF consequences are lower levels of expression of positive stimulatory molecules as well
94 as induction of anergy and Tregs upon interaction with T cells [174,175]. Disappointingly, the only dual IL 10 combination of MPs. In contrast, the only MP formulation that maintained Ilt3 surface levels on DCs was the IL 10 MP treatment. RAPA MP, and to lesser extents, RA MP and TGF MP formulations al l reduced Ilt3 expression to levels below that seen for the iDC group. These outcomes agree with studies by Fedoric et al and Vlad et al. The former indicates that rapamycin treatment downregulates Ilt3 expression on DCs and further rapamycin treated DCs inhibit T cell proliferative responses [54,176]. While Vlad and colleagues reported that IL 10 exposure induces upregulation of Ilt4, which is closely related to Ilt3, on monocytes derived from HIV patients [175,177]. Summarily, single dose agents at the c oncentrations and in the delivery format used in this study inhibit expression both inflammatory and suppressive surface molecules on DCs. This is further intensified by simultaneous delivery of cytokines by un phagocytosable MPs. Remarkably, inhibition of negative stimulatory molecules did not affect the ability of MP treated DCs to suppress proliferation of allogenic T cells in a 3 d MLR. Our results reveal that exposure of DCs to dual MP systems considerably inhibit activation of allogenic T cell populat ions. Dual MP systems certainly improve the intensity of suppression of proliferation compared to their respective single factor MPs. Indeed, the combination of RA/ IL 10 MPs dramatically reduced the API in comparison to the RA MP and IL 10 MP treatments. The significance of this result cannot be overstated as it validates our initial suppositions. Further, if we consider the full profile of results for the dual RA/IL 10 MPs group, we begin to realize the complexity and multiplicity of
95 mechanisms involved i n suppression of immunogenic T cells by DCs. DC modalities that retard T cell propagation include T cell anergy, generation of regulatory T cells (FoxP3 Tregs, Tr1 cells, TGF secreting T cells), and DC mediated T c ell apoptosis [178 180]. Given that we observe low constitutive expression of activation molecules due to dual MP and single MP treatment, it is credible to attribute this suppr ession to T cell anergy. However, the levels of suppression do not always correlate with the maturation status for our treatments. For instance RAPA/ IL10 combination treatment show a higher CMI than the RAPA MP treatment but lower API. It is therefore con ceivable that other modes of suppression are engaged. To test this theory, we repeated the allogenic MLC and probed for generation of CD25+ FoxP3+ Tregs. This class of regulatory T cells has been implicated for maintenance of local immune suppression throu gh induction of tDCs, effector T cell inactivation and apoptosis . Our results demonstrate that, with the exception of the RA/ TGF MPs group, the CD25+ FoxP3 expressing subpopulation of T cells is relatively constant for all treatments. Therefore the strong levels of suppression we observe for our MP formulations are most likely due to DC hypo stimulatory state and T cell anergy, in addition to other uninvestigated mechanisms like DC mediated T cell apoptosis.
96 Figure 3 1 MP size distribution was determined using dynamic light scattering tec hniques. A) average diameter of phagocytosable microparticles ~ 1.5 B) average diameter of non Table 3 1. MP size and loading char acterization. Table showing sizes and encapsulation efficiencies of PLGA microparticles loaded with biological and pharmacological agents. Biological/ Pharmacologic Agent Average Diameter Amount Used/PLGA (g/100 mg) Encapsulation Efficiency + SD (%) Loading + SD (ng/mg) Rapamycin 2.3 250 72.2 3.2 1804 80 Retinoic Acid 1.6 100 62.3 6.2 623 62 TGF 29.9 6.25 61.0 4.9 38 3 IL 10 30.5 5 51.9 5.1 26 3 (A) (B)
97 Figure 3 2. M P release kinetics In vitro release profiles of (A) Rapamycin, (B) Retinoic acid, (C) TGF 10 in a 2% Tween 20 PBS solution. Error bars on graphs represent standard deviation based on n = 3 measurements for each microparticle type.
98 Figure 3 3. Co culture of dual MP systems result in red uced levels of surface expression of MHC II and costimulatory markers (CD80, CD86) relative to iDCs. Phagocytosable MPs (RAPA MP, RA MP) were added at a 10:1 MP to cell ratio, while un phagocytosable MPs (TGF 10 MP) were incubated at a mass that encapsulated the effective concentration of that respective drug for the incubation media volume. Unloaded MPs and the soluble equivalent of released drugs doses were included as controls. The maturation index for each surface marker (Panels A D) is the ratio of the percent of positive cells for that treatment group to the percent of positive cells for the iDC group. The comprehensive maturation index (Panels E H) represents an average of the maturation indices of the maturation marker for each sample. For the comprehensive maturation, pair wise significant differences from: (a) iDC and Unloaded MP groups are denoted by the symbol; (b) iDC, Unloaded MP and either one of the single factor MP groups are denoted by the ** symbol; (c) iDC, Unloaded MP and both single factor
99 (C) (D) (G) (H) ** (A) (B) (E) (F)
100 Figure 3 4. Co culture of dual MP systems result in DC maturation resistance to LPS stimulation. MPs or soluble treatments were incubated with DCs for 48 h then washed to remove unbound MPs and soluble factors. Subsequently, soluble LPS was added to culture wells at a concentratio mL After 24 h incubation period, DCs were lifted and surface stained with antibodies against maturation markers MHC II, CD80, CD86 and flow cytometry used to determine the levels of expression of these markers. The maturation resistance index (Panels A D) represents an average of the maturation indices of the maturation marker for each sample. For the comprehensive maturation, pair wise significant differences from Sol LPS, Unloaded MPs + Sol LPS and either one of the single factor MP + Sol L PS groups are denoted (A) (B) (C) (D) * * * *
101 Figure 3 5. Co culture of dual MP systems result in differing levels of expression of tolerogenic marker, Ilt3, i n DCs based on combinatorial factor delivery. Phagocytosable MPs (RAPA MP, RA MP) were added at a 10:1 MP to cell ratio, while un phagocytosable MPs (TGF 10 MP) were incubated at a mass that encapsulated the effective concentration of that respec tive drug for the incubation media volume. Unloaded MPs and the soluble equivalent of released drugs doses were included as controls. Pair wise significant differences from the immature DC (iDC) group are denoted by the * (A ) (B ) (C ) (D )
102 Figure 3 6. Co culture of dual MP systems with iDCs resu lt in tolerogenic DCs with different levels of suppressive power. Briefly, different systems of MPs were incubated with C57Bl6 DCs for 2 d followed by washing to remove unbound MPs. Subsequently, freshly isolated Balbc CD4+ T cells were added to culture w ells a nd co cultured in a mixed lymphocyte reaction. T cell proliferation was then measured using a BrdU proliferation assay. Data wise significant differences are denoted by: the sy mbol for comparisons to the iDC group; the ** symbol for comparisons to the iDC and Unloaded MPs groups; the £ symbol for comparisons to the iDC, Unloaded MPs and either one of the single factor groups; the symbol for comparisons to the iDC, Unloaded MPs £ £ £ (A) ** ** ** (B) ** ** £ (C) £ £ (D)
103 Figure 3 7. Co culture of dual MP systems with DCs result in tolerogenic DCs that maintain Treg levels. Briefly, different systems of MPs were incu bated with C57Bl6 DCs for 2 d followed by washing to remove unbound MPs. Subs equently, freshly isolated BALB/c CD4+ T cells were added to culture wells and co cultured in a mixed lymphocyte coupling for 3 d. Cells were then immuno stained using antibodies against CD4, CD25 and FoxP3. Data shown represents the percent of CD4+ CD25+ Fox P3+ T cells as determined by flow cytometry. Pair wise significant differences from the immature DC (iDC)
104 CHAPTER 4 A VITAMIN D3 GM CSF AND TGF 1 ENCAPSULATING DUAL M ICROPARTICLE VACCINE SYSTEM THAT PREVENTS ONSET OF TY PE 1 DIABETES IN NOD MICE Introduct ory Remarks Type 1 Diabetes (T1D) in both humans and non obes e diabetic (NOD) mice is thought to result from a breakdown of self tolerance that is characterized by T cell cell destruction  Ultimately, glucose metabolism is interrupted resulting in the development of life threatening complications su ch as heart disease, renal failure and ketoacidosis . Recently, this failure in homeostatic regulation has been attributed to a deficiency in the number and/ or function of dendritic cells (DCs) and regulatory T cells (Tregs). Boudaly et al demonstrat ed that DCs derived from the NOD strain of mice are deficient not only in quantity but phenotypical and functional capacity compared to control strains  Additionally, Ohnmacht and associates established that constitutive ablation of DCs in mice resul t in lack of protection from spontaneous fatal autoimmunity  These studies correlate well with the commonly held view that DCs play a central role in the maintenance of peripheral self tolerance. DCs are thought to circulate throughout the body, samp ling the extracellular environments and relaying these local conditions back to effector and regulatory T cells  Moreover, these cells are professional antigen presenting cells (APCs) with the capacity to instigate either the inflammatory or anti infl ammatory wings of the adaptive immune system. This direction and magnitude of the response is dependent on the phenotype of the DC either activated for an inflammatory reaction or conversely, tolerogenic for regulatory measures  DCs influence periph eral immune tolerance via a number of modalities including effector T cell anergy and deletion, immune deviation and, expansion and induction of regulatory T
