OBESE DIABETIC MICE by ANDREW SCOTT

MUCOSAL TOLERANCE STRATEGIES FOR TREATING TYPE 1 DIABETES IN NON- OBESE DIABETIC MICE

ANDREW SCOTT NELSON

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

UNIVERSITY OF FLORIDA

2018

To my parents, my brother, and my wife Lisa

ACKNOWLEDGMENTS

I would like to thank my parents for their love and support. I would also like to thank my brother, Adam, whose standards I constantly strive to meet. To the friends I have made in graduate school, thank you for your support and help. And a big thank you to my wife, Lisa Lundgren, for working with me to not only understand my science, but to communicate it as well.

I sincerely thank my advisor, Dr. David Pascual for providing me with the opportunity to study and train in his lab, and also for his patience and guidance. Thank you David for establishing a high standard for my research, how I present my work, and how I present myself.

Thank you as well to Dr. Brusko, Dr. Nguyen, Dr. Zhou, and Dr. Abbott – my dissertation committee – for their support and guidance, without which these projects would not have been completed.

TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 8

LIST OF FIGURES ...... 9

LIST OF ABBREVIATIONS ...... 10

ABSTRACT ...... 15

CHAPTER

1 INTRODUCTION ...... 17

Historical Context ...... 18 Classification as an Autoimmune Disease...... 19 Modern Diabetes ...... 20 Genetics of Type 1 Diabetes ...... 21 Environmental factors ...... 22 The NOD mouse ...... 25 Disease Etiology ...... 26 Viruses as Disease Initiators ...... 26 β Cells as Disease Initiators ...... 27 Disease Pathogenesis ...... 28 Mechanisms of Pathogenesis ...... 30 Oral Tolerance ...... 33 Regulatory T cells ...... 34 T1D and Tolerance ...... 35 Reovirus Protein Sigma-1 ...... 36 Colonization factor antigen 1 ...... 38 Purpose of the Study ...... 40

2 ORAL LACTOCOCCAL IMMUNOTHERAPY FOR THE TREATMENT OF T1D .... 43

Background ...... 43 Methods ...... 46 NOD Mouse Husbandry ...... 46 Monitoring blood glucose levels ...... 47 Grading Insulitis ...... 47 Growing L. lactis for Oral Administration ...... 47 Neutralization of stomach acid ...... 48 Oral gavage of bacteria ...... 49 Tissue Collections ...... 49 Splenocyte isolation ...... 50

Lymphocyte isolation ...... 51 Counting cells ...... 51 Cell Culture ...... 51 Flow Cytometry ...... 52 Staining with tetramers ...... 53 Cytokine ELISA ...... 53 Statistics ...... 55 Results ...... 55 Oral Treatment with LL-CFA/I Ameliorates T1D in NOD Mice ...... 55 LL-CFA/I induced CD25+ Tregs are suppressive in vitro ...... 57 Optimization of LL-CFA/I therapy ...... 59 Phenotyping Tregs at 11 weeks ...... 60 LL-CFA/I does not induce Tr1 cells at 11 weeks ...... 63 LL-CFA/I induced Tregs are stable out to 17 weeks ...... 65 Optimized Therapy Reduces Incidence of T1D in NOD mice ...... 66 Discussion ...... 66

3 ESTABLISHMENT OF A REGULATORY MICROENVIRONMENT BY LACTOCOCCUS EXPRESSION COLONIZATION FACTOR ANTIGEN I ...... 96

Background ...... 96 Methods ...... 98 NOD Mouse Husbandry ...... 98 Growing L. lactis for Oral Administration ...... 99 Neutralization of stomach acid ...... 99 Oral gavage of bacteria ...... 100 Tissue Collections ...... 100 Splenocyte isolation ...... 100 Lymphocyte isolation ...... 101 Counting cells ...... 101 Cell Culture ...... 101 Infecting BMDCs ...... 102 Co-culture of CD4+ T cells with APCs ...... 103 Flow Cytometry ...... 104 Cytokine ELISA ...... 104 Statistics ...... 105 Results ...... 105 LL-CFA/I induces regulatory function in BMDCs ...... 105 Kinetics of LL-CFA/I induced regulatory DCs and Tregs ...... 106 LL-CFA/I reduces inflammatory potential of cDCs ...... 108 LL-CFA/I infected BMDCs do not induce Foxp3+CD4+ Tregs ...... 108 Discussion ...... 109

4 MUCOSAL TOLERANCE INDUCTION FACILITATED BY REOVIRUS PROTEIN SIGMA-1 AMELIORATES T1D IN NOD MICE ...... 129

Background ...... 129

Methods ...... 131 Design, Construction, and Purification of Pσ1 Fusion Proteins ...... 131 Quantifying and Analyzing Purified Protein ...... 133 NOD Mouse Husbandry ...... 133 P1 Treatments ...... 134 Neutralization of stomach acid ...... 134 Monitoring blood glucose levels ...... 134 Tissue Collections ...... 135 Lymphocyte isolation ...... 136 Counting Cells ...... 136 Cell Culture and T Cell Stimulation ...... 136 Flow Cytometry ...... 137 Cytokine ELISA ...... 137 Statistical Analysis ...... 138 Results ...... 138 Development of Mucosally Targeted Tolerogens for T1D ...... 138 Mucosal Pσ1 Therapy Ameliorates T1D ...... 139 Discussion ...... 140

5 CONCLUSIONS AND FUTURE DIRECTIONS ...... 150

LIST OF REFERENCES ...... 160

BIOGRAPHICAL SKETCH ...... 193

LIST OF TABLES

Table page

2-1 List of antibodies used for flow cytometry in LL-CFA/I Treg studies...... 95

3-1 List of antibodies used for flow cytometry in LL-CFA/I DC studies...... 128

4-1 List of antibodies used in flow cytometry for pσ1 based therapies...... 149

LIST OF FIGURES

Figure page

2-1 Oral treatment with LL-CFA/I ameliorates T1D in NOD mice...... 74

2-2 LL-CFA/I induced CD25+ Tregs suppress proliferation of diabetogenic Teffs. .... 77

2-3 Dose optimization of LL-CFA/I in NOD mice...... 79

2-4 Phenotyping Tregs in NOD mice...... 82

2-5 LL-CFA/I does not induce Tr1 cells at 11 wks in NOD mice...... 86

2-6 LL-CFA/I suppresses inflammatory CD8+ T cell responses in NOD mice...... 88

2-7 LL-CFA/I promotes Bregs in the spleen...... 90

2-8 LL-CFA/I maintains suppressive Tregs late in disease...... 91

2-9 Protection mediated by LL-CFA/I is stable out to 30 wks of age...... 94

3-1 LL-CFA/I induces BMDCs to function as regulatory cells...... 115

3-2 LL-CFA/I quickly induces IDO and TGF-β in the spleen and PaLNs of NOD mice...... 116

3-3 LL-CFA/I promotes Foxp3+ Tregs in the MLNs and PPs...... 122

3-4 Treatment with LL-CFA/I reduces costimulatory molecules in MLNs...... 124

3-5 LL-CFA/I infected BMDCs induce IL-10 producing Foxp3- Tregs...... 126

4-1 Development of pσ1 fusion proteins for treatment of T1D...... 144

4-2 Antigen-specific immunotherapy ameliorates T1D in NOD mice...... 145

4-3 Pσ1 fusion proteins induce Tregs to protect NOD mice from T1D...... 146

5-1 Summary of protection mediated by LL-CFA/I over time...... 158

LIST OF ABBREVIATIONS

µCi Micro-Curie

µg Microgram

µL Microliter

µm Micrometer

µM Micromolar

ACK Ammonium-Chloride-Potassium

AhR Aryl hydrocarbon receptor

APC Antigen presenting cell

BMDC Bone marrow derived dendritic cell

Breg Regulatory B cell

CD Cluster of differentiation

CFA/I Colonization factor antigen I

CFA/II Colonization factor antigen II

CFU Colony forming unit

CIA Collagen induced arthritis

CM Complete media

CTLA-4 Cytotoxic T-lymphocyte-associated protein 4

DC Dendritic cell dL Decaliter

DPBS Dulbecco’s phosphate buffered saline

EAE Experimental autoimmune encephalomyelitis

ELISA Enzyme linked immunoabsorbent assay

ER Endoplasmic reticulum

ETEC Enterotoxigenic Escherichia coli

FACS Fluorescence-activated cell sorting

FCS Fetal calf serum

Foxp3 Forkhead box p3 g Gram

GAD Glutamic acid decarboxylase 65

GAD-pσ1 GAD genetically fused to protein sigma 1

GALT Gut associated lymphoreticular tissue

GF Germ free

GM-CSF Granulocyte-macrophage colony-stimulating factor

H&E Hematoxylin and Eosin

HLA Human leukocyte antigen

HNLNs Head and neck lymph nodes

HRP Horseradish peroxidase

Hz Hertz

IA-2 Islet antigen 2

IAA Islet associated autoantibodies

IACUC Institutional animal care and use committee

IDO Indoleamine 2-3, dioxygenase

IFN Interferon

IGRP Islet-specific glucose-6-phosphatase catalytic subunit-related protein

IL Interleukin

IM Incomplete media

InsB B chain of insulin

InsB:9-23 Insulin b chain peptides 9 through 23

InsB-pσ1 Insulin B-chain genetically fused to protein sigma 1 iTreg Induced regulatory T cell

LL Lactococcus lactis

LL vector Lactococcus lactis carrying the empty pMSP3535H3 vector

LL-CFA/I Lactococcus lactis expressing CFA/I

LP Lamina propria

M Microfold

M Molar

M17-glucose M17 media with 0.5% glucose added mAb Monocolonal antibody

MALT Mucosal associated lymphoreticular tissue mg Milligram

MHC Major histocompatibility complex mL Milliliter

MLN Mesenteric lymph node mm Millimeter

MOG Myelin oligodendrocyte glycoprotein

MQ Millicule water

NALT Nasal associated-lymphoid tissue

NOD Non obese diabetic

NOR Non-obese diabetes resistant

NRP-1 Neuropilin-1

OVA Ovalbumin

PaLN Pancreatic lymph node

PBS Phosphate buffered saline

PCR Polymerase chain reaction pDC Plasmacytoid dendritic cell

PLP Proteolipid protein

PP Peyer’s Patches

PTM Post translational modification

PTPN22 Protein tyrosine phosphatase non-receptor type 22 pTreg Peripheral regulatory T cell

Pσ1 Protein sigma-1

RT Room temperature

SCFA Short chain fatty acid

SDS Sodium dodecyl-sulfate

SMQ Sterile millicule water

SPF Specific pathogen free

T1D Type 1 diabetes

T2D Type 2 diabetes

TCR T cell receptor

Teff Effector T cell

TGF-β Transforming growth factor β

TH1 T helper 1

TH17 T helper 17

TH2 T helper 2

TNF-α Tumor necrosis factor alpha

Tr1 Type 1 regulatory T cell

Treg Regulatory T cells tTreg Thymic regulatory T cells

Wks Weeks

ZnT8 Zinc transporter 8

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

MUCOSAL TOLERANCE STRATEGIES FOR TREATING TYPE 1 DIABETES IN NON- OBESE DIABETIC MICE By

Andrew Scott Nelson

December 2018

Chair: David W. Pascual Major: Veterinary Medical Science

Oral tolerance has proven effective for treating animal models of type 1 diabetes

(T1D) suggesting it may function to treat human disease. Yet, these strategies have not translated well to the clinic. One possible reason is the large amount of antigen required for tolerization in humans. Another possibility is that the heterologous nature of T1D limits the effectiveness of antigen-specific monotherapies. To overcome these obstacles, we have implemented two complimentary approaches. The first approach utilizes colonization factor antigen I (CFA/I) fimbriae from Escherichia coli. Previously, we have found CFA/I fimbriae to inhibit animal models of multiple sclerosis and arthritis.

CFA/I fimbriae initially promote bystander tolerance in an antigen-independent manner, but ultimately protection is dependent upon stimulation of antigen-specific regulatory T cells (Tregs). Initial studies with CFA/I treatment to prevent T1D showed 45% reduction in disease incidence, and an 8-fold increase in Tregs producing IL-10 and IFN-γ. CFA/I therapy was optimized and dosing with 5x107 CFUs of Lactococcus lactis expressing

CFA/I fimbriae (LL-CFA/I) every 2 weeks (wks) was shown to provide superior reduction of insulitis scores and insulin-specific T cells. Additionally, optimized therapy suppressed TH1-type inflammatory mediators Tbet and IFN-γ in T cells of the spleen

and pancreas. This suppressive phenotype was found to be stable out to 17 wks of age.

We examined the role DCs played in mediated protection and found that infection with

LL-CFA/I induced transforming growth factor-β (TGF-β) and indoleamine 2,3- dioxygenase (IDO) production by dendritic cells (DCs). This phenotype is transient and is found to evolve to reduction in costimulatory molecules CD86, CD40, and OX40L.

Furthermore, LL-CFA/I infected DCs were shown to induce IL-10-producing T cells in vitro.

Our complimentary approach utilizes reovirus protein sigma-1 (pσ1) to target microfold (M) cells of the Peyer’s patches (PPs). Antigen fused to pσ1 have been shown to induce tolerance using 1000-fold less protein than conventional methods. Mucosal administration of insulin- and GAD-pσ1 fusion proteins significantly reduced incidence of

T1D in NOD mice via induction of IL-10-producing Tregs in the spleen.

CHAPTER 1 INTRODUCTION

T1D is a chronic autoimmune disease characterized by high resting blood glucose levels or hyperglycemia. This is caused by an autoimmune attack and subsequent destruction of insulin-producing β cells in the pancreas. Insulin plays a crucial role in cellular uptake of glucose from the blood for use as energy. Left untreated, chronic hyperglycemia signals the liver to release more glucose into the blood. Cells metabolize fats in place of sugars for energy. As a result, metabolism of fats produces ketones, which can be toxic at high levels, resulting in the severe complication, diabetic ketoacidosis. Symptoms of diabetic ketoacidosis include excessive thirst, frequent urination, fatigue, nausea, vomiting, abdominal pain, dryness of mouth, and in severe cases, coma and death.