105 cells [178 180] With respect to Tregs, numerous studies have shown that this subpopulation of CD2 5+, FoxP3 expresssing T cells have functional defects and reduced frequency with progression of diabetes in NOD mice  Indeed, a study by Setoguchi et al revealed that removal of CD4+ CD25+ T cell population via IL 2 depletion in mice prone to autoim mune diseases dramatically accelerated disease onset and autoimmune pathology  Other reports detailed that Tregs control diabetogenic aut oimmunity by restraining the actions of pathogenic T cells at the site of inflammation islet cells) subsequent to initial infiltration  phenotype and other tolerogenic features have be identified as the induce rs of the CD25+ FoxP3 T cells that are implicated to protect from the onset of T1D in NOD mice  These findings have inspired a novel, personalized approach to the treatment of autoimmune diseases including T1D, known as dendritic cell based immunother apy. The general approach is that DCs are isolated from a patient and manipulated ex vivo to produce stable tolerogenic DCs which are then re introduced to the patient as cellular vaccines  Ex vivo Manipulation usually involves genetic modification of DCs or exposure to immunosuppressive agents as well as antigen [165,188,189] A particularly attractive feature of the DC based approach is that it allows for antigen specific immunotherapy, an important consideration in tolerance induction and treatment o f autoimmune disease  Although DC based immunotherapy has been developed to the point where it is currently being tested in humans  problems such as the plasticity and complexity of DC maturation, ex vivo stability, shelf life, and cost restrict the widespread application of this therapeutic approach 
106 An intelligent design strategy might focus on in vivo targeting of DCs with injectable, synthetic particulate systems that can deliver vaccine components including immunomodulatory agents. Thi s flexible approach greatly simplifies issues related to manufacturing, storage, and shipping as biomaterial encapsulation provides vaccine stability and improved shelf life  Additionally, microparticle systems can be engineered to simultaneous deliver both prime & boost doses using time release materials (e.g., poly lactide co glycolide), be modular and multifunctional, specifically target DCs, and provide both intracellular and extracellular delivery of immunomodulatory agents [19,29,86,191] Indeed, controlled release of immunosuppressive agents by poly (d lactide co glycolide) (PLGA) particulate systems have begun to be explored as immunotherapeutic tools for treatment of cancer, autoimmunity and transplant related complications [19,192 194] For ins tance, a study by Phillips et al demonstrated PLGA microspheres loaded with anti sense oligonucleotides, for co stimulatory molecule, passively targeted DCs and manipulated their immuno regulatory function. Phillips and associates successfully protected f rom T1D in NOD mice through in vivo targeting with microparticles that genetically modify APCs upon interception  Alternatively, MP systems can potentially modify in vivo DC phenotype by delivering a payload of immunomodulatoy agent(s). Of particular interest to our group, are the potent immunosuppressive agents dihydroxycholecalciferol (Vit D3), transforming growth factor beta 1 (TGF (GM CSF) for this purpose.
107 dihydroxycholecal ciferol is the active metabolite of vitamin D3. It is a steroid hormone that plays an integral role in bone formation as it regulates calcium/ phosphate metabolism  Vit D3 also has immunomodulatory effects, particularly on APCs and T cells that highl y express the vitamin D3 receptor (VDR)  When bound to this intracellular receptor, Vit D3 initiates signaling through the vitamin D responsive elements which are located in the promoter regions of target genes and therefore regulate their expression  In DCs, multi ple studies have revealed that V it D3 binding impairs DC maturation, with reduced expression of MHC II, co stimulatory molecules (CD40, CD80, CD86) and other maturation markers observed upon Vit D3 exposure. Additionally, expression of inflammatory cytokines such as IL 12, is significantly suppressed in DCs when treated with Vit D3 [148,196,197] The downstream effect of this inhibition is T cell anergy and Treg generation [198,199] Vit D3 can also act directly on T cells. In vitro it has been shown to inhibit T cell activation and induce CD25+ FoxP3+ T cells from a nave population. Further, Vit D3 treatment in vivo has been demonstrated to attenuate T cell mediated autoimmunity in animals for type 1 diabetes, experimental allergic en cephalomyelitis, collagen induced arthritis, autoimmune thyroiditis and nephritis [61,200,201] Transforming growth factor is produced and secreted in a latent form by an array of lymphoid cells, particularly DCs and T cells  Interestingly, these cel ls are not only sources for TGF Transforming growth factor beta 1 binds to either of three classes of membrane bound or soluble, cytoplasmic transforming growth factor beta receptor. This r eceptor complex signals through a serine/ threonine kinase domain to initiate translocation of SMAD
108 molecules to the nucleus. In T cells, the results of this signaling network have been well characterized and include proliferative and functional effects th at are immunosuppressive  For example, TGF STAT pathway in T cells thereby impeding the expression of the IL 2 and IL 12 receptors which are both integral to inflammatory responses by this cell type [150,151] The scope of impact of TGF discovered, but it has been demonstrated TGF immunomodulatory effects are inhibitory in nature and lead to a tolerogenic DC phenotype that is capable of inducing CD25+ FoxP3+ T cells (induced Tregs) from CD+4 nave T cell population [52,53] Notably, TG F 1 DCs produce indoleamine 2,3 di oxgenase (IDO), an enzyme involved in tryptophan catabolism and responsible for the generation of kynurenines . Granulocyte macrophage colony stimula ting factor is another plei o trophic cytokine with huge impact on a number of immune cells, particularly APCs. GM CSF is secreted by peripheral tissues under pathological conditions and influences DC recruitment, phagocytic activity, antigen presentation ca pacity and proliferation  Additional, DCs exposed to GM CSF in vitro have impaired maturation capacity in response to stimul ation with TNF  Further evidence that GM CSF plays a role in maintenance of peripheral tolerance was given by studies performed by Ino et al that revealed GM CSF mobilized monocytes inhibited T cell proliferation through IL 10 produ ction and apoptosis of activated T cells [205,206] Indeed, researchers have begun to investigate the ability of GM CSF to modulate DC phenotype and thereby promote peripheral tolerance against autoimmune diseases. Studies by Cheatem et al
109 and Gaudreau et al demonstrated that GM CSF treated semi mature DCs play an integral role in prevention of T1D in NOD mice and further, suggest that they induce IL 10 secreting CD4+ CD25+ Tregs that suppress diabetogenic T cells that promote diabetes development [69,207 ] Herein, we exploit the versatility of PLGA to develop a dual microparticle system for protection from T1D in NOD mice. This dual MP system always comprised of combinations of two phagocytosable MPs either of Vit D3 loaded MPs, antigen loaded MPs (insu lin B peptide, human insulin), or unloaded MP s and two un phagocytosable MPs either of TGF loaded MPs, GM CSF loaded MPs or unloaded MPs. This is novel attempt to develop an easily injectable system that targets DCs and delivers combinatory drug therapy to these specialized cells that play an im portant role in the development of autoimmune diseases as T1D. The primary goal of this study is to prevent T1D in NOD mice using this MP formulation. Experimental Procedure Experimental Animals Female NOD/Ltj, C57Bl/6 and B ALB /c aged 4 8 weeks old were purchased from either The Jackson Laboratories (Bar Harbour, ME) or The University of Florida Animal Care Services (ACS) (Gainesville, FL). All animals were housed in specific free environment conditions in University of Florida ACS facilities and used in accordance with detailed experimental protocols approved by The University of Florida Institutional Animal Care and Use Committee (IACUC). Microparticle Preparation A 50:50 polymer composition of poly(d lactide co glycolide) (PLGA) (MW ~ 44,000 g/mol or 65,000 [Study I only]) in methylene chloride (Purac) was used to generate
110 MPs. Poly vinyl alcohol (PVA) (MW ~ 100,000 g/mol) was purchased from Fisher Scientific (NJ, USA) and was used as an emulsion stabilizer. Distilled water (DiH2O) was used as the aque ous phase to form the emulsions while methylene chloride (Fisher Scientific, NJ, USA) was used as the organic solvent to dissolve PLGA polymer. Microparticles were formed using a standard oil water solvent evaporation technique. Phagocytosable MPs were fa bricated using either single or double emulsion solvent evaporation techniques based on the solubility of the desired drug in organic solvent. Briefly, 100 mg of PLGA polymer was dissolved in methylene chloride at 5% w/v ratio. Vit D3 in dimethyl sulfoxid e (DMSO) (Fisher Scientific, NJ, USA) was loaded into 2 mL of 5% PLGA solution. This solution was added to 2 mL of 5% PVA solution in DiH2O and homogenized at 35,000 rpm for 180 s using a tissue miser homogenizer (Fisher Scientific, NJ, USA) to form a prim ary emulsion. This was added to 30 mL of 1% PVA solution. The particles thus formed were agitated using a magnetic stirrer (Fisher Scientific, NJ, USA) for 24 h to evaporate residual methylene chloride. The remaining solution was centrifuged at 10,000 x g for 10 minutes to collect MPs which were subsequently washed three times with DiH2O. The water was aspirated from the centrifuged MPs, which were then flash frozen in liquid nitrogen and kept under vacuum in dry ice overnight. The MPs were stored at 20 o C until used. For insulin encapsulated MPs, 100 mg of PLGA polymer was dissolved in methylene chloride at 5% w/v ratio. 0.1 mL of Insulin solution (1mg/ mL ) was then added the 5% PLGA solution and homogenized at 35,000 rpm for 120 s to form a primary emuls ion. This emulsion was added to 2 mL of a 5% PVA solution and homogenized again at 19,500 rpm for 60 s to form the secondary emulsion which was
111 transferred to a beaker containing 30 mL of 1% PVA. The particles thus formed were agitated using a magnetic sti rrer (Fisher Scientific, NJ, USA) for 24 h to evaporate residual methylene chloride. The remaining solution was centrifuged at 10,000 x g for 10 minutes to collect MPs which were subsequently washed three times with DiH2O. The water was aspirated from the centrifuged MPs, which were then flash frozen in liquid nitrogen and kept under vacuum in dry ice overnight. The MPs were stored at 20 o C until used. Un phagocytosable MPs (TGF and GM CSF) were fabricated using by a double emulsion solvent evaporation technique similar to that described above but using a vortexter (Fisher Scientific) instead of the tissue miser homogenizer. Either DMSO or distilled water was used to make unloaded MPs. Microparticle Characterization Sizing, Loading, Release Kinetics T he size distributions of MP were measured by the Beckman Coulter LS13320 (Beckman Coulter Inc., Brea, CA) and the Microtrac Nanotrac Dynamic Light Scattering Particle Analyser (Microtrac, Montgomery, PA). The MP diameter is reported as mean standard devi ation (SD). The loading efficiency of Vit D3 MPs was measured by dissolving 100 mg of MPs into 2 mL MC and re precipitating the PLGA with a known volume of methanol (Acros Organics). The suspension was centrifuged and the supernatant removed to a new tube. Following evaporation, residue remaining in the tube is concentrated in a known, small quantity of DMSO and the solution concentration measured by spectrophotometer. For the other MPs (Insulin, TGF CSF), loading efficiency was measured using a solv ent evaporation technique followed by spectrophotometric analysis.
112 The in vitro release kinetics of all MPs were determined as described. Briefly, a known mass of MPs were resuspended in a known volume of phosphate buffer saline (PBS) (Hyclone) containing 2% tween 20 (Acros Organics). These samples were vortexed and placed in a shaking incubator at 37 o C. At regular intervals MPs were pelleted, the supernatant collected and MPs resuspended in an equal, fresh volume of release media. Drug content of the sup ernatant was determined through spectrophotometric detection. Bone Marrow derived DC Culture and MP incubation 12 week old, female, C57BL6/j mice in accordance with guidelines approved by University of Florida using a modified 10 day protocol. For DC culture, mice are euthanized by CO2 asphyxiation followed by cervical dislocation and tibias and femurs are harvested for isolating marrow cells. The marrow cells are obtained by flushing the shaft of the bones wit h a 25g needle using RPMI medium (MP Biomedicals, OH, USA) containing 1% fetal bovine serum (Lonza, Walkersville, MD) and 1% penicillin streptomycin (Hyclone) and mixed to make a homogenous suspension. The suspension is then strained using 70 m cell strai ners (Becton Dickinson, NJ, USA) and cells were collected at 1300 rpm for 7 min. The red blood cells (RBCs) are removed by lysing with ACK lysis buffer (Lonza, Walkersville, MD) followed by centrifugation at 1500 rpm for 5 min to recover leucocytes. Leucoc ytes were then re suspended in DMEM/F 12 with L glutamine (Cellgro, Herndon, VA), 10% fetal bovine serum, 1% sodium pyruvate (Lonza, Walkersville, MD), 1% non essential amino acids (Lonza, Walkersville, MD), 1% penicillin streptomycin (Hyclo ne) and 20 ng/ mL GM CSF (R&D systems, MN, USA) (DC media) and plate on tissue culture flasks for 2 days in order to remove adherent cells. At day 2 the floating cells are
113 transferred to low attachment plates and cultured in fresh DC media for expansion of DC precursor cells. At day 7, cells are transferred to tissue culture plates to allow for DC adhesion and proliferation. At day 10, they are lifted from tissue culture plates and used for various studies. For these studies, MPs were incubated at 37 o C for a period of 48 h prior to analysis or addition of T cells. Phagocytosable MPs (VIT D3 MP) were added at a 10:1 MP to cell ratio, while un phagocytosable MPs (TGF mass that encapsulated the effective concentration of that respecti ve drug for the incubation media volume. Unloaded MPs and the soluble equivalent of released drugs doses were included as controls. Bone Marrow derived Dendritic Cell Phenotype Maturation and Tolerogenic Markers Dendritic cell maturation and tolerogenic molecule expression were quantified by measuring cell surface marker and intracellular cytokine levels by flow cytometry. Following MP incubation, DCs were lifted by incubating with a 5 mM Na2EDTA in PBS solution at 37 o C for 20 min. Dendritic cells were then washed with 1% fetal bovine o C to and then stained with antibodies against CD80 (clone 16 A/I E (clone PD L2 (clone TY25, IgG2 30 min at 4 o C. For intracellular molecules, cells were fixed, permeabilized and stained with antibodies against IDO (clone 10.1, IgG3) (eBiosciences), IL 10 (JES5 16E3, IgG2b) and IL 12 (clone C15.6, IgG1) (BD Pharmingen). Appro priate isotypes were
114 used for each antibody species as negative controls. Data acquisition was performed using (FACScalibur, Becton Dickinson, NJ, USA) flow cytometry and the geometric fluorescent intensities as well as percent of positively stai ned cells determined. More than 10,000 events were acquired for each sample and data analysis was performed using FCS Express version 3 (De Novo Software, Los Angeles, CA). Cell culture supernatants were collected after 48 h of MP incubation, centrifuged t o remove any cell debris and stored at 20 o C until analysis. The IL 12 cytokine subunit, IL 12p40, IFN 10 cytokine production was analyzed using sandwich enzyme linked immunosorbant assay (ELISA) kits (Becton Dickinson, NJ, USA) according to manufacturer's directions. CD4+ T cell Isolation Mouse CD4+ T cells were purified from splenocyte suspensions by negative instructions. The purity of CD4+ T cells as det ermined by flow cytometry was in the 90 92% range. Mixed Lymphocyte Coupling (MLC) T cell Suppression and Treg Induction For suppression studies, bone marrow derived C57Bl6 DCs (2.5 x 10 4 / well) were co incubated with the different combinations of MPs as well as the relevant soluble and control treatments, in a 96 well tissue culture plate for 48 h at 37 o C in culture media. After thoroughly washing away all un phagoctyosed and unbound MPs, BALB/c CD4+ T cells (1.25 x 10 5 / well) were added to each wel l and incubated at 37 o C for 3 d. Bromodeoxyuridine (BrdU) (kit from Beckton Dickinson) was pulsed into the culture media for the last 4 h. T cells were then immunofluorescently stained for BrdU
115 s then used to quantify T cell proliferation for the different treatments. A similar method was employed for Treg induction studies with purified CD4+CD25 BALB/c T cells. However, following DC T cell co culture for 72 h, T cells were immunofluorescent ly stained using antibodies against CD4 (clone RM4 5, 16s, Novo Software, Los Angeles, CA) Antigen Presentation by Dendritic Cells after Microparticle Phagocytosis NOD DCs (2.5 x 10 4 / well) were co incubated with 1040 55 mimetope loaded MPs, as well as the relevant control treatments, in a 96 well tissue culture plate for 4 h at 37 o C. MPs outnumbered NOD DCs by 10:1. After thoroughly washing away all un phagocytosed and unbound MPs, NOD BDC2.5 CD4+ T cells (1.25 x 10 5 / well) were added to each well and incubated at 37 o C for 3 d. Bromodeoxyuridine (BrdU) (kit from Beckton Dickinson) was added to the cu lture for the last 4 h. T cells were then cytometry was then used to quantify T cell proliferation for the different treatments. Diabetes Prevention Studies Study I : A c ohort of 4 week old, female NOD mice were divided in 5 groups (n = 13). The groups were given MP formulation injections as follows: Group A Unloaded MPs only; Group B GM CSF MPs + Ins B MPs; Group C Vit D3 MPs + TGF + Ins B MPs; Group D Vit D3 MPs + TGF CSF MPs + Ins B MPs; Group E Ins B MPs. All injections c onsisted 10 mg of MPs in 0.2 mL PBS (Unloaded MPs were added to formulations where there was a deficit in mass). Mice were injected