Certain cells and tissues are especially vulnerable to high blood glucose levels, demonstrating that chronic hyperglycemia itself can be dangerous. Cells in the retina are unable to downregulate glucose uptake in the context of high extracellular glucose.

This results in increased production of superoxide which leads to oxidative stress (1, 2).

Diabetic retinopathy can impair blood flow in the vessels of the retina, which induces proliferation of new vessels. However, these vessels are fragile and leaky, and retinal perfusion may ultimately lead to blindness. Cells of peripheral nerves and kidneys can be similarly damaged leading to loss of normal sensation (diabetic neuropathy) and kidney failure (diabetic nephropathy), respectively.

These medical complications create an intense burden for individuals with T1D and necessitates insulin replacement therapy, which monitoring blood glucose levels and self-administering exogenous insulin to manage the symptoms of disease.

Diagnosis of T1D occurs most frequently in children and the resulting lifelong burden of managing this disease must be shared with family and their community. This in conjunction with rising incidence rates globally over the last 30 years demonstrates the need for new therapeutic options that can address the cause of the disease, the autoimmune attack on β cells, and ultimately the prevention or reversal of T1D altogether.

The studies described in this dissertation aim to prevent the development of T1D by blocking the autoimmune attack occurring in the pancreas. Specifically, these studies aim to demonstrate the feasibility of oral, food based, therapeutics in inducing tolerance towards antigens associated with T1D. The oral route of administration was selected because of its excellent safety profile and ability to stimulate the mucosal compartment of the immune system, which has been shown to influence and modulate the systemic immune system.

Historical Context

Diabetes is a Greek term, meaning “to go through”, and was initially considered a disease of the kidneys, characterized it by frequent urination and the observation that urine could attract ants and flies. Though it was not until 1776 that a British physiologist named Matthew Dobson identified the cause of this observation being sugar in the urine

(3). In 1815, Eugene Chevreul was able to show that this sugar was glucose and not long after, a German chemist, Hermann von Fehling, developed a solution to test the levels of glucose in the urine (4). Using pancreatectomized dogs as models of diabetes, researchers Frederick Banting and Charles Best were able to identify a protein extracted from the pancreas that could lower blood glucose levels, which they called insulin (5). A third member joined their team shortly thereafter, Bertram Collip, who

focused efforts to purify insulin for testing in humans. In 1922, in Toronto, Canada, the team tested their insulin solution on a severely diabetic 14-year-old boy who was near death. Shortly after injection, the boy rapidly regained strength and appetite. They would later test their solution on volunteers with diabetes. The discovery of insulin to treat the symptoms of diabetes was momentous, where the disease was once a death sentence it could now be controlled, provided the patient continued to receive insulin. The Nobel

Prize in Physiology or Medicine was awarded to Best and the leader of their lab group,

John Macleod, in 1923.

Classification as an Autoimmune Disease

By the 1950s, researchers recognized that there were at least two different forms of diabetes, which they differentiated based on clinical observations such as age of diagnosis, presence of obesity, and insulin dependence (6). Work continued to define aspects of these diseases and the term ‘juvenile diabetes’ was used for many years to describe diabetes diagnosed in children who also required insulin replacement therapy.

Over a century ago, an inflammatory lesion was described in the pancreatic islets of

Langerhans of a young boy who died of diabetes. These lesions were later described as

‘insulitis’ and provided the first clue that diabetes may have an immune component (7).

In 1974, Bottazzo et. al was able to show that diabetic patients possessed autoantibodies specific to the islets of Langerhans; however, their methods did not allow them to identify the antigens (8). Autoantibodies towards the self-antigens insulin, glutamic acid decarboxylase 65 (GAD), and islet antigen 2 (IA-2) were later described, further supporting the notion of an autoimmune cause of disease (9–11).

Modern Diabetes

Since the wide spread use of exogenous insulin began in the 1920s, it remains the most common and safest way to manage the symptoms of diabetes. Managing blood glucose levels through use of exogenous insulin is commonly done by careful monitoring of blood glucose levels and injecting insulin. Currently, patients with T1D may opt for an insulin pump, which dispenses insulin through a catheter placed under the skin with dosing amounts controlled through patient set programming. Insulin replacement therapy manages the symptoms of T1D, but does not provide a cure.

Indeed, even with insulin replacement therapy, the life expectancy of patients type 1 diabetes is nearly 10 years lower than that of a person without the disease (12).

An estimated 415 million adults or 8.8% of the world’s population have diabetes

(13). The majority of these cases are of type 2 diabetes, with 10-15% of the total cases being of T1D. However, T1D is the most common form of diabetes in children under 15 years of age with 86,000 children being diagnosed each year (14). Recent data from diabetic patients in Sweden show a peak in the diagnosis of disease in children aged

10-12 years of age (15). T1D’s association with childhood informs the common name, juvenile diabetes, though it should be noted it can be diagnosed at any age. T1D is one of the few autoimmune diseases that does not exhibit a gender bias, effecting males and females, on average, equally (16). Globally, the incidence of disease is increasing and has been for several decades. Incidence rates vary greatly between countries, being highest in Scandinavian countries such as Sweden and Finland and lowest in

Asian countries such as China, Korea, and Japan (17). This variation may be due in part to prevalence of genetic risk factors. However, drastically different incidence rates have been observed in Finland and Estonia, which are separated by only 120

kilometers, supporting the view that the environment plays a role in determine the risk of developing T1D (18).

Genetics of Type 1 Diabetes

In the 1970s, groups of researchers performing genome-wide association studies observed a correlation with certain human leukocyte antigen (HLA) haplotypes and T1D

(19–21). These results represented definitive evidence that T1D and type 2 diabetes

(T2D) were distinct and separate diseases. It’s been demonstrated that HLA class II genes are responsible for 30%-50% of the genetic risk for T1D (22). Specifically, the

HLA haplotypes that confer the highest risk of developing disease are DR3 and DR4; at least one of these haplotypes is present in 95% of patients with T1D (22). The HLA loci is a strong indicator of disease susceptibility, with different HLA haplotypes conferring different levels of risk of disease development and some even conferring protection. For example, the HLA-DQA1*0102, DQB10602 haplotype is protective against T1D, even when autoantibodies towards insulin are present (23). A patient’s HLA haplotype is a powerful predictor of disease risk, however, is not conclusive evidence that a patient will progress to overt disease. Patients with the HLA-DR3/4 genotype have about a 1 in 15 chance of developing T1D, which is 20 times more likely than the general population

(24). However, in recent years the likelihood that a newly diagnosed patient with T1D has a high risk HLA haplotype has been decreasing, even as the incidence of T1D increases worldwide (25, 26).

A growing list of non-HLA loci have been identified and include genes for insulin, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), protein tyrosine phosphatase non-receptor type 22 (PTPN22), and the interleukin 2 receptor (27–30). The expression level of insulin expression in the thymus, in particular, plays an important role in disease

susceptibility. There are a variable number of tandem repeats upstream of the proinsulin gene that control thymic insulin levels. Experiments utilizing mice genetically modified to express graded thymic insulin levels showed that greater thymic proinsulin expression led to greater deletion of autoreactive T cells by central tolerance (31).

Siblings of patients with T1D are 15 times more likely to develop disease than the general population, affirming the importance of genetic risk in T1D development (32).

However, it is apparent that T1D does not follow a normal inheritance pattern, as monozygotic twins are often discordant for T1D (33, 34). Also, given the rapid increase in cases of T1D over the last 40 years, genetic drift cannot account for the change.

Others have reported that children born to migrant families tend to have the same risk as the endogenous population (35, 36).These observations have prompted researchers to probe for environmental factors that add risk to developing T1D.

Environmental factors

Environmental factors capable of modulating the immune system include diet, pollution, and sunlight. Incidence reporting of T1D showed higher incidences in economically well off countries, prompting some to postulate that the hygiene hypothesis affects T1D. The hygiene hypothesis states that autoimmune diseases may become more common due to lower frequency of childhood infections (37). Indeed, observations have been made showing that as the geographical distribution of infectious diseases wanes that the incidence of autoimmune diseases, including T1D, increases (37, 38). This is thought to be due to multiple underlying mechanisms including decreased education or maturation of T cell subsets which puts an individual at risk for aberrant immune responses (39). At odds with viral infections causing T1D, a

study following children in the United Kingdom found that T1D was less prevalent in children that had experienced more infections during childhood (40).

Early observations of viral infections or signs of viral infections in patients with

T1D led some to think that viruses caused the disease (41, 42). Enteroviruses have been shown to be able to infect human pancreatic islets and signs of chronic enterovirus infection have been detected in patients recently diagnosed with T1D (42,

43). However, studies with children at high risk for developing T1D showed no correlation between development of islet autoantibodies and infection with enterovirus

(44). Further complicating matters, some enterovirus studies in the non-obese diabetic

(NOD) mice have shown protective effects after infection (45, 46). Thus it is currently unclear if viral infections can cause T1D.

Looking again at the geographical distribution of T1D incidence there is also an observed bias towards countries farther north of the equator, such as Scandinavian countries. A big difference in the environment between northern and southern countries is hours of sunlight each year, which has prompted some researchers to probe the role of vitamin D. As latitude increases, individuals experience light starvation to varying degrees. At 42°N, synthesis of vitamin D via sunlight exposure to the skin is negligible from November through February (47, 48). Diagnosis of T1D in humans observes seasonal peaks, favoring winter months (49). Vitamin D has been shown to upregulate

Treg responses while suppressing T helper 1 (TH1) type responses, providing a potential mechanism for protection against autoimmune diseases (50). However, vitamin D supplementation during pregnancy was shown not to affect incidence of T1D in offspring (51). Timing the treatment may be an important factor here as having higher

serum levels of vitamin D late in pregnancy, but not early in pregnancy, correlates with lower disease incidence (52, 53).

Changes in lifestyle, diet, antibiotic usage, and hygiene can all alter a person’s microbiota, which is quickly being recognized as a factor determining T1D risk. Early observations of T1D incidence in NOD mouse colonies across different centers showed large center to center variations and variations in specific pathogen free (SPF) versus non-SPF colonies, suggesting a role for the microbiota in disease development(54). To elucidate the role of the microbiota colonies of germ-free (GF) NOD mice have been established. While some studies showed exacerbation of T1D under GF conditions, more recent studies demonstrated that GF conditions do not overtly affect T1D development (55, 56). These data suggest that microbiota diversity or the abundance of certain bacteria modulate disease risk. To that end, researchers have turned attention towards mapping the microbiome of both diabetic patients and those at high risk and found that the microbiome of patients with T1D are less diverse (57, 58). More specifically, the abundance certain genera of bacterium shifts. In both NOD mice and humans, decreases in Firmicutes and increases in Bacteroidetes have been observed

(57–59). Commensal microorganisms play multiple supportive roles in the human gut including the production of essential metabolites such as vitamins and short-chain fatty acids (SCFAs). SCFAs such as butyrate, lactate, and propionate have been shown to play protective roles in some models of inflammatory diseases, such as for multiple sclerosis, hypertension, and T1D (60–62). In children with detectable islet autoantibodies, there are lower abundances of lactate- and butyrate-producing bacteria,

suggesting an aberrant microbiome plays a role or is associated with T1D development

(63).

The NOD mouse

Type 1 diabetes gained classification as an autoimmune disease after the discovery that patients carried autoantibodies specific for insulin prior to dosing with exogenous insulin. Additionally, physicians and researchers had noted inflammatory lesions in the islets of Langerhans of recently deceased patients with T1D. Studying the pathogenesis of T1D in humans has proven difficult due to extreme challenges associated with safely acquiring pancreatic tissue from live patients. Thus, animal models have been relied upon to answer many questions regarding the pathogenesis of the disease. The most commonly used animal models are the NOD mice and the bio breeding rat models. The NOD mouse model shares many similarities with human T1D including the presence of autoantibodies including insulin, GAD, IA-2, zinc transporter 8

(Znt8), and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)

(64). Both also show a cellular infiltrate of the pancreas – known as insulitis. In the NOD mouse, as in humans, insulitis appears prior to dysglycemia. However, the bio breeding rat shows the insulitis after dysglycemia, suggesting the pathogenesis of disease differs from humans. Additionally, NOD mice and humans at risk for T1D share many genetic risk factors. Like humans, the primary genetic factor determining disease risk in the

NOD mouse is the major histocompatibility complex (MHC), known as idd1 in mice or

IDDM1 in humans. NOD mice carry I-Ag7, which is the ortholog of human HLA-DQ (65,

66).

Disease Etiology

In 1986, George Eisenbarth described a model of T1D that is close to what we know today. In this model, humans are born with sufficient functional insulin-producing β cells. After an initial insult, APCs are recruited to the pancreas, where they take up and present self-antigens before migrating to the pancreatic lymph nodes. There, they activate CD4+ and CD8+ T cells that have escaped deletion in the thymus, which in turn migrate to the islets of Langerhans to destroy β cells and further promote inflammation

(67).

Viruses as Disease Initiators

Originally, this initiating insult was thought to occur because β cells carried the autoantigens associated with this disease. However, now there are at least two hypotheses about why β cells are targeted. The most attractive option is that a viral infection of the pancreas or a direct infection of the β cells is responsible for attracting the initial immune infiltrates. Many viruses, including enteroviruses, have shown the capacity to infect β cells (68, 69). Such an infection may lead to direct targeting of β cells by macrophages or dendritic cells, leading to the presentation of autoantigens to T cells. Data is mounting showing that infection with enteroviruses such as coxsackievirus is a risk associated with developing T1D (43, 70–72). However, the data are unclear on whether viruses are capable of initiating T1D or just aiding the progression to overt disease. A recent study in humans showed no association of enteroviral infection with

T1D in children (44). Additionally, animal models have shown that viral antigen may accelerate progress to disease, but is insufficient to trigger the disease alone (73, 74).