116 twice at a subcutaneous site anatomically proximal to the pancreatic lymph nodes, once at 4 weeks old then again at five wee ks of age. The blood glucose levels of mice were then monitored once weekly for the next 27 weeks. Once the blood glucose level is over 250 mg/dl for two consecutive days, diabetes was diagnosed and the mouse sacrificed. Study II : A cohort of 8 week old, f emale NOD mice were divided in 9 groups (n = 13). The groups were given MP formulation injections as follows: Group A Unloaded MPs only; Group B GM CSF MPs + hInsulin MPs; Group C TGF MPs; Group D Vit D3 MPs + hInsulin MPs; Group E Vit D3 MPs + TGF hInsulin MPs; Group F Vit D3 MPs + TGF CSF MPs + hInsulin MPs; Group G hInsulin MPs; Group H Vit D3 MPs + TGF CSF MPs; Group I Soluble equivalent doses of all factors (Vit D3 + TGF CSF + hInsulin). All injections consisted 10 mg of MPs in 0.2 mL PBS (Unloaded MPs were added to formulations where there was a deficit in mass). Mice were injected six times, once weekly beginning at 8 weeks old, at a subcutaneous site anatomically proximal to the pancreatic lymph nodes. Diabetes monitoring was performed as described above for 24 weeks after the first injection. Study III : A cohort of 8 week old, female NOD mice were divided in 8 groups (n = 13). The groups were given MP formulation injections as follows: Group A Unloaded MPs only; Group B GM CSF MPs + hInsulin MPs; Group C TGF MPs; Group D Vit D3 MPs + hInsulin MPs; Group E Vit D3 MPs + TGF GM CSF MPs + hInsulin MPs; Group F hInsulin MPs; Group G Vit D3 MPs + TGF CSF MPs; Group H Soluble equivalent doses of all factors (Vit D3 + TGF CSF + hInsulin). All injections consisted 10 mg of MPs in 0.2 mL PBS
117 (Unloaded MPs were added to formulations where there was a deficit in mass). Mice we re injected three times in the first week of the study (8 weeks) and once monthly thereafter. All injections were at a subcutaneous site anatomically proximal to the pancreatic lymph nodes. Diabetes monitoring was performed as described above for 24 weeks after the first injection. Midpoint Mechanistic Studies Histopathology, APC and Treg Analysis At 15 weeks of age, 3 mice for each treatment group were rando ml y removed from the study, sacrificed and organs (spleen, pancreas) excised for analyses at th is time point. Histopathology : Pancreata were fixed with formalin, embedded using paraffin, sectioned, mounted and stained with Hematoxylin and Eosin (H&E). Stained sections were blind scored for Islet infiltration using the following grading system: 0 = h ealthy islet, 1 = peri insulitis, 2 = < 25% leucocytic infiltration of islet area, 3 = 25 75% leucocytic infiltration of islet area, 4 = <20% of islet area healthy. At least 50 islets were examined for each group. APC Maturation and Differentiation : Us ing the cell surface staining protocol described above, splenocytes (1 x 10 6 cells from each mouse) were stained with (eBiosciences) and Gr 1 (clone RB6 alyzed using the FCS express V3 software (De Novo Software, Los Angeles, CA) Treg Analysis : Splenocytes (1 x 10 6 cells from each mouse) were immunofluorescently stained using antibodies against CD4 (clone RM4 Pharmingen), CD25 (clone PC61.5
118 (eBioscience). Cells were analyzed using the FCS express V3 software (De Novo Software, Los Angeles, CA) Statistical Analysis Statistical analyses were performed using general linear nested model ANOVA, followed by posthoc assessment using Tukey test to make pair wise comparisons. Differences were considered significant when equal or less than p = 0.05 using Systat (Version 12, Systat Software, Inc., San Jose, CA). For the diabetes prevention studies, statistical analysis of survival curves was done in SYSTAT using Kaplan Meier Non parametric Survival analysis model. P values were determined by comparisons with the Results We successfully prepared MPs using either single or double em ulsion (depending on organic solvent solubility), solvent evaporation techniques for phagocytosable MPs and a double emulsion, solvent extraction method for the un phagocytosable MPs. These MPs were characterized to determine their size distribution, agent encapsulation efficiency and agent release kinetics. The size range of phagocytosable MP was Figure 4 1A shows the size distribution graph for Vit D3 MPs. It demonstrates tha t fabricated phagocytosable MPs, in general, have an average diameter of approximately CSF MPs, as determined by impedance measurements using a Beckmann Coulter Counter, was approximately 30 un distribution plot, representative of un phagocytosable MPs, is displayed for the TGF MP in Figure 4 1B.
119 Table 4 1 reports the encapsulation efficiencies of each of the prepared MPs used for the t hree diabetes prevention studies. These entrapment efficiencies for the phagocytosable MPs were determined to be 65.5 3.0%, 9.9 0.7%, and 34.9 1.1% for Vit D3 MPs, hInsulin MPs and Ins B MPs (Study I only) respectively. The encapsulation efficiencies for TGF CSF MPs were revealed to be 61.0 4.9% and 65.6 3.5% which corresponded to loadings of 38 3 ng/mg and 131 7 ng/mg respectively (Study III). The in vitro release kinetics for all MPs was studied at pH 7.4 in a 2% Tween20 PBS so lution at 37 o C. It should be noted that these release profiles were for the pharmaceutical and biological agents loaded into 50:50 PLGA polymer, where the polymer average molecular weight was approximately 44,000 g/mol. Release profiles for the heavier po lymer (average M.W. = 65,000 g/mol) are likely to be different but were not characterized. Both phagocytosable and un phagocytosable MPs showed sustained release over a period of approximately 30 d (Figure 4 2) for MPs fabricated with the lower M.W. PLGA. Both types of MPs show an initial burst release within the first 5 d of incubation. For the phagocytosable MPs, this is followed by a more linear release over the next 25 d (Figure 4 2A). The un phagocytosable MPs exhibited a period of plateau following th e initial burst then final third of the release curve shows a slower release of agent. However, at the end of the 30 d period, the cumulative release for the GM CSF MP is only about 49%. Similarly, for TGF d was only 61%. Contrastingly, the Vit D3 MP emitted the bulk of its entrapped drug by day 30 with its cumulative release determined to be approximately 77%. The Vit D3 release kinetics profile suggest that long after being taken up by APCs, MPs will be
120 continuously delivering Vit D3 to its engulfing DC, provided that polymer degradation rates are unchanged in the mildly acidic phagolysosome  Similarly, based on the release profiles for the un phag ocytosable MPs, we expect slow release of biological factors into the extracellular environment for a protracted time following the initial burst period. All data shown on in vitro biological characterization of MPs were determined using MPs prepared fo r use in Study III (See Table 4 1). We proceeded to characterize the immunomodulatory effects of MPs on DC phenotype in vitro More specifically, we studied how the combination of Vit D3 MPs and TGF controls including single fa ctor MPs and the soluble equivalent doses of these factors, affected expression of the stimulatory molecules MHC II, CD80 and CD86 on DCs (Figure 4 3A). The levels of expression of each of these maturation markers for each treatment were compared to that on iDCs for determination of the marker maturation index. Flow cytometric analysis revealed that C57Bl/6 bone marrow derived DCs treated with the Vit D3/ TGF indices for CD80 and MHCII compared to iDCs while CD86 expression levels are also reduced. This overall effect on DC phenotype is efficiently exhibited through the comprehensive maturation index (CMI). This value represents an average of the individual maturation marker indices for each treatment (Figure 4 3B). Interestingly, the CMI for the soluble equivalent dose of combined Vit D3 and TGF than that of the corresponding MP treatment, corroborating that the observed phenotype is agent induced. Further, combination treatments (MP and sol uble) substantially eclipsed the reduction of maturation state achieved by single factor MPs or single agent
121 equivalent doses, suggest that this effect is cumulative. We also examined expression levels of CD86 and MHC II on DCs treated with MPs then subse quently incubated in mL ) for 24 h. The immature phenotype of Vit D3/ TGF dual MP treated DCs were not reversed upon LPS exposure (Figure 4 3C, D). Comparatively, neither the soluble dosage of combined Vit D3 and TGF er relevant control treatments resisted upregulation of positive stimulatory molecules following LPS exposure. In addition to examining the modulation of stimulatory molecules on DCs by MP treatment, we explored the expression of tolerance inducing molecul es by DC in response to MP administration, particularly indoleamine 2, 3 dioxygenase (IDO). The induction of IDO has been describe in human and murine DC subsets and has been  Flow cytometric analysis revealed that application of Vit D3/ TGF number of IDO expressing DCs above that seen for iDCs (Figure 4 4). Administration of TGF level of significance (p < 0.1) than the Vit D3/ TGF show no appreciable modulation of IDO expression with the exception of the Sol Vit D3 group, which reduced IDO expression levels considerably. The capacity of th e dual Vit D3/ TGF allogenic T cells was evaluated. Following MP exposure for 2 d, as well as relevant controls, C57Bl/6 DCs were co cultured with purified, splenic CD4+ T cells from BALB/c mice for 3 d. BrdU inco rporation was used as a measure of T cell proliferation. We found that following incubation with combined Vit D3 and TGF
122 inhibit proliferation of CD4+ to T cell only negative control levels, without allogenic stimulus (Figure 4 5). Th e extents of suppression by Vit D3 MP, Sol Vit D3, TGF and Sol Vit D3/ TGF by at least 3 fold compared to untreated DCs. The potency of these factors to influence DC inhibition of allo stimulatory proliferative responses has been well documented on an individual basis [61,64] However, our results imply that coupling these to inhibitory agents could generate DCs with even greater suppressive capacity, especially through microsphere technology. Additionally, we examined CD4 T cells for expression of CD25 and FoxP3 to determine if Tregs were responsible for the dampening of allogenic proliferative responses observed. As shown in Figure 4 6A, coupling of Vit D3 MPs with TGF expressing T cells. Conspicuously, soluble equivalent dosages of Vit D3 and TGF combination, to DCs generated greater numbers of Tregs than any of the MP formulations. To further substant iate these observations, we measured the levels of cytokines in the supernatants from these MLR cultures (Figure 4 6B). The amounts of IL 4 secreted in MLR co cultures were not considerably different between either of the treatments and that of the non tre ated control. The amount of TGF supernatant from MLR cultures with soluble Vit D3/TGF treated DCs was substantially greater than that of the non treated control. Further, TGF also increased for mixed cultures with DCs pre e xposed to TGF
123 We employed a similar mixed lymphocyte coupling method to demonstrate the functional presentation of MP encapsulated antigen b y DCs. Briefly, NOD DCs were cultured with either MPs loaded with 1040 55 mimitope or soluble peptide (as a control, Subsequently, freshly isolated BDC2.5 CD4+ T cells were a dded to culture wells and co cultured in a 3 day mixed lymphocyte reaction. T cell proliferation was then measured using a BrdU proliferation assay as a measure of functional antigen presentation. The NOD BDC2.5 mouse is engineered with T cell receptors th at are specifically engaged by the 1040 55 mimetope. Upon binding the 1040 55 peptide (e.g. when presented on APCs), T cells are stimulated to proliferate. Figure 4 7 shows that DCs exposed to 1040 55 loaded MPs are capable of stimulating antigen dependent T cell proliferation to levels comparable to that of direct loading of peptide on DC MHC II complexes via soluble administration. This outcome is a valuable contribution for the design of an antigen specific tolerance inducing microparticle system for tre atment of type 1 diabetes. The results of the in vitro studies were very promising and inspired further investigation of whether this dual MP system could effectively prevent the onset of diabetes in live NOD mice. We performed three different mouse studie s to determine the efficaciousness of the Vit D3/ TGF CSF/ antigen (Insulin B peptide or whole human insulin) loaded MP system in abrogation of this disease that develops spontaneous in female NOD mice between the ages of 12 and 15 weeks. In the f irst study, 4 week old mice were injected twice at a subcutaneous site anatomically proximal to the pancreatic lymph nodes, once at 4 weeks old followed by a
124 booster at five weeks of age, with MP formulations consisting of either Unloaded MPs only, GM CS F MPs + Ins B MPs, Vit D3 MPs + TGF TGF CSF MPs + Ins B MPs, Ins B MPs. Mice were monitored for diabetes over a 32 week period. Results showed that injection of neither of the GM CSF MPs + Ins B MPs, Vit D3 MPs + TGF protected from diabetes development with diabetes incidences at 80%, 70% and 80% respectively. Administration of the full complement of MPs (Vit D3 MPs + TGF GM CSF MPs + Ins B MPs) did, however, prevent onset of diabetes in 40% of mice tested for that treatment. Kaplan Meier analysis for this treatment group reveals a p value of 0.025 in comparison to survival proportions for the unloaded MP group (Figure 4 8). Mechanistic assessments were perfor med on tissues harvested from mice at the half point of the study to elucidate the possible conduits on a cellular level, responsible for the observed prevention of diabetes in this study. A number of reports have linked diabetes progression with decreas ing numbers of functional FoxP3+ regulatory T cells [185,187] To ascertain whether Treg numbers are conserved by injection of the MP formulations, we enumerated the percentage of FoxP3 expressing, CD4+ T cells in the splenocyte population for mice from t he different treatments. Flow cytometric analysis reveals that splenic Treg numbers are increased marginally for mice injected with GM CSF MPs + Ins B MPs, Vit D3 MPs + TGF Vit D3 MPs + TGF CSF MPs + Ins B MPs (Figure 4 9A). Additionally, we evaluated the expr ession of Gr 1 on splenic APCs (CD11b+ and CD11c+ representing macrophages and DCs respectively). Studies have shown that Gr 1+ monocytes, derived from the bone marrow, can differentiate in CD11b+ and CD11c+ cells in the
125 presence of GM CSF and TGF her, these cells have been shown to have immunosuppressive capabilities and prevent autoimmune diabetes in 4 week old NOD mice  Enumeration of Gr 1expression on CD11b+ cells revealed slight increases for mice injected with Vit D3 MPs + TGF ns B MPs and Vit D3 MPs + TGF CSF MPs + Ins B MPs. Injections of the former formulation also prompted a subtle increase in levels of splenic CD11c Gr 1+ cells in comparison to mice treated with unloaded MPs (Figure 4 9B). The final mechanistic assessment looked at the maturation state of splenic DCs. Specifically, the expression of MHC II, CD80 and CD86 were examined for splenic CD11c+ cells. FACS analysis shows that the expression of none of these stimulatory markers varied significantly for th e different treatments. However, it should be noted that MHC II expression levels are slightly decrease in mice injected with Vit D3 MPs + TGF + TGF CSF MPs + Ins B MPs when compared to the Unloaded MPs cohort of mice. Additionally, CD86 levels for mice treated with Ins B MPs only are moderately higher than those in mice treated with Unloaded MPs (Figure 4 9C). Currently, tests for diagnosis of type 1 diabetes in humans rely on the detection of islet auto antige ns, including insulin, and glutamic acid decarboxylase (GAD), via auto antibodies. Unfortunately, prediction of diabetes onset through identification of these metabolic markers is not accurate until the disease has progressed to a later stage [209,210] Th erefore, any new therapy under development would have to effectively abrogate type 1 diabetes at the point where signs of the disease are just becoming overt, to be clinically relevant. In NOD mice this time point correlates to about 8 weeks of age. For th e continued development of the dual MP vaccine, it was therefore
126 expedient to test the efficacy of these MPs in mice of age corresponding to a human age at which type 1 diabetes is detectable by current standards. We tested the following formulations of MP s in 8 week old, female, NOD mice: Group A Unloaded MPs only; Group B GM CSF MPs + hInsulin MPs; Group C TGF Group D Vit D3 MPs + hInsulin MPs; Group E Vit D3 MPs + TGF MPs; Group F Vit D3 MPs + TGF Ps + GM CSF MPs + hInsulin MPs; Group G hInsulin MPs; Group H Vit D3 MPs + TGF CSF MPs; Group I Soluble equivalent doses of all factors (Vit D3 + TGF CSF + hInsulin). The number of booster shots for this trial was also increased t o 5 injections. Figure 4 10 shows that none of the formulations, under this dose regiment, prevented diabetes onset in NOD mice. Mice that received injections of either TGF TGF CSF MPs + hInsulin MPs, Vit D3 MPs + TGF CSF MPs or soluble equivalent doses of all factors (Vit D3 + TGF CSF + hInsulin) had a 3 4 week delay of onset when treatment was initiated at 8 weeks as demonstrated by the mean survival time. Midpoint analysis of splenocy tes recovered revealed that Treg numbers are consistent for all treatments (Figure 4 11A). We looked at the levels of Gr1 on CD11b and CD11c splenocytes. Although, there are apparent fluctuations in the expression of Gr1 on these cells between treatments, these differences are not noteworthy (Figure 4 11B). The same conclusions were drawn when we examined expression of positive stimulatory molecules on CD11c cells (Figure 4 11C). However, it should be noted that MHC II, CD80 and CD86 expression levels were lowered in groups that showed early promise to prevent type 1 diabetes in this study, particularly groups E, F and I.