Viruses may serve to accelerate disease in a non-specific manner by initiating an inflammatory milieu to attract T and B cells specific for islet-associated antigens.

β Cells as Disease Initiators

There is also evidence that β cells play an active role in their own destruction.

The idea of β cell suicide was proposed by Bottazzo in the 1980s when he questioned whether β cell death in T1D was primarily due to direct killing through the immune response or through apoptosis triggered by cellular stress and inflammatory signals (75,

76). Local inflammatory responses include the production of cytokines such as IL-1β and tumor necrosis factor alpha (TNF-α) by APCs, and have been shown to have negative effects on β cell survival either through induction of free radicals or the upregulation of FAS expression on the surface of β cells, furthering their susceptibility to killing by cytotoxic cells (77, 78). Furthermore, β cells are able to directly interact with immune cells through production of cytokines and expression of MHC class I and II molecules (79, 80).

β cells are optimized for production of insulin, capable of making 1 million molecules per minute in the endoplasmic reticulum. Due to this specialization, β cells become especially prone to self-directed destruction after experiencing endoplasmic reticulum (ER) stress (76, 81, 82). ER stress can alter the transcriptome and proteome of β cells, causing them to produce novel variants of native proteins associated with disease. Post transcriptional modifications of IGRP in the β cells leads to novel, immunogenic peptides, and T cells specific to these neo-antigens are detectable in patients with T1D (83). Post-translational modifications (PTMs) such as citrullination, glycosylation, or deamidation, have been shown to generate peptides able to stimulate inflammatory immune responses (84). T cell responses to modified epitopes of insulin and GAD have been detected in humans with T1D (85, 86). Additionally, autoantibodies

specific for modified insulin have been detected in some T1D patients that tested negative for autoantibodies specific for native insulin peptides (87).

Recent reports detail multiple forms of islet neo-antigens that can stimulate autoreactive T cells. Hybrid insulin peptides formed by covalent cross-linking of proinsulin peptides to other peptides present in β cell secretory granules can stimulate autoreactive T cells and have been detected in NOD mice (88). Additionally, β cells are able to naturally exocytose different and novel inulin peptides into the blood in response to inflammatory cytokines or insulitis, providing a means of making immunogenic insulin peptides systemically available (89). It is not yet clear if these responses are the cause of T1D or if they appear as a part of the disease pathogenesis. However, because these antigens are modified outside of the thymus there is no central tolerance towards them, providing a potential means of breaking tolerance towards other islet antigens.

Disease Pathogenesis

Autoantibodies specific for islet antigens appear early in life and can be present for decades prior to progression to overt T1D. While the presence of antibodies towards a single autoantigen is not a useful predictor of disease risk, individuals with antibodies against two or more islet antigens develop T1D with near certainty in the long term (90–

94). The role autoantibodies play in the development of T1D remains unclear; however, as studies utilizing NOD mice with B cells unable to secrete antibodies were shown to develop diabetes and insulitis to a significantly greater extent than B cell-deficient NOD mice (95). Additionally, autoantibodies specific for islet antigens are all intracellular antigens, which suggests that antibody-dependent cell-mediated cytotoxicity does not play a role in the destruction of β cells. On the other hand, depleting B cells from NOD

mice using an anti-CD20 antibody can reduce incidence of disease (96, 97). It is now thought that B cells play a role as APCs in T1D.

Other APCs such as DCs and macrophages also play important roles in the pathogenesis of T1D. Innate cells, such as DCs, macrophages, and neutrophils have been shown to infiltrate the pancreas of NOD mice as early as 3 wks of age (98–100).

DCs have been shown to be able to present islet self-peptides to autoreactive T cells in the pancreatic lymph nodes (PaLN) (101, 102). Macrophages can also serve as APCs to activate diabetogenic T cells, and both DCs and macrophages have been shown to be important for recruitment and retention of T cells in the islets of Langerhans (103).

Additionally, acute depletion of DCs and macrophages in NOD mice with insulitis cured insulitis, and significantly slowed progress to overt T1D (103). Macrophages also have a pathogenic effector function through production of inflammatory cytokines such as IFN-β and IL-1β, which can induce apoptosis in β cells (104). DCs, macrophages, and neutrophils are all found among the cells infiltrating human islets, suggesting they play similar roles in human T1D.

Though unproven, consistent presence of autoantibodies in humans and mice suggests ongoing immune activity in the pancreas. Kinetic studies of infiltrating immune cells in NOD mice show that macrophages and DCs precede other cell types, infiltrating the pancreas between 3 and 4 wks of age (105, 106). Around 8 wks of age, T and B cells infiltrate and expand, dominating the cellular infiltrate. Adoptive transfer experiments have shown that both CD4+ and CD8+ T cells are required for transfer of disease to T cell-deficient NOD.SCID mice (107–110). It is thought that CD4+ T cells are critical for the recruitment and licensing of CD8+ T cells, which are thought to serve as

the final killers of β cells. CD8+ T cells are the primary component of the pancreatic infiltrate in both humans and the NOD mice. In humans, it has been shown that the antigen repertoire increases in diversity the longer a person has had the disease, suggesting that epitope spreading continues after diagnosis (111).

Mechanisms of Pathogenesis

The NOD mouse model has allowed researchers to probe the immune system to identify potential mechanisms of T1D pathogenesis. Early observations in the NOD mouse showed the importance of activated CD4+ and CD8+ T cells in causing T1D (112,

113). Thus, attention was turned to the cytokines, chemokines, and other effector molecules capable of being made by T cells. IFN-γ gained attention early after it was demonstrated that naïve T cells with a diabetogenic TCR could cause T1D in neonatal

NOD mice after these T cells were differentiated to TH1 not T TH2 cells (114).

Additionally, blocking or suppressing IFN-γ production through administration of the TH2 cytokine, IL-4, was shown to protect NOD mice from developing diabetes (115). It is thought that IFN-γ contributes to T1D development via activation of macrophages and promoting the recruitment of diabetogenic T cells to the islets of Langerhans (116). IFN-

γ may also play a role in directly inducing β cell death as β cells lacking either IFN-γ or the IFN-γ receptor are resistant to cytokine induced killing, including death induced by

IL-1β (117). However, the role of IFN-γ remains unclear as NOD mice deficient in IFN-γ only experience a mild delay in T1D development, and the administration of IFN-γ does not accelerate the disease and in some contexts can prevent disease (118–120).

Additionally, some Treg phenotypes have been reported to require IFN-γ to protect mice from T1D (121).

Inhibiting IL-17 has also been shown to be protective in NOD mice, potentially implicating TH17 cells in T1D (122, 123). TH17-polarized cells bearing a diabetogenic

TCR have been shown to adoptively transfer T1D; however, they appear to differentiate towards a TH1-like phenotype first (124, 125). Further complicating conclusions on the role of IL-17 is the observation that oral dosing of Lactobacillus johnsonii in bio breeding rats protects against T1D and is associated with promoting TH17 cells (126). One possibility is that both IL-17 and IFN-γ are required for development of T1D at different stages. NOD mice lacking both IFN-γ and IL-17 develop T1D significantly less than mono-deficient mice (127).

Given that both CD4+ and CD8+ T cells are required to cause T1D, it is thought that CD4+ T cells’ role is primarily to help CD8+ T cells and activate macrophages. Mice lacking MHC class I expression do not develop T1D, suggesting CD8+ T cells are the final effectors in T1D (128). CD8+ T cells are capable of directly killing β cells through contact dependent mechanisms including granzyme B, perforin, and the Fas pathway

(129, 130). The absence of perforin significantly reduces incidence of T1D in NOD mice

(131). However, granzyme B deficiency has little effect of disease incidence, suggesting either the presence of compensatory granzyme molecules or that it is a secondary effector molecule (132, 133).

Macrophages and DCs have been reported to be crucial for the initiation of insulitis in animal models. Myeloid cells are some of the first cells to infiltrate the pancreas, and in addition to IL-1β and TNF-α, can produce type 1 interferons (T1-IFNs) such as IFN-α and IFNβ. In NOD mice, a T1-IFN signature is detectable in the pancreas at 4 to 6 wks of age, prior to T cell infiltration (134). Additionally, dosing with poly I:C, a

potent stimulator of T1-IFNs, accelerates T1D in NOD mice (135, 136). Plasmacytoid

DCs (pDCs) have been observed in the pancreas as potent T1-IFN producers, potentially playing a role in initiating disease (100, 137). However, pDCs also can act as suppressors of T cell-mediated inflammation through production of IDO, which catabolizes tryptophan and effectively starves Teffs; thus, the role pDCs play is unclear in T1D pathogenesis (137, 138). Transgenic mouse studies have sought to concisely determine if T1-IFNs are pathogenic by overexpressing them under the insulin promoter. Some mice, such as the C57BL6/SJL mice did not develop disease, but did experience hyperglycemia, whereas non-obese diabetes resistant (NOR) mice quickly developed T1D (139–142). These data suggest that although T1-IFNs may trigger autoimmune attack on β cells, other risk factors are required for the initiation of disease.

These data demonstrate that the exact etiology of T1D in humans or NOD mice is not known, and it is possible and likely that the initiation of the disease is heterogeneous. To clarify some aspects of the disease, researchers and health care providers consider the disease in stages. The three stage model currently used was originally steeped in Eisenbarth’s model and updated with more recent findings. In stage

1, patients have developed two or more islet autoantibodies and are normoglycemic. In stage 2, both autoantibodies and dysglycemia are present, but honeymoon periods may occur here as well. In the third and final stage of T1D, patients present additional clinical symptoms such as polyuria, polydipsia, weight loss, fatigue, and potentially ketoacidosis. This model of the disease illustrates two distinct points that therapies could intervene to prevent or treat T1D. In stage one, the patient still has normal levels of functioning β cells and early detection of disease markers, such as autoantibodies,

may enable prevention of T1D. In stage 2, enough β cell mass and function remains that halting the autoimmune attack may allow recovery. However, in stage 3 additional treatment would be required as halting the immune response would not restore normal blood glucose levels.

Oral Tolerance

An attractive means of halting autoimmune attack is through the induction of tolerance. Tolerance is defined as a state of immunological unresponsiveness towards an antigen. To ensure lymphocytes do not recognize self antigens, the host uses two mechanisms to enforce tolerance. Central tolerance refers to negative selection or the elimination of cells that recognize self antigen. However, the T cell receptor (TCR) repertoire is virtually unlimited, resulting in lymphocytes specific for food or other innocuous antigens. A secondary mechanism of tolerance, termed peripheral tolerance, compensates through inducing antigen-specific tolerance outside of the thymus and bone marrow. Peripheral tolerance can be induced towards specific autoantigens through mucosal administration of protein, engaging the gut-associated lymphoreticular tissue (GALT). In humans, the GALT samples over 30 kg of food proteins each year, and must manage interactions between the immune system and the gut microbiota – approximately 1012 microorganisms per gram of stool (143). Undue inflammatory responses towards food antigens or the colonizing microbiota would keep a human in a perpetual state of inflammation, e.g., diarrhea and illness. It is thought then, that the primary function of the GALT is enforcing tolerance towards dietary antigens and commensal bacteria while protecting the host from pathogens (144). Therefore, one can surmise that the GALT specializes in inducing tolerance towards the antigens that it encounters. Antigens from the intestinal lumen are constantly being sampled by

microfold (M) cells for presentation to DCs and macrophages. Studies have shown that targeting antigen for uptake by M cells can induce Tregs (145, 146). DCs associated with the can also sample luminal antigens by extending dendrites through M cell- specific transcellular pores or through tight junctions in the lamina propria (LP) (147,

148). Antigen-loaded DCs can migrate to mucosa draining lymph nodes such as the mesenteric lymph nodes (MLNs) to present antigens to T cells, either inducing Tregs or anergy in Teff cells to enforce tolerance.

Regulatory T cells

There are at least two distinct mechanisms for the induction of tolerance, which depend on the amount of antigen. High doses of antigen preferentially induce anergy or deletion of antigen-specific T cells, whereas low doses induce Tregs. Tregs were initially described as CD4+ T cells expressing IL-2 receptor alpha, CD25, on their surface in addition to the transcription factor Forkhead box P3 (Foxp3) (149, 150). However, more recent studies have demonstrated the phenotypic breadth of these anti-inflammatory cells. Natural or thymic Tregs (tTregs) develop in the thymus, express CD25, Foxp3, and can express Helios and neuropilin-1 (151–153). Tregs can be induced outside of the thymus by exposing naïve T cells to IL-2 and TGF-β (154). When done in vitro, these cells are known as induced Tregs (iTregs), and when done in vivo these are known as peripheral Tregs (pTregs).

In humans with T1D and NOD mice, there is a growing body of evidence to suggest Tregs lose their function as the disease progresses. Notably, different groups have been able to show that Tregs from humans with T1D are less able to suppress proliferation of autologous Teffs than Tregs from healthy patients (155, 156). This begs the question of whether Tregs are defective or if Teffs are resistant to suppression. Data

from human and mouse studies has shown that Teffs from diabetic patients are resistant to suppression by Tregs from healthy subjects, suggesting that the defect lies with Teffs (157–159). However, it is likely that both cell types have defects, either intrinsic or as a result of chronic inflammation, that play roles in the pathogenesis of

T1D. The defect in suppressive activity of Tregs has multiple potential causes. Tregs in patients with T1D have reduced sensitivity to IL-2, which plays a crucial role in the development and maintenance of Tregs (160–163). Additionally, Tregs from diabetic patients have been shown to co-produce inflammatory cytokines, suggesting that they differentiate towards an effector phenotype as the disease progresses (164, 165).

These data suggest that restoring or promoting Treg function and stability is a potential therapeutic strategy for treating T1D.