127 Disappointingly, the second live animal prevention study failed miserably. This prompted a reformulation of our MP vaccines to more close ly mimic dosages used in in vitro testing. The vaccination schedule was also changed to three injections in the 8th week followed by monthly booster shots for mice remaining non diabetic. The following formulations of MPs were tested in 8 week old, female, NOD mice: Group A Unloaded MPs only; Group B GM CSF MPs + hInsulin MPs; Group C TGF MPs; Group D Vit D3 MPs + hInsulin MPs; Group E Vit D3 MPs + TGF GM CSF MPs + hInsulin MPs; Group F hInsulin MPs; Group G Vit D3 M Ps + TGF MPs + GM CSF MPs; Group H Soluble equivalent doses of all factors (Vit D3 + TGF CSF + hInsulin). As shown in Figure 4 12, 70% of the mice treated with Vit D3 MPs + TGF CSF MPs + hInsulin MPs remained normoglycemic until th e completion of the study. This is quite significant compared to the group treated with unloaded MPs that showed 90% diabetes incidence at 28 weeks (p value = 0.045). Interestingly, mice treated with only TGF significant p rotection from diabetes (p value = 0.093) in comparison to the cohort of mice from Group A. Finally, Figure 4 13 displays the results of tests done to resolve the mechanisms involved for diabetes protection. We first examined the expression of CD25 and Fox P3 in CD4+ splenic T cells from treated mice. Treg frequencies in mice treated with Vit D3 MPs + TGF CSF MPs + hInsulin MPs and TGF MPs + hInsulin MPs are elevated, although, not significantly compared to those treated with Unloaded MPs (Fig ure 4 13A). Gr1 expression levels on CD11b and CD11c cells, although lowered groups C and E, were statistically comparable with the other groups
128 (Figure 4 13 B). Likewise, results from analysis of maturation markers on CD11c+ splenocytes were inconclusive (Figure 4 13C). Impact of Study immunity and more recently, its role in the induction of central and peripheral tolerance has been elucidated [41,95] Unsurprisingly, the applica tion of manipulated DCs to restore homeostatic conditions from autoimmune dysregulation has exploded over the past decade [189,207,208] Although this approach has been successfully demonstrated to abrogate autoimmune complications in mice, it has been rec ognized that broad scale application is untenable due to the complexity of the approach, which involves ex vivo DC manipulation, as well as other logistic disadvantages  Polymeric microparticulate systems are emerging immunotherapeutic tools for effic ient delivery of immunomodulatory factors to DCs. MPs can be fashioned to desired size ranges with encapsulated tolerance inducing agents, are easily injectable and, therefore can be utilized for in vivo conditioning of DCs [19,29,191,192] In this study, we developed a dual microparticle vaccine system that significantly protects NOD mice from the onset of T1D. This dual MP system is comprised of combinations of two phagocytosable MPs for intracellular targeting either of Vit D3 loaded MPs, antigen loade d MPs (insulin B peptide, human insulin), or unloaded MPs, and two un phagocytosable MPs for extracellular delivery either of TGF loaded MPs, GM CSF loaded MPs or unloaded MPs. This is novel attempt to develop an injectable system that targets APCs an d delivers drug combinatorially in a spatiotemporal manner to these specialized cells that play an important role in the development of autoimmune diseases as T1D. Others, including Phillips et al and
129 Yeste et al ., have reported on the formulation of bioa ctive agents into polymeric particulate matter as a means of modulating DC phenotype and ultimately, curtailing autoimmunity in mice [38,211] However, this dual MP system represents the first multi modal approach to deliver multiple pharmaceutical and bio logical agents simultaneously to DCs and other APCs, in an attempt to abrogate T1D development. The princi pal outcome of this study is that this formulation of MPs encapsulating dihydroxyvitamin D3, GM CSF and TGF week old and 8 w eek old NOD mice. Disappointingly, analysis of cells recovered from MP treated mice, including Treg frequency, failed to elucidate the mechanism of action by which the observed prevention of T1D is facilitated. However, in vitro results provide clues that indicate induction of tolerogenic DCs by MPs may subsequently invoke downregulation of T cell immunogenic responses. Further, there is evidence from the survival proportions of the final live animal study that suggest this effect is antigen specific. We translocated to draining LNs within 48 h  Other reports have documented that intraperitoneal injection of t hese particles result in trafficking to the spleen and other internal organs, including the pancreas, by DCs and other phagocytic cells following interception  Therefore, it is safe to assume that, at some level, APC interaction with MPs resulted in t he demonstrated T1D protection. Especially for DCs, given that one of the non phagocytosable MPs contained GM CSF. Ali et al demonstrated that
130 GM CSF is a powerful recruiter of CD11c+ cells  The other un phagocytosable MP encapsulated TGF  In this dual MP system, these two chemokines are encased in large, un phagocytosable gradually release th eir contents into the surrounding extracellular milieu. This design aspect of the vaccine system is crucial as it allows for initial recruitment of DCs, coinciding with the initial burst release of factor in the first five days, but not long term retention at the injection site. Both GM CSF and TGF encourage DC recruitment, but they can also significantly alter DC phenotype [52,64,207] dihydroxyvitamin D3) downreg ulates the levels of stimulatory molecules on DCs  Through in vitro testing, we corroborated these findings and further, elucidated their cumulative effects on DC expression of positive stimulatory molecules MHCII, CD80 and CD86, when coupled toget her in microparticulate and soluble dosage forms. GM CSF was already included in the culture media as a DC differentiation factor, therefore further supplementation for in vitro tests was redundant. Predictably, DC exposure to different combinations of the se bioactive agents in either dosage form resulted in reduced expression of MHCII, CD80 and CD86. The levels of expression for these positively stimulatory molecules were dramatically lowered when a combination of all three factors, both in MPs and soluble bolus. Steinman et al and Banchereau et al have reported on the requirement of MHC (signal 1) in the presence of costimulatory molecules (signal 2) for activation of T cells by DCs [3,95] Immediately, a plausible
131 mechanism for the dampening of immunity by Vit D3/ TGF CSF MPs becomes apparent. In the absence of either one of those signals, DCs are unable to stimulate immunogenic T cell responses, resulting in T cell anergy  We investigated the impact of LPS stimulation on expression of posi tive stimulatory molecules on DCs, subsequent to exposure to the different combinations of Vit D3 and TGF inflammation affected sites, like the pancreatic islets during diabetes development, are i noculated with soluble mediators that can dictate activation of immature DCs [9,215,216] Therefore, it is imperative that if MP treated DCs are to reverse autoimmunity directly at the site of inflammation, these DCs must be immune to inflammatory conditio ns. Treatment with either TGF the only formulations that bestowed resistance to LPS induced maturation upon DCs. This result advances the claim that microsphere vehicles through local and/or direct delivery of immunomodulat ory factors can promote increased functional effects unattainable with comparable soluble boluses. This is reemphasized when we consider the effects of MP incubation versus equivalent soluble bolus on IDO expression in DCs. For instance, packaged TGF s ignificantly influences upregulation of IDO in DCs after 48 h, whereas expression remains unchanged by Sol TGF time where IDO expression maybe greatest for DCs treated with the soluble equivalent dosage. Perhaps, the most pertinent result from this in vitro assay is that combined Vit D3 and TGF
132 dioxygenase is a tryptophan catabolizing enzyme that is implicated for tolerance induction by tryptophan starvation and production of catabolites called kynurenines  crosstalk b etween tolerogenic DCs and regulatory T cells as well T cell apoptosis [55,217,218] Extrapolating these observations, it becomes evident that multiple mechanisms that lead to tolerance induction may be triggered by DCs interception of Vit D3/ TGF n the presence of GM CSF. This theory is given further credence when we take into account the downstream effects of Vit D3/ TGF treated DCs on T cells in co culture. Mixed lymphocyte coupling of T cells with DCs pretreated with either of TGF Sol TGF TGF concentrations of TGF effects of this cytokine on both DCs and T cells have been well documented and include the induction of regulatory CD25+ FoxP3+ T cells, in addition to blockading the activation and proliferation of effector CD4+ T cells [64,151,202] Our studies on Treg f requency in MLR co cultures are also consistent with these reports, as well as, our data on TGF secretion, to an extent. Sol Vit D3 treated DCs influence the induction and expansion of CD25+ FoxP3+ T cells without the assistance of TGF ively, the soluble equivalent doses all increased Treg numbers above that of their corresponding MP treatments. One possible explanation for this is that DCs are affected by the degradation of the polymer. PLGA particles degrade by hydrolysis of the ester bonds in the polymer backbone producing the monomeric units of lactic acid and glycolic acid
133  Researchers have shown that bacteria derived lactic acid reduces constitutive expression of positively stimulatory molecules on DCs [172,173] As Fedoric et al revealed, downregulation of stimulatory markers can be accompanied by reduction in inhibitory molecules, like Ilt3, that play a role in Treg generation  However, this neration the influence of Vit D3/ TGF treated DCs on T cell alloreactivity. The unloaded MPs control was able to reduce allogenic T cell proliferative responses to D Cs by approximately 50%. In spite of this fact, the combination of Vit D3 and TGF the most potent in inhibiting T cell proliferation. Incubation of these MPs with DCs result in suppression of T cell proliferation to levels only seen when there i s no allogenic stimulus. Finally, we confirmed the ability of DCs to process MP encapsulated antigen, load it onto MHC II complexes and functionally present to T cells, in vitro This is an important consideration for antigen specific DC therapy. Althou gh bystander, non specific suppression of immunity is an effective therapeutic method for autoimmune diseases and transplant related complications, its efficacy, as well as safety, would be greatly improved if responses are auto antigen specific [211,220] Herein, we begin to demonstrate the impact of co delivery of immunosuppressive agents and antigen by MPs. For instance, the survival proportions for mice treated with Vit D3 MPs + TGF MPs + GM CSF MPs + hInsulin MPs in Study III is at 60%. When the hIn sulin MP is removed from the formulation, survival proportions drop to 20%. These results suggest that antigen is required for suppression of autoimmunity in this model.
134 Summarily, we have packaged a number of immunosuppressive factors as well as antigen i nto polymeric microparticles for efficient delivery to DCs. Upon DC MP interaction, these immunomodulatory agents appear to act through different modalities, including anergy, T cell apoptosis, and Treg generation, based on in vitro studies. The downstream effect is T cell hyporesponsiveness and ultimately, reduced autoimmunity in animals. However, elucidation of the precise in vivo mechanism(s) involved in protection from T1D in NOD mice remains a major objective.
135 Figure 4 1 Microparticle characterization: Size Distribution. Microparticle size was determined using dynamic light scattering techniques (A) average diameter phagocytosable microparticles ~ 30 Table 4 1. Microparticle characterization: Encapsulation Efficiency. Table showing sizes and encapsulation efficiencies of PLGA microparticles loaded with biological and pharmacological agents for mouse studies. Biological/ Pharmacologic Agent Amoun t Used/PLGA (g/100 mg) Encapsulation Efficiency + SD (%) Loading + SD (ng/mg) Vit D3 (Studies I, II &III) 5 65.5 3.0 33 2 Ins B (Study I only) 1000 34.9 1.1 3494 110 hInsulin (Studies II & III) 4000 9.9 0.7 3958 286 TGF (Studies I & II ) 1 62.4 5.2 6 0.5 TGF (Study III only) 6.25 61.0 4.9 38 3 GM CSF (Studies I & II) 2.5 71.7 5.6 18 2 GM CSF (Study III only) 20 65.6 3.5 131 7 (A) (B )
136 Figure 4 2. Microparticle characterization: R elease Profile In vitro release profiles of A) Vit D3, B) TGF C) GM CSF in a 2% Tween 20 PBS solution. Error bars on graphs represent standard deviation based on n = 3 measurements for each microparticle type. (B) (C) (A )
137 Figu re 4 3. Co culture of the Vit D3/ TGF levels of surface expression of MHC II and costimulatory markers (CD80, CD86) relative to iDCs (A, B). Further, co culture of Vit D3/ TGF System result in DC maturation re sistance to LPS stimulation (C, D). The maturation index for each surface marker (Panel A, C) is the ratio of the percent of positive cells for that treatment group to the percent of positive cells for the iDC group. The comprehensive maturation index (Pan el B, D) represents an average of the maturation indices for each sample. For the comprehensive maturation, pair wise significant differences from iDC and * (C) (D) (A) (B)
138 Figure 4 4. C o culture of the Vit D3/ TGF expression of Indoleamine 2,3 dioxygenase. P air wise significant differences **
139 Figure 4 5. Co culture of Vit D3/ TGF allogenic CD4+ T cells. Briefly, MPs were incubated with C57Bl/6 DCs for 2 d followed by washing to remove unbound MPs. Subsequently, freshly isolated BALB/c CD4+ T cells were added to culture wells and co cul tured in a mixed lymphocyte reaction. T cell proliferation was then measured using a BrdU proliferation assay. Unloaded MPs and the soluble equivalent of released immunomodulatory agent doses were included as controls. Data shown in (A) represent the mean wise significant differences are denoted by: the symbol for comparisons to the iDC group; the ** symbol for comparisons to the iDC and Unloaded MPs groups. (B) Representative dot plots of various treat ments for T cell suppression studies.
140 iDC Unloaded MPs Vit D3 MP Sol Vit D3 TGF B1 MP Sol TGF B1 Vit D3/ TGF B1 MPs Sol Vit D3/ TGF B1 PMA/ Ionomycin T Cell Only (B) * (A)
1 41 Figure 4 6. Co culture of Vit D3/ TGF treated DCs with CD4+ T cells results in (A) expansion and induction of Tregs and (B) increased secretion of t he suppressive cytokine, TGF for 2 d followed by washing to remove unbound MPs. Subsequently, freshly isolated BALB/c CD4+ T cells were added to culture wells and co cultured in a mixed lymphocyte reaction. (A) Following DC T cell co culture for 72 h, T cells were immunofluorescently stained using antibodies against CD4, CD25 and FoxP3 (n = 3). (B) Additionally, IL 4 and TGF from MLR wells were detected using standard ELISA kits. P air wise
142 (A) * (B)
143 Figure 4 7. Antigen encapsulated in MP can be processed an d loaded onto MHC II complexes by DCs and functionally presented to T cells. NOD DCs were cultured with either MPs loaded with 1040 55 mimitope or soluble peptide (as remove unbound M Ps. Subsequently, freshly isolated BDC2.5 CD4+ T cells were added to culture wells and co cultured in a 3 day mixed lymphocyte reaction. T cell proliferation was then measured using a BrdU proliferation assay as a measure of functional antigen presentation (A) Data shown represent the mean proliferation percentages standard deviation and (B) representative dot plot s for the various treatments iDC 1040 55 loaded MP Soluble 1040 55 Unloaded MP (A) (B)
144 Figure 4 8. Study I : Vit D3/ TGF CSF/ Ins B MPs injections into NOD mice at 4 weeks of age prevents diabetes onset. Five groups of NOD female mice (4 weeks old) were given two subcutaneous injections of combinations of agent loaded MPs at a site anatomically proximal to the pancreatic lymph nodes. Each injection contained 10mg of MPs in PBS. Tail vein blood glucose was >240 mg/dl. The graph shows cumulative survival for the different MP injections. Kaplan Meier analysis reveals significance in comparison t o the Unloaded MP control. INJECTIONS
145 Figure 4 9. Study I : Vit D3/ TGF CSF/ Ins B MPs injections into NOD mice at 4 weeks of age influences immune cell phenotype. Five groups of NOD female mice (4 weeks old) were given two subcutaneous injections of combina tions of agent loaded MPs at a site anatomically proximal to the pancreatic lymph nodes. At age 15 weeks, 3 non diabetic mice were randomly chosen from each group and sacrificed for spleen harvest. Single cell suspensions of splenocytes were stained with antibodies against CD4, FoxP3, CD11c, CD11b, Gr1, MHC II, CD80 and CD86 antigens. FACS analysis reveals the proportions of (A) regulatory T cells, (B) Gr1 the spleen as well as (C) MHC II, CD80 and CD86 expressed on DCs for the different MP injections.
146 (B) (A) (C)
147 Figure 4 10. Study II : (A) Vit D3/ TG F CSF/ hInsulin MP injection into NOD mice at 8 weeks of age delays diabetes onset. Nine groups of NOD female mice (8 weeks old) were given a six subcutaneous injections of agent loaded MPs or relevant controls at a site anatomically proximal to the pancreatic lymph nodes. Each injection contained 10mg of MPs in PBS or an equivalent soluble dose of respective agents. Tail vein blood glucose was measured mg/dl. The graph shows cumula tive survival for the different treatments. Kaplan Meier analysis reveals significance in comparison to the Unloaded MP control. INJECTIONS
148 Figure 4 11. Study II : Vit D3/ TGF CSF/ hInsulin MPs injections into NOD mice at 8 weeks of age influences immune cel l phenotype. Nine groups of NOD female mice (8 weeks old) were given six subcutaneous injections of combinations of agent loaded MPs at a site anatomically proximal to the pancreatic lymph nodes. At age 15 weeks, 3 non diabetic mice were randomly chosen fr om each group and sacrificed for spleen harvest. Single cell suspensions of splenocytes were stained with antibodies against CD4, CD25, FoxP3, CD11c, CD11b, Gr1, MHC II, CD80 and CD86 antigens. FACS analysis reveals the proportions of (A) regulatory T cel ls, (B) Gr1 express ing expressed on DCs for the different MP injections.