T1D and Tolerance

The capability of reinstating tolerance towards a self-antigen without immunocompromising the patient is an attractive treatment option. The NOD mouse has been used extensively to research the feasibility of this strategy. Oral or nasal administration of insulin or GAD has been shown to significantly reduce incidence of disease and delay onset (166–168). Additional studies have shown that oral insulin and

GAD based oral therapeutics induce regulatory cells that can adoptively transfer protection against T1D (169–173). Given these promising results and the low risks of complications with oral therapies, insulin was selected for testing in humans. The first oral insulin clinical trial, the Diabetes Prevention Trial-Type 1 enrolled patients at increased risk for developing T1D, but who were not dependent on insulin replacement therapy. The study lasted six years, and found no delay in onset of disease, but a post hoc analysis revealed a delay in patients with higher titers of insulin autoantibodies

(174, 175). These results prompted a follow up study, called the TrialNet study, and followed participants for over 9 years. However, this trial similarly did not show prevention or an overall delay in onset, but confirmed a significant delay in disease diagnosis in a small subset of patients (176). A recent pilot study using a higher dose of oral insulin in children without islet autoantibodies, called the Pre-POINT study, found induction of regulatory cytokines and Tregs responsive towards proinsulin (177). Human trials with GAD have met with similar results, potentially delaying onset of T1D in small subgroups of patients (178–180). It should be noted that the GAD trials utilized a GAD- alum vaccine and administered it subcutaneously, as opposed to orally as in the inulin trials.

Collectively, these data demonstrate some hurdles that must be overcome to successfully treat or prevent T1D in humans. In both humans and mice, the disease is heterogeneous in terms of age of onset, rate of β cell destruction, and autoantibody profile. This heterogeneity suggests the T cell response to insulin-producing β cells is also varied, complicating the effectiveness of an antigen monotherapy. Additionally, translating dosing amounts from mice to humans is difficult to estimate. A new trial has recently started, called the Immune effects of Oral Insulin, to explore multiple dose sizes in humans. However, new therapies capable efficiently of inducing antigen-specific tolerance could address these issues.

Reovirus Protein Sigma-1

In this dissertation, we will describe two complimentary approaches that take advantage of the natural preference for tolerance induction by engaging the GALT. To overcome challenges in dosing amounts and frequencies, we utilize the reovirus adhesin, protein sigma 1 (pσ1). This adhesin is specific to M cells of the PPs in the gut

and nasal associated-lymphoid tissue (NALT), and is important for reovirus entry into the body (181). PPs have been shown to play a crucial role in inducing tolerance towards food antigens (182–184). Previous work in our lab has shown that by genetically fusing the model antigen ovalbumin (OVA) to pσ1, induces tolerance to the fused antigen by stimulating antigen-specific Tregs, and that their suppressive capability is mediated by IL-10 and TGF-β (146, 185, 186). In the context of the experimental model of multiple sclerosis – experimental autoimmune encephalomyelitis (EAE) – pσ1 fused to either myelin oligodendrocyte glycoprotein (MOG) or proteolipid protein (PLP) was able treat and prevent EAE induced by the respective protein (185, 186). However, when PLP was fused to OVA-pσ1, it was unable to protect mice from MOG-induced

EAE, demonstrating the importance of antigen-specificity for the induction of tolerance

(186).

There has been much discussion over the identity of the initiating antigen in T1D.

Insulin is a likely candidate; given its expression is specific to β cells, and that GWAS show there is a strong risk associated with polymorphisms in the insulin gene and T1D

(187). In the NOD mouse, many of the islet-infiltrating T cells are reactive towards insulin, specifically the insulin B-chain peptide 9-23 (InsB:9-23) (188, 189). Additionally, tolerance studies using the NOD mouse have shown that tolerance induced towards whole insulin, but not InsB:9-23, is able to tolerize mice in late stages of disease (190).

Still, other antigens, such as GAD and IGRP, have shown similar efficacy in treating

T1D in mouse models, and autoantibodies specific for them can appear before insulin autoantibodies in humans (92, 168, 171, 178, 191–193). These data support the idea that T1D does not develop in a homogenous pattern in beginning its attack on insulin-

producing β cells. Thus, inducing tolerance to multiple antigens may be required to effectively treat the disease in humans. We will describe strategies for dosing with different and combinations of antigen-pσ1 fusions for preventing the onset of T1D.

However, the heterogeneous pathogenesis and epitope spreading to non-insulin and

GAD antigens may still limit the effectiveness of a combination treatment. To complement pσ1-based therapies, we will also describe a therapeutic capable of inducing tolerance in an antigen-independent manner.

Colonization factor antigen 1

Towards this end, we have selected CFA/I fimbriae as a candidate therapeutic for T1D. CFA/I fimbriae are derived from enterotoxigenic Escherichia coli (ETEC), and are important for ETEC to colonize the human gut (194). CFA/I fimbriae initially gained attention as a potential vaccine target to protect children against travelers’ diarrhea; however, initial attempts of treating humans orally with CFA/I or colonization factor antigen II (CFA/II) fimbriae met with limited success (195, 196). This was believed to be due to stomach acidity, even when neutralized, altering the immunogenicity of the fimbriae (197). In an effort to overcome this hurdle, CFA/I fimbriae were expressed in a vaccine vector. A live attenuated strain of Salmonella was selected based on its capability of inducing immunity towards Salmonella as well as to heterologous passenger antigens (198, 199). This efficient ability to immunize because the vector allows delivery of antigens to both the mucosal and systemic immune compartments

(200).

Expression of CFA/I is controlled by the four-gene operon, cfaABCE (201–203). cfaA, cfaB, cfaC, and cfaE encode for CfaA, CfaB, CfaC, and CfaE, respectively. CfaA is responsible for chaperoning the other components of the operon to the outer

membrane of the bacteria. CfaB is the major fimbral subunit of the fimbriae, and it along with CfaE, are the main extracellular components, forming a long, pilus structure composed of 1000:1 CfaB to CfaE ratio. CfaC acts as a platform to orchestrate proper assembly on the surface of the microbe. Expression of the CFA/I operon in a live, attenuated Salmonella vector appears visibly similar to expression in ETEC, with the same long, hair like protrusions on the surface of the microbe (204). Further facilitating expression of CFA/I, both ETEC and Salmonella are rod-shaped, Gram-negative bacteria, helping to facilitate the surface expression of the fimbriae on Salmonella.

Initial findings with Salmonella-CFA/I showed an increase in mucosal IgA and serum IgG antibodies specific for the fimbriae (205, 206). A surprising finding was that

Salmonella-CFA/I induced a biphasic T helper response with TH2 cells being rapidly induced after vaccination, followed by TH1 cells, likely to destroy the remaining salmonellae (206). These results were surprising as Salmonella vaccines typically induce a TH1 response (207, 208). Also, surprising was the finding that this altered T cell response did not affect a mouse’s ability to survive against a wild-type Salmonella challenge (209). To further elucidate the mechanism of Salmonella-CFA/I, in vitro infection of the RAW264.7 macrophage cell line was done. These experiments showed that infection with Salmonella-CFA/I induced little to no IL-1α, IL-1β, IL-6, and TNF-α even at high infection ratios whereas a Salmonella vector lacking CFA/I expression induced a proinflammatory cytokine response with as little as 1 bacterium to 80 macrophages (210). It is thought, then, that CFA/I fimbriae acts as an anti-inflammatory vaccine.

To further explore this, studies were carried out using animal models of autoimmune diseases. Using the EAE model for multiple sclerosis, oral dosing with

Salmonella-CFA/I prevented and reversed the disease (211, 212). Additionally, this protection was shown to be mediated by TGF-β-producing Foxp3+CD25+CD4+ Tregs

(212, 213). It is important to note that experiments showed that Tregs from Salmonella-

CFA/I-treated mice adoptively transferred protection against EAE to naïve mice, suggesting that knowledge of Treg antigen specificity was not needed and Salmonella-

CFA/I protects against EAE in a bystander manner.

Additional studies have explored Salmonella-CFA/I’s efficacy in treating the collagen-induced arthritis (CIA) model for rheumatoid arthritis. These studies showed that Salmonella-CFA/I can prevent or reverse development of CIA (214, 215).

Protection in this model was dependent upon two separate populations of Tregs expressing CD39 on their surface: a Foxp3+ subset producing IL-10 and a Foxp3- subset producing TGF-β (215). These data showed that Salmonella-CFA/I induced

Tregs with anti-inflammatory functions specific to disease they are treating. Collectively, these findings suggest that CFA/I fimbriae acts as an anti-inflammatory therapeutic capable of treating broad range of inflammatory diseases.

Purpose of the Study

Nearly 100 years ago, the discovery of insulin and its use to control the symptoms of T1D were heralded as a miracle of modern medicine. Patients with a once fatal disease can now safely and effectively manage their symptoms, and live relatively full lives. Even so, T1D poses an incredible burden for the patient, their loved ones, and society. Novel treatment strategies are required that can address the cause of the disease – the autoimmune attack on the pancreas. However, new therapies must be as

safe and effective as insulin replacement therapy for patients to use and must not substitute the symptoms of T1D with additional complications

The last century of research has seen great leaps in understanding of T1D.

Insights from animal models like the NOD mouse have shown the therapeutic potential of tolerance inducing strategies. However, the lack of satisfying outcomes in human trials have forced reexamination of approaches to treat this disease. There is a growing appreciation for the complex etiology and pathogenesis of T1D. By studying the gut microbiota of patients with T1D, we further our understanding of how the environment and genetics control risk of developing the disease. Additionally, we recognize new immunogenic insulin peptides, formed through PTMs, peptide fusion, or other means and how they may serve to initiate or develop T1D in humans and mice. All these data point to an understanding of T1D as a disease with a heterogeneous pathogenesis, potentially occurring through unique means on a patient to patient basis. This heterogeneity means we will have to consider therapies capable of treating a broader spectrum of aberrant immune responses.

Strategies to induce oral tolerance have been shown to be incredibly safe and their ease of use makes them excellent therapy candidates. However, tolerance strategies to treat autoimmune diseases that have proven effective in animal models have yet to yield positive outcomes for humans. Even so, disease in the NOD mouse more closely mimics human T1D than any other animal model and continues to further our understanding of the disease. The difference in observed results between mice and men demonstrates the both the drawbacks of the model and of our understanding of the disease, and thus full utilization of the NOD mouse requires we appreciate the lessons

learned from human trials. Reproducibility and understanding of protective mechanisms will be of the highest priority.

The purpose of these described studies is to show the potential of antigen- specific and antigen-independent oral tolerance strategies to prevent T1D development in both animals and humans. Selected therapeutics have individually shown effectiveness in inducing tolerance towards model antigens and in the context of autoimmunity. Additionally, previous work suggests these therapeutic candidates have the potential to overcome the hurdles that human clinical trials have illuminated; those of antigen delivery and selection. By overcoming these hurdles, these studies seek to identify immunologic mechanisms of protection in the context of T1D. Additionally, through utilization of a well-studied model of autoimmunity, these studies involve investigation how bystander suppression mediated by CFA/I fimbriae is mediated.

CHAPTER 2 ORAL LACTOCOCCAL IMMUNOTHERAPY FOR THE TREATMENT OF T1D

Background

T1D is a chronic autoimmune disease characterized by a patient’s lack of insulin and consequent inability to control blood glucose levels. This is due to autoreactive T cells that infiltrate the pancreas and destroy insulin-producing β cells. Current treatment options are limited to insulin replacement therapy, which has proven safe and generally efficient at controlling blood glucose levels. However, even with insulin replacement therapy, patients still remain susceptible to a number of severe complications including kidney disease, nerve damage, and blindness (13). It’s now recognized that T1D raises likelihood of cardiovascular disease and the average lifespan of an individual with T1D is 10 years lower than the average (216, 217). Thus, a robust therapy is needed that can halt the autoimmune attack on β cells, allowing for prevention or reversal of established disease. However, such a treatment must maintain the excellent safety standard that insulin replacement therapy has set and not replace the symptoms of t1D with novel complications.

Tolerance is an attractive means of achieving this goal. Defined as immunological unresponsiveness towards an antigen, tolerance can be induced orally, and historically has been shown to be very safe for humans and animals (144, 200,

218). Oral tolerance strategies involve administering an antigen orally and inducing tolerance either through the induction Tregs or through the induction of anergy in Teff cells, often dependent upon the dose used (219). This strategy requires knowledge of the target or initiating antigen. Studies have shown that oral tolerance strategies targeting insulin protect NOD mice from T1D (170–172). Additionally, insulin specificity

dominates the TCR repertoire in the PaLN and pancreas of pre-diabetic NOD mice and insulin autoantibodies are frequently used to help track disease progression in mice and humans (11, 188). These finding suggest a critical role for insulin in initiating T1D.

Several antigens have been described in the pathogenesis of T1D. Oral tolerance strategies targeting GAD protect NOD mice from T1D with similar efficacy to insulin based therapies (168, 171). Autoantibodies specific to GAD, IA-2, and ZnT8, have been detected in humans (9, 193, 220, 221). Autoantibodies are used as a sign of progression to T1D. However, the presence of multiple islet associated autoantibodies

(IAAs), not insulin alone, serves as an indicator that a patient will progress to overt T1D, suggesting that a broader autoimmune response is required for T1D (92, 94, 193). The first detected IAA suggests that that antigen is important for the initiation of T1D, and the appearance of additional IAAs suggests epitope spreading has occurred. When they are the first autoantibody detected, the appearance of insulin autoantibodies peaks in humans around 2 years of age, while autoantibodies towards GAD appear later, around

3 to 5 years of age (222). Notably, an INS gene risk allele was found to be associated with autoantibodies specific for insulin appearing first (222). These findings suggest that the initiation of T1D is a heterogeneous process that may be controlled by genetic factors.

This picture is further complicated by recent findings detailing neo-antigens formed through post-translational modifications or partially formed insulin protein secreted by β cells into the blood stream. Because these alterations to host native proteins occurred outside of the thymus central tolerance does not provide its usual protection against targeting self. CD4+ and CD8+ T cells that have escaped central and

peripheral mechanisms of tolerance are required for the development of T1D in mice

(107, 223). This finding is supported by the therapeutic effect anti-CD3 therapy has demonstrated in human clinical trials (224). However, the pathogenesis of T1D has yet to be fully detailed. Experiments that limit or block inflammatory mediators such as IFN-

γ, IL-17, or granzyme B do not show complete protection in the NOD mouse (132, 225,

226). Taken together, these data suggest the pathogenesis of T1D is complex and heterogeneous, potentially requiring individualized therapy from patient to patient.

An attractive means of overcoming this hurdle is the utilization of bystander tolerance. CFA/I fimbria is a virulence factor of ETEC important for its ability to colonize the gut (202). ETEC is the leading cause of diarrhea in children, and is responsible thousands of deaths each year (227). Thus, CFA/I fimbriae have been scrutinized as a target antigen for an anti-ETEC vaccine. However, a Salmonella vector expressing

CFA/I fimbriae (Salmonella-CFA/I) was shown to induce an anti-inflammatory cytokine response, demonstrating its potential as an anti-inflammatory vaccine (206, 211). This potential was further explored with experiments utilizing Salmonella-CFA/I to prevent and treat animal models of multiple sclerosis and arthritis (212, 214). In both models,

Salmonella-CFA/I was shown to initially induce a Treg response in a bystander fashion; however, the protection was shown to be mediated by antigen-specific Tregs.

Interestingly, the observed Treg phenotypes were shown to be unique to the disease they were treating. In the EAE model of multiple sclerosis, TGF-β-producing

Foxp3+CD25+CD4+ Tregs were shown to play a definitive protective role (212).

Protection in the CIA model of arthritis was mediated by two distinct populations of

Tregs expressing CD39+ on their surface; an IL-10-producing Foxp3+ population and a

TGF-β-producing Foxp3- population (215). These data suggest that CFA/I induces tolerance independent of the initiating antigen, but specific to the context of inflammation. Additionally, animals treated with Salmonella-CFA/I were shown to be able to protect themselves from Salmonella infection, suggesting that treatment with

CFA/I fimbriae do not have a globally immunosuppressive effect and maintains the excellent safety profile characteristic of oral tolerance therapies (209).

In an effort to develop CFA/I fimbriae as an oral, food-based therapy, fimbriae were expressed on Lactococcus lactis (LL). LL-CFA/I was shown to have the same protective profile that Salmonella-CFA/I had in the CIA model of arthritis (228). We hypothesize that LL-CFA/I will act as an antigen-independent, anti-inflammatory therapeutic, capable of treating a wide range of autoimmune diseases. The work detailed in this report explores this hypothesis in the context of T1D. The NOD mouse was selected to study T1D due to the many similarities with human disease, including the presence of insulitis prior to the onset of disease, insulin autoantibodies, and insulin- specific CD4+ and CD8+ T cells in the PaLNs and peripheral blood (65). Finally, the

NOD mouse provides a spontaneous model of T1D in which multiple antigens and multiple pathogenic mechanisms play roles.

Methods

NOD Mouse Husbandry

Female NOD/ShiLtJ mice (The Jackson Laboratory, Bar Harbor, ME, USA) were

3 or 5 wks of age upon arrival. Mice were housed under SPF conditions with food and water available ad libitum. Mice were allowed to acclimate to the facility for at least 5 days prior to handling. All animal procedures were approved by the University of Florida

Institutional Animal Care and Use Committee (IACUC).

Monitoring blood glucose levels

Blood glucose levels of NOD mice were checked regularly starting at 10 wks of age. Mice were restrained using a stationary cone and the tail was wiped with 70% ethanol before pricking the tail with the tip of a 23 gauge needle to draw blood. Blood was applied to an AlphaTRAK 2 (Abbott Animal Health, Abbott Park, IL, USA) glucose test strip. A reading higher than 250 mg per dekaliter (dL) is considered hyperglycemic, and the mouse was set to be screened again the following day. Diabetes was diagnosed after 2 consecutive blood glucose readings above 250 mg/dL or one reading at 500 mg/dL – the upper limit of the glucose test strip

Grading Insulitis

Freshly isolated pancreata were formalin-fixed, paraffin-embedded, deparaffinized, rehydrated, and cut in 5 µm sections for staining with H&E. Two sections were cut from each pancreata, 150 µm apart, to provide two distinct views inside the pancreas and ensuring enough islets were present for analysis. Prepared slides were analyzed under a Nikon Eclipse E200 microscope (Nikon, Minato, Tokyo, Japan). At least 20 islets from each pancreas were analyzed. Observed islets were given a score ranging from 0 to 3 based on degree of cellular infiltration as previously described (118).

Briefly, 0 corresponded to no infiltration; 1 corresponded to peri-insulitis or infiltration limited to the boundary of the islet; 2 corresponded to less than 50% of the islet infiltrated; and 3 corresponded to greater than 50% of the islet infiltrated.

Growing L. lactis for Oral Administration

L. lactis lactis IL1403 was engineered to carry the pMSP3535H3 vector in its empty state or with a synthetic operon for CFA/I fimbriae as described previously (228).

Liquid M17 media with 0.5% glucose added (M17-glucose) was used as growing

medium for all L. lactis strains. Sterile M17-glucose media were made by suspending

37.25 g of Difco M17 Broth (BD, Franklin Lakes, NJ) and 5 g of glucose (ThermoFisher,

Waltham, MA, USA) in 950 mL of deionized water. M17-glucose media was sterilized and allowed to cool to room temperature before use.

Starter cultures of LL-CFA/I and LL vector were started the day before dosing by adding 50 µL of LL-CFA/I or LL vector glycerol stock to a Falcon 14 mL Polystyrene

Round-Bottom Tube (Corning, Corning, NY, USA) with 6 mL of M17-glucose and 60 microgram (µg) of erythromycin (MilliporeSigma, Burlington, MA, USA). Starter cultures were incubated overnight at 30°C with no shaking. The following morning 2 mL of starter culture were added to 48 mL of pre-warmed M17-glucose media along with 500

µg of erythromycin (MilliporeSigma). Starting 1 hour after growth culture inoculation, OD was monitored using a BioMate spectrophotometer (ThermoFisher). L. lactis mutants were allowed to grow to an OD of 0.19 to 0.21 before adding 25 μg of nisin

(MilliporeSigma) to induce expression CFA/I fimbriae. Bacteria were incubated at 30°C for 4 hours after induction. After incubation, bacteria were collected by centrifugation and washed with DPBS to prepare for administration to mice.

Neutralization of stomach acid

Prior to oral gavage of bacteria, NOD mice were treated with a 10% sodium bicarbonate solution to neutralize stomach acid. The sodium bicarbonate solution was made fresh the day of dosing by dissolving 3 g of sodium bicarbonate (ThermoFisher) in deionized water. This solution was then diluted 1:1 in DPBS (Genesee Scientific) and filtered through an Olympus sterile 0.22 micrometer (µm) syringe filter (Genesee

Scientific). Mice were orally gavaged with 200 µL of the diluted sodium bicarbonate

solution using a reusable AFN 20 gauge, 1.5’’ long, 2.25 mm-curved gavage needles

(Cadence Science, Inc.) 5 to 10 minutes before administration of bacteria.

Oral gavage of bacteria

Four hours after nisin induction, LL-CFA/I or LL vector were collected by centrifugation. Bacteria were washed twice with DPBS before being resuspended at the concentration required for the study. Concentration was adjusted such that mice were orally dosed with 5x109, 1x108, 5x107, or 1.5x107 CFUs in 100 – 200 µL of DPBS. 10 minutes after neutralization of stomach acid with sodium bicarbonate LL-CFA/I, LL vector, or DPBS was loaded into a 1 mL syringe. Mice were orally gavaged with a reusable AFN 20 gauge, 1.5’’ long, 2.25 mm-curved gavage needles (Cadence Science,

Inc.).

Tissue Collections

At specified study endpoints mice euthanized by carbon dioxide asphyxiation.

For necropsy, mice were pinned to a small dissection stage using 23 gauge needles.

Mice were sprayed with 70% ethanol, and sterile surgical instruments were used to harvest organs. Tissues were kept in incomplete media (IM) until necropsy was complete. IM was prepared by adding 5 mL of HEPES solution (Genesee Scientific) and

5 mL of Penicillin-Streptomycin solution (100x) (Genesee Scientific) to 500 mL of RPMI

1640 Medium (Genesee Scientific).

Pancreata were collected using sterile forceps before either being fixed for staining with hematoxylin and eosin (H&E) or processed for isolation of lymphocytes.

For H&E staining, pancreata were spread evenly upon a thin sponge within a tissue cassette (University of Florida Molecular Pathology Core; Gainesville, FL, USA).

Tissues were incubated in 10% neutral buffered formalin (Leica, Wetzlar, Germany) for

at least 24 hours before being washed and stored in 70% ethanol. Processing, paraffin embedding, and cutting of slides were performed by the University of Florida Molecular

Pathology Core (University of Florida, Gainesville, FL, USA). Two 5 µm sections were cut at least 150 µm away from each other to provide two distinct points of reference for histological analysis.

Lymphocytes from the islets of Langerhans were purified as previously described

(229). Briefly, a 1 mg/mL type IV collagenase (MilliporeSigma) solution was made in IM and perfused into the pancreas through the common bile duct. After perfusion with 2 mL of collagenase solution the pancreas was removed from the animal and placed into a 50 mL conical on ice. Once all pancreata were pulled in this way conical tubes containing tissue were placed into a hot water bath set to 37°C for 15 minutes. Pancreata were mechanically dissociated by pipetting with a 10 mL serological pipette and washed 5 times with IM. After the final wash, the pellet was resuspended in 5 mL of CM and transferred to a petri plate. 5 mL of CM was then used to rinse the inside of the 50 mL conical and subsequently added to the same petri plate. Islets were purified by hand using a p200 pipette (Eppendorf) and StereoZoom 5 Dissecting microscope (Leica). To purify lymphocytes from islets, islets were first added to a 24 well plate and incubated at

37°C overnight. T cells were harvested the following day by centrifugation.

Splenocyte isolation

Spleens were mechanically dissociated using a Tissuelyzer II (Qiagen, Hilden,

Germany) at 20 hertz (Hz) for 1 minute, and then filtered through sterile nytex (Sefar

Inc.). Cells were washed with IM and resuspended in 1 mL of IM. 1 mL of 2X

Ammonium-Chloride-Potassium (ACK) lysis buffer was added to lyse red blood cells.

Cells were incubated with ACK lysis buffer at room temperature for 2 minutes. Cells were washed with IM and resuspended in CM.

Lymphocyte isolation

During necropsy, PaLNs, MLNs, and PPs were collected and placed into 2 mL

Safe-Lock Tubes (Eppendorf) tubes with 1 mL of IM and a sterile, 5 mm, stainless-steel bead. The tissue was first mechanically disrupted using a Tissuelyzer II set to 15 Hz for

1 minute. Cells were then filtered through sterile nytex (Sefar Inc.) and washed with IM and resuspended in CM.

Counting cells

Cells were counted on a Cellometer Auto T4 (Nexcelom). Single cell suspensions of cells from different tissues were isolated as described above. A Trypan Blue solution was prepared by diluting Trypan Blue Solution, 0.4% (ThermoFisher) 1:1 in DPBS. For spleens, 10 µL of the cell suspension was added to 90 µL of Trypan Blue-DPBS. 20 µL of this mixture was added to a Cellometer Disposable Counting Chamber (Nexcelom) and counted. For other tissues or blood the process was the same, except 20 µL of single cell suspensions were diluted in 20 µL of Trypan Blue-DPBS.

Cell Culture

Mixed and purified T cell cultures were used to observe changing T cell phenotypes. Single cell suspensions of cells from different tissues were isolated and counted as described above. 200,000 cells were added to each well of 96 well, round bottom, tissue culture treated plates (MilliporeSigma) plates coated with anti-CD3 (clone

17A2; Invitrogen, Carlsbad, CA, USA). Anti-CD28 (clone 37.51; Invitrogen, Carlsbad,

CA, USA) was then added to a final concentration of 2.5 µg/mL and samples incubated for 48 hours at 37°C. Stimulated cells meant for FACS analysis were treated with

Brefeldin A (Biovision, San Francisco, CA, USA), at a working concentration of 5 µg/mL, for the last 4 hours of culture. Supernatants of cell cultures meant for cytokine analysis by ELISA were collected by centrifugation and stored at -20°C until analysis.

Treg suppressor assay To observe the suppressive capacity of Tregs induced by LL-CFA/I, Tregs were co-cultured with Teff cells from PBS-treated mice. CD25+CD4+ Tregs and CD25-CD4+

Teffs were purified from spleens, MLNs, HNLNs, and axial lymph nodes as described above using magnetic bead kits. Teffs were taken from 19 wk-old NOD females. 96- well, round bottom, tissue culture treated plates (MilliporeSigma) were coated with 10

μg/mL of anti-CD3 mAb (clone 17A2, Invitrogen, Carlsbad, CA, USA) and stored at 4°C overnight. Cells were counted and plated at a 1:1 Treg to Teff ratio, with a maximum cell count of 200,000 cells per well in 200 µL of CM. 5 µg/mL of anti-CD28 mAb (clone

37.51, Invitrogen, Carlsbad, CA, USA) was added to each well and plates were incubated at 37°C for 72 hours. For the last 12 hours of culture 0.5 micro-Curie (µCi) of

3H-thymidine (PerkinElmer, Waltham, MA, USA) was added to each well. After incubation, cells were harvested using a Perkin Elmer Filtermate Cell Harvester (Perkin

Elmer) and radioactivity in DNA was measured using a Perkin Elmer Micro JET –

Microplate Scintillation and Luminescence Counter (Perkin Elmer).