149 (B) (A)
151 Figure 4 12. Study III : (A) Vit D3/ TGF CSF/ hInsulin MP injection into NOD mice at 8 weeks of age prevents diabetes onset. Eight groups of NOD female mice (8 weeks old) were given a seven subcutaneous injections of agent loaded MPs or relevan t controls at a site anatomically proximal to the pancreatic lymph nodes. Each injection contained 10mg of MPs in PBS or an equivalent soluble dose of respective agents. Tail vein blood glucose was ve readings of >240 mg/dl. The graph shows cumulative survival for the different treatments. Kaplan Meier analysis reveals significance in comparison to the Unloaded MP control. INJECTIONS
152 Figure 4 13. Study III : Vit D3/ TGF CSF/ hInsulin MPs injections into NOD mice at 8 weeks of age influences immune cell phenotype. Eight groups of NOD female mice (8 weeks old) were given seven subcutaneous injections of combinations of agent loaded MPs at a site anatomically proximal to the pancreatic lymph nodes. At age 1 5 weeks, 3 non diabetic mice were randomly chosen from each group and sacrificed for spleen harvest. Single cell suspensions of splenocytes were stained with antibodies against CD4, FoxP3, CD25, CD11c, CD11b, Gr1, MHC II, CD80 and CD86 antigens. FACS anal ysis reveals the proportions of (A) regulatory T cells, (B) Gr1 expressing expressed on DCs for the different MP injections.
153 (B) (A)
155 CHAPTER 5 CONCLUSIONS AND FUTU RE DIRECTIONS The overall goal of this thesis was to develop a dendritic cell targeting, biomaterial microparticle vaccine for the prevention of Type 1 Diabetes (T1D). We aimed to ac complish this by engineering a DC targeting, two component synthetic microparticle system with encapsulated antigen and immuno modulatory agents that direct tolerogenic dendritic cell phenotypes and promotes antigen specific protection from T1D. Our centra l hypothesis was that guided co delivery of diabetogenic epitopes and immunosuppressive molecules to DCs in a non inflammatory mode would drive antigen specific immuno suppression and mitigate developing autoimmunity. Our supposition was based on the succe ss seen in treating autoimmune disease using dendritic cell based therapy. DC based immunotherapy has been dev eloped to the point where it is currently being tested in humans, problems such as the plasticity and complexity of DC maturation, ex vivo stabili ty, shelf life, and high costs restrict the widespread application of this therapeutic approach [25,221] A biomaterial particle approach attempts to apply the same concept of DC conditioning to modulate immune responses, but instead DC manipulation occurs in situ This preliminary development of a DC targeting, biomaterial, microparticle vaccine to prevent T1D was streamlined into three major studies, as follows: 1) an evaluation of the ability of ligand functionalized microparticles to improve DC specific phagocytosis, in a non activating manner ; 2) an investigation of combinatorial delivery by microsphere technology to enhance DC suppressive capacity and; 3) an assess ment of the ability of the multi component vaccine particle formulation to prevent the on set of insulin dependent diabetes in non obese diabetic (NOD) mice
156 For our first aim, we investigate d targeting of DCs using surface functionalized PLGA MPs, in a non stimulat ory manner under in vitro and in vivo conditions We modified MPs by surface immobilizing DC receptor targeting molecules antibodies (anti CD11c, anti DEC 205) or peptides (P D2, RGD), where anti CD11c antibody, P D2 and RGD peptides target integrins and anti DEC 205 antibody targets the c type lectin receptor DEC 205. Our resul ts demonstrate d that the modified MPs are neither toxic nor activating, and DC uptake of MPs in vitro is improved by the anti DEC 205 antibody, the anti CD11c antibody and the P D2 peptide modifications. The P D2 peptide MP modification significantly impro ved DC antigen presentation in vitro both at immediate and delayed time points. Notably, MP functionalization with P D2 peptide and anti CD11c antibody increased the rate and extent of MP translocation in vivo by DCs and D2 peptide modified MPs demonstrating the highest translocation. This is work informative for the design of non activating polymeric microparticulate applications such as vaccines for autoimmune diseases. Our second study tested the hypothesis that targeted, local delivery o f combinations of these immunosuppressive factors can be achieved through polymeric microsphere technology, which would further result i mmuno regulatory effects not realized by equivalent single factor doses. Additionally, this study would help to identify microparticle formulations. W e separately enca psulated RAPA and RA in phagocytosabl e microparticles (~1 10 in un phagocytos co incubated bone marrow derived murine DCs with microparticle couples consisting of one of either type of MP and assessed their ability to modulate DC im mune function. The major finding from this
157 study was that DCs exposed to combinations of these two immunosuppressive agent loaded MPs considerably depressed proliferation of allogenic T cells, in comparison untreated controls as well as some relevant singl e MP treatments. In attempting to discern the plausible mechanism(s) by which the observed suppression is occurring, we examined expression levels of stimulatory molecules (MHC II, CD80, 86), inhibitory markers (Ilt3), and regulatory T cell generation. Our results indicate that Treg numbers remain unchanged, in comparison to untreated controls, when MP treated DCs are co cultured with allogenic T cells. Although we have yet to determine if the functionality of these tregs is altered, based on the unchanged frequency in culture wells we believe that their role in suppression is minimal. Alternatively, MPs with encapsulated agents generally effect greater decrease in DC activation markers than their soluble equivalent counterparts This observation suggests DC mediated T cell anergy due to low levels of stimulatory signals may be involved in T cell hyporesponsiveness. However, this is not the only mechanism at play, as expression levels of stimulatory molecules on DCs treated by MP combinations does not always correlate with T cell suppression levels. The combination of Vit D3 and TGF the best formulation to be tested in vivo The dual MP system for this study always comprised of combinations of two phagocytosable MPs either of Vit D3 loaded MPs, antigen loaded MPs (insulin B peptide, human insulin), or unloaded MPs and two un phagocytosable MPs either of TGF loaded MPs, GM CSF loaded MPs or unloaded MPs. The primary goal wa s to prevent T1D in NOD mice using this MP formulation. Summarily, we packaged a numb er of immunosuppressive factors as well
158 as antigen into polymeric microparticles for efficient delivery to DCs. Upon DC MP interaction, these immunomodulatory agents appear to act through different modalities, including anergy, T cell apoptosis, and Treg g eneration, based on in vitro studies. The downstream effect is T cell hyporesponsiveness and ultimately, reduced autoimmunity in NOD mice at ages 4 and 8 weeks at commencement of treatment However, the precise in vivo mechanism(s) involved in protection f rom T1D in NOD mice is yet to be elucidated Evidently, this project is still only at a nascent stage. But, given the promising results thus far it should come as no surprise that we intend to further explore and optimize this microparticle vaccine system for prevention and reversal of T1D. Ultimately, we want to develop a product that is translatable to humans. However, to accomplish this distant goal we will first have to improve the efficacy of the current formulation in the NOD mouse test bed. More spec ifically, we want to first determine the mechanisms involved in the apparent protection from T1D in NOD mice. For this, we will examine the expansion and functionality of suppressor cell populations including Tregs, Tr1 cells and myeloid Gr1+ suppressor ce lls. Once the mechanism is elucidated we want to take measures to further increase protected proportions in live animal studies. In addition to any incite gained from mechanistic studies, we will incorporate targeting moieties onto phagocytosable MPs, incr ease the scope of antigen, as well as manipulate drug dosages in an attempt to improve non diabetic proportions. Another immediate aim is to reduce the number of MP injections required to induce protection from T1D in mice. Based on our animal studies resu lts, we surmise that our particle formulation do not provoke immune memory to the insulin antigen encapsulated in the
159 MP formulations. We intend to investigate various means to activate memory cell populations by including stimulatory agonists such as CpG oligonucleotides. Regardless of future reincarnations, this microparticle vaccine system represents the first multi modal approach to deliver multiple pharmaceutical and biological agents simultaneously to DCs and other APCs in situ in an attempt to abr ogate T1D development.
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179 BIOGRAPHICAL SKETCH Jamal Lewis was born in 1984 in New Amsterdam, Guyana. He completed his primary school education in Georget own, Guyana and shortly thereafter, moved to for five years. Upon completion of his secondary level education at age 16, Jamal had history both academically and extracurricularly, was on the move again for his tertiary education. He attended Florida A&M University and North Carolina State Univer sity for his Bachelor of Science in Chemical Engineering and Master of Science Biomedical Engineering degrees respectively. In August 2007, Jamal transferred from North Carolina State University to the University of Florida in Gainesville, Florida to read for his doctoral degree under the mentorship of Dr. Benjamin Keselowsky. In the course of his doctoral studies, Jamal has undertaken many multi disciplinary projects includ ing the use of echistatin and other integrin binding peptides to mitigate the foreig n body response to implanted materials (e.g. poly ethylene) in mice as well as the surface modifying gold coated surfaces through alkane thiol chemistry with integrin binding peptides to modulate dendritic cell di fferentiation. His dissertation project is a novel attempt to abrogate and reverse auto inflammation seen in type 1 diabetes through the use of multifunctional, dendritic cell targeting, controlled release microparticles. The success of this project has lead to patent protection of this invention a nd p ublications that document the extraordinary result s are currently in preparation. This multifaceted research and development program has afforded Jamal an integrated understanding of mechanisms that can be redesigned, improved, and used to create
180 new k nowledge. In the future, he hopes to continue working on novel and exciting biotechnologies with huge impact on human healthcare.