Flow Cytometry

To determine the phenotype of Tregs induced by pσ1 fusion proteins, cells were washed with PBS and stained for viability using a LIVE/DEAD Fixable Blue Dead Cell

Stain Kit, for UV excitation (ThermoFisher). Cells were then washed with FB and labeled with mAbs specific for CD4, CD8α, TCR-β, CD25, CD39, CD44, CD62L, Tigit,

Tim-3, CTLA-4, PD-1, CXCR3, CD103, and TGF-β. Then cells were washed and fixed

with 1X fixation buffer from the True-Nuclear Transcription Factor Buffer Set

(BioLegend, San Diego, CA). Cells were then washed with 1X permeabilization buffer from the True-Nuclear Transcription Factor Buffer Set (BioLegend) and labeled with mAbs specific for IL-10, IFN-γ, IL-17, Foxp3, Tbet, and RORγt. Cells were labeled with antibodies diluted according to Table 2-1.

Staining with tetramers

MHC class II tetramers specific for InsB:9-23 and GAD:206-220 along with MHC class I tetramers specific to InsB:15-23 were provided by the NIH Tetramer Core Facility

(Emory University, Atlanta, GA, USA). Single cell suspensions of cells from tissues or whole blood were prepared as described above. One million cells were added to FACS tubes for preparation of compensation controls. Cells were washed and supernatants were discarded before being resuspended in 200 µL of FB. To block non-specific staining via Fc receptors, 1.25 µg of anti-mouse CD16/32 (ThermoFisher) was added to each tube. Samples were incubated for 10 minutes at RT, protected from light. Without washing, 0.63 µg of a given tetramer (NIH Tetramer Core Facility) was added to each tube. Samples were incubated at RT for 45 minutes, protected from light. Without washing, cells were stained for surface antigens as described above. Samples were then washed and analyzed on a Fortessa flow cytometer (BD) without fixation.

Cytokine ELISA

Spleens and MLNs were aseptically removed at 11 or 24 wks of age from PBS-,

LL vector-, or LL-CFA/I-treated groups of mice. Lymphocytes were prepared, as described above, and resuspended in CM. Lymphocytes were cultured at 5x106 cells/mL on 96 well, round bottom, tissue culture treated plates (MilliporeSigma) in the presence of 5 µg/mL anti-CD3 (clone 17A2; Invitrogen, Carlsbad, CA, USA) and 2.5

µg/mL anti-CD28 (clone 37.51; Invitrogen, Carlsbad, CA, USA) in a total volume of 200

µL. The supernatants were collected by centrifugation and stored at -80°C. Capture

ELISA was employed to quantify, on duplicate sets of samples, the levels of IFN-γ, IL-

10, TGF-β, and IL-17 produced by lymphocytes.

For IFN-γ, 10 µg/mL rat anti-mouse IFN-γ monoclonal antibody (mAb) (clone R4-

6A2, ThermoFisher) was used as the capture antibody, and 0.5 µg/mL of biotinylated rat anti-mouse IFN-γ mAb (clone XMG1.2; BD) was used as detecting antibody.

For IL-10, 2 µg/mL rat anti-mouse IL-10 mAb (clone JES5-2A5; eBioscience, San

Diego, CA, USA) was used as capture antibody, and 1.5 µg/mL of biotinylated rat anti- mouse IL-10 mAb (clone SXC-1; BD) was used as detecting antibody.

For TGF-β, 2 µg/mL rat anti-mouse TGF-β mAb (clone A75-2; eBioscience, San

Diego, CA, USA) was used as capture antibody, and 5 µg/mL of biotinylated rat anti- mouse TGF-β mAb (clone A75-3; BD, Franklin Lakes, NJ, USA) was used as detecting antibody.

For IL-17, 2 µg/mL rat anti-mouse IL-17 mAb (clone TC11-18H10; BD) was used as capture antibody, and 1.5 µg/mL of biotinylated rat anti-mouse IL-17 mAb (clone

TC11-8H4; BD) was used as detecting antibody.

The color reaction was developed using a horseradish peroxidase (HRP) conjugated goat anti-biotin Ab (Vector Laboratories, Burlingame, CA, USA) and ABTS peroxidase substrate (Moss, Inc., Pasadena, ME, USA). Cytokine concentrations were extrapolated from standard curves generated by recombinant murine cytokines IFN-γ

(Peprotech, Rocky Hill, NJ, USA), IL-10, TGF-β, and IL-17 (R&D Systems, Minneapolis,

MN, USA).

Statistics

All presented data are the mean ± standard error of the mean (SEM). Statistical significance was tested using GraphPad Prism 7 (Prism). One-way ANOVA with

Tukey’s multiple comparisons test were used to compare FACS data, cell counts, cytokine production, and insulitis scores. Log-rank (Mantel-Cox) tests were used to compare incidence of T1D. All results were discerned to the 95% confidence interval.

Results

Oral Treatment with LL-CFA/I Ameliorates T1D in NOD Mice

Previously, CFA/I fimbriae expressed by a Salmonella vector was shown to prevent and treat EAE and CIA in mice. In an effort to develop therapy with CFA/I fimbriae as an oral, food based therapy for humans, it was expressed on the Gram- positive bacteria LL. To test the hypothesis that LL-CFA/I could prevent T1D, NOD mice were dosed with PBS, LL carrying the empty pMSP3535 vector (LL vector), or LL-CFA/I.

Two doses of LL-CFA/I were tested; a high dose of 5x109 CFUs and a low dose of

5x107 CFUs. The first dose was given at 6 wks of age, and additional doses were given every 3 wks. The low dose regimen significantly (p<0.05) reduced incidence of T1D in

NOD mice. In fact, 52% of mice given the low dose of LL-CFA/I were protected from

T1D at 24 wks of age, as compared to PBS- and LL vector-treated mice where 16.67% and 25%, respectively showed no incidence of disease (Fig. 2-1A). For mice given the high dose of LL-CFA/I 37.5% were diabetes-free at the end of the study, which, as it turns out, was not significantly different than PBS or LL vector treatments (Fig. 2-1A).

For further study, additional groups of NOD mice were dosed with 5x107 CFUs of LL-

CFA/I or LL vector.

At 24 wks of age, the study was terminated, pancreata were collected for histological analysis. Over 44% of the observed islets from LL-CFA/I low-dose group had no lymphocytic infiltration compared to only 18% of islets from the PBS control group. However, neither LL-CFA/I high-dose nor LL-CFA/I low-dose significantly reduced insulitis scores in NOD mice as compared to PBS or LL vector groups (Fig. 2-

1B). This apparent contradiction is due to a small number of LL-CFA/I treated mice that showed no lymphocytic infiltration. One potential explanation for high insulitis scores in mice protected from T1D by LL-CFA/I is that LL-CFA/I has induced Tregs that are actively suppressing Teffs in the islets.

Previous studies utilizing CFA/I based therapies show a critical role for Tregs in protecting in animal models of autoimmunity (213–215). To determine if Tregs were induced by LL-CFA/I in the context of T1D, splenocytes were isolated and stimulated with anti-CD3 plus anti-CD28 mAbs. FACS analysis showed a 2.45-fold increase in

Foxp3+CD25+CD4+ Tregs (Fig.2-1C-D). Additionally, these Tregs were found to produce both IFN-γ and IL-10 (Fig. 2-1C-D). Cytokine ELISAs of culture supernatants support the FACS findings with a greater than 2-fold increase in IL-10 production and a significant (p<0.05) increase in IFN-γ production (Fig. 2-1E).

IFN-γ has been shown to have both protective and pathogenic effects in the pathogenesis of T1D (117, 118). One potential protective mechanism of IFN-γ is the ability to suppress IL-17 responses (118). To test whether LL-CFA/I suppressed IL-17 responses, we examined IL-17 production from CD4+ T cells by FACS analysis and cytokine ELISA. LL–CFA/I significantly decreased IL-17-producing cells in the spleens

of treated animals (p<0.05), however, IL-17 and TGF-β production by whole splenocytes was unaffected (Fig.2-1F).

LL-CFA/I induced CD25+ Tregs are suppressive in vitro

Our initial results suggested a crucial role for CD25+Foxp3+CD4+ Tregs in LL-

CFA/I mediated protection. This inference is supported by previous studies with CFA/I fimbriae where CD25+ Tregs were required for protection against EAE (213). Treg based therapy to treat T1D is complicated by observations that Tregs lose their suppressive capability and Teffs become resistant to Treg mediated suppression as the disease progresses (157, 158, 230).

To determine the role of CD25+ Tregs in LL-CFA/I mediated protection against

T1D, we queried whether they could suppress Teffs from NOD mice at a late stage of disease. Tregs were generated by dosing 7 wk-old female NOD mice with 5x107 CFUs of LL-CFA/I, LL vector, or PBS. One week later, Tregs were purified based on expression of CD25+ from the spleens, MLNs, HNLNs, PaLNs, and axial lymph nodes of treated mice. Concurrently, Teffs were purified from the spleens, MLNs, HNLNs, PaLNs, and axial lymph nodes of 19 wk-old female NOD mice. Tregs and Teffs were co- cultured at a 1:1 ratio in the presence of anti-CD3 and anti-CD28 mAbs and proliferation was measure via 3H-thymidnine uptake. CD25+ Tregs from LL-CFA/I treated mice reduced 3H-thymidine uptake in co-culture to the level of Tregs only (Fig. 2-2A). There was no observed difference in 3H-thymidine uptake in cultures with Teffs only and Teffs with Tregs from PBS or LL vector treated mice (Fig. 2-2A-B). These data indicate that

Tregs induced by LL-CFA/I therapy can suppress resistant diabetogenic Teffs.

To test whether CD25+ Tregs were responsible for LL-CFA/I mediated protection against T1D, an adoptive transfer model of T1D was utilized. Others have shown that

splenocytes from 16 wk-old, or older, NOD mice efficiently induce T1D into T cell- deficient NOD.SCID mice after adoptive transfer (107, 231). Additionally, this disease model is accelerated when splenocytes are depleted of CD25+ Tregs (232). To test the hypothesis that LL-CFA/I induced CD25+ Tregs protected NOD mice from T1D, CD25+

Tregs from groups of NOD mice treated with PBS, LL vector, or LL-CFA/I were adoptively transferred in a 1 to 20 ratio with splenocytes from 19 wk old NOD mice depleted of CD25+CD4+ Tregs into NOD.SCID recipients. Tregs were generated as before, treating 7 wk-old female NOD mice with 5x107 CFUs of LL-CFA/I, LL vector, or

PBS. Splenocytes from 19 wk-old female NOD mice were depleted of CD25+CD4+ T cells and used as a source of Teffs. Blood glucose levels were monitored 3 times each wk for 6 wks after adoptive transfer (Fig 2-2C). Tregs from LL-CFA/I treated NOD mice protected 3 of 8 mice from hyperglycemia, as compared to only 1 of 8 mice in PBS and

LL vector treated mice. However, survival curve analysis showed no significant differences between groups (Fig. 2-2D). We inferred that the CD25+CD4+ T cell population included both Teffs and Tregs. Others have demonstrated that CD25+CD4+

Tregs maintain low proliferative capacities under conditions that stimulate proliferation of CD25-CD4+ T cells (233, 234). Therefore, high 3H-thymidine uptake observed in

CD25+CD4+ Treg cultures suggest that the population includes Teffs (Fig 2-2A).

Additionally, the initial test of LL-CFA/I’s ability to protect against T1D showed a 50% reduction in disease incidence, suggesting that treatment efficacy can be optimized. It was hypothesized that optimized LL-CFA/I therapy would allow easy distinction between

Teff and Tregs.

Optimization of LL-CFA/I therapy

To optimize LL-CFA/I therapy, the presence and severity of insulitis was considered the primary measure of protective clinical effect. Insulitis has been shown to begin in NOD mice as early as 3 wks of age and is dominated by CD4+ and CD8+ T cells by 11 wks of age. Thus, experiments were designed with an 11 wk endpoint in mind. To support insulitis data, CD4+ and CD8+ T cells reactive towards insulin, which have been shown to be diabetogenic, were examined (188, 235, 236). An MHC class II tetramer specific for InsB:9-23 and a MHC class I tetramer specific for InsB:15-23 were provided by the NIH Tetramer Core Facility (Emory University, Atlanta, GA, USA) to detect CD4+ and CD8+ T cells, respectively.

Female NOD mice were dosed at 4 wks of age with 5x107 CFUs of LL-CFA/I or

PBS and additional doses administered every 3 wks, every 2 wks, or weekly.

Administering LL-CFA/I every 2 wks, not every 3 wks or weekly significantly (p<0.05) reduced the average insulitis score as compared to PBS treated mice, suggesting that increasing dosing frequency to every 2 wks provides superior protection (Fig. 2-3A).

LL-CFA/I therapy was further optimized by testing different doses of bacteria.

Previous data showed that dosing with 5x109 CFUs every 3 wks did not provide long- term protection against T1D, providing an upper limit to dose optimization (Fig. 2-1A).

Thus, 4 wk-old female NOD mice were dosed with 1x108, 5x107, or 1.5x107 CFUs of LL-

CFA/I or PBS vehicle only. Additional doses were given every 2 wks and mice were euthanized at 11 wks of age, one wk after their final dose. Only by dosing every 2 wks with 5x107 CFUs of LL-CFA/I, but not by any other treatment regimen, significantly

(p<0.05) reduced insulitis scores as compared to the PBS group (Fig.2-3B). In support of these data, both CD4+ and CD8+ T cells from the PaLNs were examined for

specificity towards insulin by tetramer analysis. The tetramer binding data are consistent with insulitis data, showing a 2-fold reduction (p<0.05) of insulin-specific CD4+ and CD8+

T cells in PaLNs of treated mice (Fig. 2-3C-E).

Together, these data show that dosing with 5x107 CFUs of LL-CFA/I every 2 wks provides superior protection against T1D. To confirm these findings, we tested whether this dosing regimen provided better protection than LL vector. No differences between

PBS and LL vector treatment were found in insulitis scores or insulin specific CD4+ and

CD8+ T cells. LL-CFA/I significantly reduced insulitis scores (p<0.005) by 1.6-fold, and insulin specific CD8+ T cells (p<0.005) by 3.1-fold as compared to the PBS group (Fig.

2-3G). These data confirm that LL-CFA/I therapy is optimized for treating T1D in the

NOD mouse.

CFA/I fimbriae have been shown to protect mice from induced models of autoimmunity through induction of antigen-specific Tregs. These Tregs were found to express CD25 or CD39, depending on the context of disease (213, 215).Thus, it was hypothesized that LL-CFA/I would induce CD25+ or CD39+ Tregs specific to InsB:9-23,

InsB15-23, or GAD:206-220 in the PaLNs of treated mice. However, no differences in

CD25 or CD39 expression by insulin-specific CD4+ T cells were not found (Fig. 2-3H).

GAD-specific CD4+ T cells were not detected at the 11 wk time-point, however, other studies suggest GAD may play a role later in T1D pathogenesis in the NOD mouse model (Fig. 2-3F) (190).

Phenotyping Tregs at 11 weeks

We next sought to utilize this optimized therapy to discern the phenotype of protective Tregs induced by LL-CFA/I. It was originally hypothesized that optimized LL-

CFA/I therapy would allow easy distinction between Tregs and Teffs. Tregs are

canonically identified using Foxp3 and CD25, however, results from the Treg suppression assay indicate CD25 is present on both Teffs and Tregs in NOD mice (Fig.

2-2A) (237). To identify Tregs induced by LL-CFA/I expression of co-inhibitory molecules PD-1, CTLA-4, Tigit, and Tim-3 were examined. T cells from the spleen,

PaLNs, and pancreas were examined for Treg presence subsequent treatment to assess if Tregs are induced at the site of inflammation and induced systemically.

Additionally, MLNs and PPs were examined as the mucosal inductive sites were the likely initiation points for oral LL-CFA/I therapy. Eleven wks was selected instead of 25 or 30 wks to ensure Treg phenotypes were not compromised by hyperglycemia and cachexia. NOD mice, like human patients with T1D, are known to experience a wasting disease, called cachexia (238). Cachexia is associated with weight loss, muscle atrophy, lymphopenia, and chronic immune activation (239). This chronic immune activation includes both inflammatory and regulatory signals and complicates immunophenotyping. Additionally, we examined Tregs expressing Foxp3 with Tbet or

RORγt, as these Tregs have been shown to specialize in suppressing TH1 and TH17 responses, respectively (240, 241).

Four wk-old female NOD females were treated according to the optimized therapy, and euthanized at 11 wks of age, 1 wk after their final dose. Treatment with LL-

CFA/I did not affect expression of co-inhibitory molecules PD-1, CTLA-4, Tigit, or Tim-3 on splenic or MLN CD4+ T cells (Fig. 2-4A-B) or in Foxp3-CD4+ T cells (data not shown).

However, LL-CFA/I significantly (p<0.05) increased Foxp3 expression by PP CD4+ T cells (Fig. 2-4B, right side). Notably, LL-CFA/I shifted the Treg to Teff ratio in the islets of Langerhans to favor Tregs (Fig. 2-4E). However, this was due to a reduction in Teffs,

as marked by Tbet, rather than an increase in Foxp3+ Tregs. This led to the hypothesis that LL-CFA/I stimulated the function of Foxp3+ Tregs. To test this hypothesis, expression co-inhibitory molecules PD-1, CTLA-4, Tigit, and Tim-3 were examined.

Additionally, expression of CD39 on Foxp3+ Tregs was examined as CD39 has been shown to regulate inflammatory cells by hydrolyzing ATP and ADP (215, 242). However,

LL-CFA/I did not affect expression of CD39 or co-inhibitory molecules in the spleen (Fig.

2-4C, left side), PaLN (Fig. 2-4C, right side), or MLN (Fig. 2-4D, left side). Splenic

Foxp3+ Tregs from LL-CFA/I treated mice did show significantly reduced (p<0.05) co- expression of RORγt and Tbet (Fig. 2-4C, left side). This finding was interesting as others have shown that Foxp3, when co-expressed with Tbet or RORγt – the signature transcription factors of TH1 and TH17, respectively – that the resulting phenotype is specialized at suppressing the type of inflammation denoted by the signature cytokine

(240, 241). However, the observation that Tbet and RORγt were co-expressed in mice with higher insulitis cores (Fig. 2-3B) and higher prevalence of insulin specific T cells

(Fig.2-3E) suggest that, in this context, these were not Tregs specialized in suppressing

TH1 or TH17 responses. Cytokine responses from splenocytes and cells from the MLNs were examined by ELISA. LL-CFA/I therapy significantly increased IL-10 production in the MLN (p<0.05) (Fig. 2-4H, right side). Here, as in the spleen and islets, Foxp3 was not increased, but inflammatory mediators were suppressed.

One potential explanation is that LL-CFA/I interferes with formation of T effector- memory. To test this hypothesis, we examined expression of CD44 and CD62L in the spleen and MLN. However, we did not observe significant changes in T effector memory cells at 11 wks (Fig. 2-4I-J). Since LL-CFA/I affected the Treg to Teff ratio in the islets of

treated mice, we queried whether LL-CFA/I was interfering with the migration of Teffs to the sites of inflammation. To test this hypothesis we examined the expression of

CXCR3, which has been shown to play an important role in trafficking to the islets on

CD4+ and CD8+ T cells (243, 244). LL-CFA/I therapy significantly reduced (p<0.05)

CXCR3 expression on Foxp3+ Tregs, but not for CD4+ or CD8+ T cells of the spleen

(Fig. 2-4C and Fig. 2-6A).

LL-CFA/I does not induce Tr1 cells at 11 weeks

Data from 24 wk old mice showed Tregs expressing both IFN-γ and IL-10, which is indicative of Tr1 cells (245). Tr1 cells are identified by expression of CD49b and Lag-

3 and are frequently Foxp3- (246). To test if LL-CFA/I induced Tr1 cells at 11 wks of age expression of CD49b and Lag-3 were examined on CD4+ T cells. However, LL-CFA/I did not induce co-expression of CD49b and Lag-3 in splenic nor MLN T cells, ex vivo

(Fig. 2-5A). Tr1 cells are plastic pTregs that can express a variety of inhibitory molecules in addition to producing IL-10 and IFN-γ (245). To further test if LL-CFA/I induced Tr1 cells at 11 wks splenocytes and lymphocytes from the MLNs and PaLNs were stimulated with anti-CD3 (Invitrogen) and anti-CD28 (Invitrogen) as previously described, and expression of CD39, PD-1, CTLA-4, IL-10, and IFN-γ were examined.

LL-CFA/I suppressed expression of Tbet in splenic (Fig. 2-5B) and PaLN (Fig. 2-5D)

CD4+ T cells after stimulation. However, expression of CD25, CD39, IL-10, IFN-γ, PD-1, and CTLA-4 were unaffected in the spleen, MLNs, and PaLNs (Fig. 2-5B-D). Since Tr1 cells were detected at 24 wks, it was hypothesized that Tr1 cells are important for protection later in disease.

These data suggest that LL-CFA/I may induce a non-CD4+ Treg. Both regulatory

B cells (Bregs) and more recently described CD122+CD8+ Tregs have been shown to

play protective roles in the context of autoimmunity (247–250). We hypothesized that

LL-CFA/I induced CD8+ Tregs in the spleen and PaLNs of NOD mice. Additionally, the

MLNs and PPs were examined for CD8+ Tregs expressing CD122, CD25, CD39, or the co-inhibitory molecules PD-1, Tigit, Tim-3, and CTLA-4. Treatment with LL-CFA/I, however, did not significantly affect these markers, suggesting that LL-CFA/I does not induce CD8+ Tregs to treat T1D (Fig. 2-6A-B). Interestingly, treatment LL-CFA/I significantly (p<0.05) suppressed Tbet expression in stimulated CD8+ T cells as well as production of inflammatory cytokines IFN-γ (p<0.05) and TNF-α (Fig. 2-6C-D)

(p<0.005). These data are consistent with observations from CD4+ T cells, showing that

LL-CFA/I restrains IFN-γ- and Tbet-mediated inflammation in 11 wk-old NOD mice.

We next queried if LL-CFA/I induced Bregs to suppress T cell-mediated inflammation. Breg phenotypes were examined in the spleens, MLNs, and PaLNs of 11 wk-old female NOD mice that were given the optimized LL-CFA/I therapy. LL-CFA/I induced CD1d+CD5+CD19+ Bregs in the spleens (p<0.05), but not the MLN or the PaLN of treated mice (Fig. 2-7A). Additionally, these Bregs were found to express the co- inhibitory molecule PD-L1 (Fig. 2-7A). However, since we did not observe Bregs in the

PaLNs, we surmised that Breg induction may play a lesser role in LL-CFA/I treatment and not the primary protective mechanism.

Taken together, data from 11 wk-old mice show LL-CFA/I suppresses Tbet and

IFN-γ mediated inflammation. The mechanism through which this suppression is mediated is not related to Bregs or CD8+ Tregs. Instead, the data show Foxp3 induction in the PPs (Fig. 2-4B, right side) and increased IL-10 production in the MLNs (Fig. 2-4H, right side). As LL-CFA/I did not increase Foxp3+ Tregs in the MLNs, it is possible that

multiple Treg phenotypes are crucial to LL-CFA/I-mediated protection, similar to what was observed in CFA/I fimbriae mediated protection against CIA (215). Our observations show that LL-CFA/I suppressed Tbet+CD4+ T cells in the islets (Fig. 2-4E-

F) as well as Tbet and RORγt expression in Tregs of the spleen (Fig. 2-4C). Since

Tbet+Foxp3+CD4+ T cells were associated with more diseased mice, we concluded that these were not protective Tregs specialized in suppressing TH1 responses as others have seen (240). In this case, we infer that this is indicative of Tregs losing their suppressive capability. Others have demonstrated that Tregs losing their suppressive capability and Teffs becoming more resistant to suppression as the T1D progresses

(155, 157, 158). The data show that Tregs induced by LL-CFA/I are capable of suppressing resistant Teffs (Fig. 2-2A-B) and are not becoming ex-Tregs as marked by co-expression of Tbet or RORγt (Fig. 2-4C and Fig. 2-4E-F). Alternatively, Foxp3+Tbet+

Tregs in PBS and LL vector treated mice may be induced by inflammatory IFN-γ- and

TNF-α producing CD8+ T cells (Fig. 2-5C, right side).

LL-CFA/I induced Tregs are stable out to 17 weeks

To test whether LL-CFA/I induced Tregs are stable we examined Treg phenotypes at 17 wks of age. Seventeen wks was selected because our incidence studies showed the high frequency of T1D diagnosis occurs between 16 and 20 wks of age, thus providing us with a time-point just prior to overt diabetes. We dosed 4 wk-old female NOD mice with the optimized LL-CFA/I therapy, giving additional doses every 2 wks. Mice were euthanized at 17 wks of age, 1 wk after their final dose and Treg phenotypes were examined in the spleen, MLNs, and PaLNs. There were no observed differences in Treg or T effector-memory phenotypes in the spleens or MLNs taken ex vivo (Fig. 2-8A-C). Notably, LL-CFA/I reduced IFN-γ-producing splenic CD4+ T cells 10-

fold (p<0.005) (Fig. 2-8D-E). IFN-γ-producing splenic CD8+ T cells were also significantly reduced (p<0.05) (Fig. 2-8D-E). Additionally, LL-CFA/I suppressed expression of Tbet and IFN-γ by Foxp3+ Tregs of the PaLNs (p<0.05), supporting our hypothesis that LL-CFA/I induces stable, suppressive Tregs that protect against T1D

(Fig. 2-8F-G).

Optimized Therapy Reduces Incidence of T1D in NOD mice

To test whether LL-CFA/I optimized LL-CFA/I therapy protected NOD mice against T1D, 4 wk-old NOD mice (n=10/group) were orally dosed with 5x107 CFUs of

LL-CFA/I, LL vector, or 200 µL PBS. As done previously, blood glucose levels were monitored 3 times/wk starting at 10 wks of age. At 30 wks, 40% of LL-CFA/I treated mice remained normo-glycemic and significantly different compared to 100% PBS control mice (p<0.05), but not to LL vector-treated mice (Fig. 2-9A-B). Additionally, treatment with LL-CFA/I, but not LL vector, significantly (p<0.05) suppressed Tbet expressing CD4+ T cells in the pancreas (Fig. 2-9C). These results are consistent with disease incidence findings at 24 wks as well as TH1 specific suppression at 11 and 17 wks of age. These results show that LL-CFA/I induces varied Foxp3+CD4+ Treg responses at different stages of disease as well as consistent suppression of pathogenic TH1 mediated inflammation (Fig. 2-10A-B). Additionally, these data show the protective effect of LL-CFA/I therapy is stable in the long-term.

Discussion

The global incidence of T1D is expected to increase by 3% each year for the foreseeable future (17). Insulin replacement therapy may be the closest thing to a medical miracle that we have seen in the last 100 years. However, while insulin

replacement therapy is safe and effective at controlling blood glucose levels it does little to address the underlying cause of the disease, the autoimmune attack on β cells.

In the NOD mouse, development of T1D requires both CD4+ and CD8+ T cells

(223). While this finding has not been conclusively proven in humans, the presence of T cells specific to insulin and GAD in the peripheral blood and islets of patients with T1D suggest a similar autoimmune pathology (251–254). In support of this, treatment with anti-CD3 antibody to deplete T cells protects NOD mice from T1D and slows progression to overt T1D in humans (243, 255–257). Together, these findings have prompted several studies to elucidate how CD4+ and CD8+ T cells cause T1D. The IFN-

+ γ-producing TH1 phenotype dominates islet infiltrating CD4 T cells in humans and in

NOD mice, blocking IFN-γ can protect against T1D (117, 258–260). However, IFN-γ has

+ also been shown to be protective in some cases, either by suppressing TH17 or CD8 T cell responses (118, 261). CD4+ T cells are thought to be required for attracting and activating CD8+ T cells, which have been implicated as the primary killer of β cells (262,

263). However, studies targeting cytolytic pathways such as granzyme B do not show protection in the NOD mice (132, 133). Additionally, β cells are prone to apoptosis induced by proinflammatory cytokines such as T1-IFNs, IL-1β, and TNF-α (77, 78, 264).

Additionally, a recent report studying samples from humans just prior to or after disease onset found differential T cell cytokine profiles in patients with different risk of developing T1D (265). Further studies in humans have shown that the age of appearance of the first IAA is dependent upon IAA specificity, with insulin IAA appearing at 2 years of age and GAD IAAs appearing at 3-5 years of age, suggesting heterogeneity in the initiation of T1D (222). These findings describe a disease with

multiple, potentially compensatory, mechanisms of pathogenesis, potentially requiring individualized approaches to treatment.

Reviews of therapeutic interventions against T1D in NOD mouse reveal patterns of efficacy in respect to the age of the mouse (266). Different therapies utilizing IL-10 demonstrated that dosing mice younger than 10 wks of age provided a strong protective effect, but was unable to protect mice older than 15 wks of age from T1D (267, 268).

Another report showed that dosing 2 wk-old NOD mice with TNF-α accelerated disease, but giving the same therapy to 4 wk-old mice slowed progression to overt T1D (269).

Most notably, therapies utilizing anti-CD3 mAb are effective at preventing T1D when given early and can induce remission from disease. However, there is a range of ages from 4 to 12 wks in the NOD mouse where anti-CD3 mAb seemingly is not protective

(266). Though NOD mouse is a spontaneous model of autoimmunity, studies detailing the transcriptional landscape and populations of islet-infiltrating lymphocytes show that the disease progresses at a regular pace (106, 134). Collectively, these findings indicate that the pathogenesis of T1D is heterogeneous and evolves as the disease progresses. Additionally, they suggest that therapies targeting a single antigen or inflammatory pathway will have limited protective efficacy.

To overcome these hurdles the induction bystander suppression via CFA/I fimbriae may offer an approach to prevent development T1D in the NOD mouse. CFA/I fimbriae were shown to prevent disease in induced models of multiple sclerosis and arthritis (211, 214). Protection was ultimately shown to be dependent upon antigen- specific Tregs. Notably, the phenotypes of these Tregs varied between the models of

EAE, and CIA. In the EAE model of multiple sclerosis, TGF-β-producing Foxp3+CD25+

CD4+ Tregs were shown to play a definitive protective role (212). Protection against

CIA, however, was mediated via two distinct populations of Tregs, both expressing

CD39+ on their surface: an IL-10-producing Foxp3+ population and a TGF-β-producing

Foxp3- population (215). These findings indicate that CFA/I-induced bystander suppression results in the stimulation of different Treg populations uniquely equipped to suppress inflammation in the context of different inflammatory diseases. Additionally, treatment with Salmonella-CFA/I does not affect a mouse’s ability to respond to wild- type Salmonella challenge, suggesting that CFA/I fimbriae offer uniquely powerful means of treating autoimmune diseases without risking immunosuppression or immunocompromising the patient (209).The mechanistic repertoire of Tregs induced by

CFA/I fimbriae includes inflammatory cytokines IL-10 and TGF-β as well as expression of CD25 and CD39. Expanding Tregs independent of antigen in the NOD mouse has proven to be effective at treating and reversing the disease and IL-10 and TGF-β are critical for protection in Treg mediated therapies (270–272). These findings suggest that

LL-CFA/I is an excellent therapeutic candidate for treating T1D.

Therapy with LL-CFA/I was optimized by examining insulitis as the primary clinical marker for T1D. Insulitis has been shown to begin as early as 3 wks in the NOD mouse and by 10 wks of age, the lymphocytic infiltrate is dominated by CD8+ and CD4+

T cells, both of which are required for T1D development (106, 107, 112, 113). The data show that after optimization, LL-CFA/I protects NOD mice from T1D, preventing disease onset in 40% of mice. It is possible that therapy with LL-CFA/I therapy could be further optimized. Recent tolerization studies with L. lactis expressing insulin, GAD, or heat shock protein show the potential for L. lactis based therapies for the treatment of T1D

(273–276). Moreover, studies utilizing L. lactis expressing proinsulin or GAD in tandem with IL-10 showed the capability to reverse the disease when preceded by systemic treatment with a suboptimal dose of anti-CD3 mAb (273–275). Both therapies utilized a treatment regimen where NOD mice were treated each day for 1 wk. It has also been shown that LL does not colonize the gut, which we interpreted as a strength showing that LL based therapy would not induce a permanent immunoregulatory effect (277).

Viewed differently, it may demonstrate a need for short term frequent dosing to promote antigen visibility to the muscosal associated lymphoreticular tissue (MALT). Additionally, these studies with LL suggest that LL-CFA/I is capable of reversing T1D in NOD mice when combined with anti-CD3 mAb therapy.

We utilized the optimized LL-CFA/I therapy at the 11 wk time-point to examine the phenotype of Tregs induced by LL-CFA/I. This disease time-point was selected to study as others have shown that T cells have become the dominant component of insulitis by this age, providing a window of opportunity to study how LL-CFA/I induced

Tregs suppress diabetogenic Teffs (106). Additionally, this time-point was chosen over a terminal one to control for aberrant immune responses due to cachexia, hyperglycemia, or survivor bias (238). However, no significant changes were observed in Foxp3 expression or co-inhibitory molecules such as CTLA-4, PD-1, Tigit, or Tim-3 in

CD4+ T cells of the PaLN or pancreas. Treatment with LL-CFA/I did stimulate Foxp3+

Tregs in the PPs as well as IL-10-producing cells in the MLNs of treated mice. IL-10 has shown to be protective in animal models of autoimmunity, including T1D (278).

Additionally, LL-CFA/I has been shown to induce IL-10-producing Tregs in CIA (228).

Since there was no concurrent increase in Foxp3+ Tregs in the MLNs of treated mice it

is possible that LL-CFA/I is inducing Foxp3- Tr1 cells. Tr1 cells have previously been described as Tregs that produce IL-10 and IFN-γ, similar to what was observed in 24 wk-old NOD mice treated with LL-CFA/I (245). While IFN-γ is canonically an inflammatory cytokine it has been implicated in suppressing the activity of diabetogenic

CD8+ T cells, suggesting it may be protective in the context of T1D (261). Tr1 cells were not detected at 11 wks when CD49b, Lag-3, or expression of IFN-γ and IL-10 were examined. However, CD49b is also expressed by memory T cells and Lag-3 activates

DCs when it becomes soluble, limiting their use as identifying markers (245, 246, 279–

281). IL-27 has been shown to play an important role in differentiating Tr1 cells, activating aryl hydrocarbon receptor (AhR) and c-Maf to transactivate Il10 transcription, and thus providing potentially more accurate markers for Tr1 cells (282–284).

CFA/I fimbriae have previously been shown to induce multiple Treg phenotypes to protect mice from CIA (215). Thus, the possibility was considered that LL-CFA/I induced Bregs or CD8+ Tregs – marked by PD-1 and CD122. While CD8+ Tregs were not observed, CD19+CD5+CD1d+ Bregs were detected in the spleen of treated mice.

Additionally, these Bregs were found to express PD-L1. PD-L1 expression has been found to be low in the NOD mouse, and enhancing expressing through genetic overexpression was shown to reverse disease (285). Since Bregs were found in the spleen, and not in the PaLNs or pancreas, it is thought that they are not the main contributor to protection mediated by LL-CFA/I, though they may be responsible for the observed suppression of Tbet and IFN-γ expression in splenic CD4+ and CD8+ T cells at

11 wks, respectively. This would suggest they are capable of suppressing autoimmunity in the periphery.

In our colony, most female NOD mice become diabetic between 16 and 20 wks.

To further elucidate the mechanism of LL-CFA/I Treg phenotypes were examined at 17 wks. This time-point offered a window to examine Treg phenotypes at a late stage in disease. Data from 11 wks indicate that systemic suppression of Tbet and IFN-γ- producing CD4+ and CD8+ T cells is a sign of protection, providing a means to determine if the protective effect of LL-CFA/I is stable at this late disease time-point.

The data from 17 wk-old NOD mice showed that LL-CFA/I mediated suppression of

IFN-γ in the spleen was stable. Additionally, examination of Foxp3+ Tregs from the

PaLNs revealed that Foxp3+ Tregs from PBS or LL vector-treated mice expressed Tbet and IFN-γ after stimulation but that LL-CFA/I prevented this co-expression. These data suggest Tregs in T1D become ex-Tregs or otherwise lose their suppressive function as the disease progresses. These findings are consistent with other reports suggesting that

Tregs in T1D are defective, or lose function as the disease progresses (286).

Additionally, data from the Treg suppression assay shows that Tregs from healthy NOD mice are unable to control proliferation of Teffs from a late stage in disease. Taken together, these data support suppression resistant Teffs and dysfunctional Tregs both play a role in the pathogenesis of T1D.

These data, when considered collectively, suggest that LL-CFA/I induces multiple cell types to protect NOD mice from diabetes. At 11 wks, LL-CFA/I induces IL-10- producing cells and Foxp3 Tregs in the MLNs and PPs, respectively. PD-L1 expressing

Bregs are concurrently induced in the spleen. Later in disease, Foxp3+Tbet-IFN-γ- Tregs in the PaLNs are important. Finally, data from 24 wk-old mice show that IL-10- and IFN-

γ-producing Tr1 cells are critical to protection mediated by LL-CFA/I. Induction of Tr1

cells is notable as others have shown that Tr1 cells can be induced in the periphery and suppress diabetogenic T cell responses to prevent development of T1D (287).

Additionally, in humans, Tr1 cells, but not Foxp3+CD25+CD4+ Tregs, correlate with ease of blood glucose control after diagnosis and Tr1 cells are reduced in adults with T1D, suggesting that Tr1 cells play an important role in controlling the symptoms of disease after diagnosis (288–290). Collectively, these data show that LL-CFA/I stimulates multiple regulatory mechanisms at different time-points to protect NOD mice from the heterogeneous, evolving pathogenesis of T1D.

Figure 2-1. Oral treatment with LL-CFA/I ameliorates T1D in NOD mice. Six wk-old female NOD mice were orally dosed with 5x107 CFUs or 5x109 CFUs of LL- CFA/I, 5x107 CFUs LL vector, or 200 µL of sterile PBS. Data are representative of 2 experiments. A) Incidence of T1D in mice given indicated treatments. Dotted line represents final disease incidence in mice treated with LL-CFA/I low dose. B) Pancreata were stained with H&E for examination of insulitis. Individual islets were scored on a scale of 0 to 3, left side. Frequency of observed scores, middle, and the average score per mouse, right, are shown. At the termination of the study splenocytes were stimulated with anti- CD3 and anti-CD28 for 72 hours. C) Representative plots of CD4+ T cells, upper, and Foxp3+CD25+CD4+ Tregs, lower. D) Total CD4+ cells expressing CD25+ and Foxp3+, left side, or IL-10 and IFN-γ, right side, after stimulation with anti-CD3 and anti-CD28. E) Concentration of IL-10, left side, and IFN-γ, right side in supernatants of splenocytes stimulated with anti-CD3 and anti- CD28. F) Percentage of splenic CD4+ T cells expressing IL-17, left side, and concentration of IL-17, middle, and TGF-β in supernatants of splenocytes stimulated with anti-CD3 and anti-CD28. *p<0.05 as compared with PBS and LL vector groups, #p<0.05 as compared with LL vector group, +p<0.05 as compared with PBS group. Data are representative of 2 experiments.

PBS LL vector LL-CFA/I

CD25

Foxp3

- IL

IFN-γ

+ + + + +

CD25 Foxp3 Tregs IL -1 0 IF N - F o x p 3 T re g s r 05 0 5 r 410 e 2 .5  1 0

e *

b * b 0 5 05 m 2 .0  1 0

m 310

u n

0 5

n l

1 .5  1 0

l l

l 05

210 e

e 0 5

C 1 .0  1 0

l 05

110 a 0 4 t a 5 .0 1 0

t 

o T T 0 0 r I S r /I S o / B to A B t A P c F c F e C P e v - v C L L - L L L L L L

Figure 2-1. Continued.

5 5 0 +

) *

) l

4 4 0

/ g

3 g 3 0

(

 0 2 0

2 -

- F

L 1 0 I

I 1

0 0 I I r / S r / S o o A B t A B t c F P c F P e C e C v - v - L L L L L L L L

Figure 2-1. Continued.

Teff (Disease) + - - - + + + + Treg (Disease) - + - - - + - - Treg (Vector) - - + - - - + - Treg (Vaccine) - - - + - - - + CD3.CD28 + + + + - + + + B

6 0

n o i 4 0

s *

e r

p 2 0

u S

0 %

-2 0

s s s g g g e re r re T T T r I S o / t A B c P e F C v - L L L L

Figure 2-2. LL-CFA/I induced CD25+ Tregs suppress proliferation of diabetogenic Teffs. 7 wk-old female NOD mice were orally dosed with 5x107 CFUs of LL-CFA/I, LL vector, or 200 µL of DPBS. 1 wk later, CD25+ Tregs were purified from treated each treatment group. Tregs were co-cultured with CD25-CD4+ Teffs from 19 wk-old untreated NOD mice at a 1:1 ratio and stimulated with anti- CD3 and anti-CD28 in triplicate. Proliferation was measured by 3H-thymidine uptake. A) 3H-thymidine counts of individual cultures and co-cultures. B) Suppression index of Treg/Teff co-cultures. C) NOD.SCID mice adoptively transferred diabetogenic splenocytes and Tregs from NOD mice treated with the indicated treatment (n=8/group). D) Summary incidence of T1D of NOD.SCID mice adoptively transferred diabetogenic splenocytes and Tregs from indicated sources. *p<0.05 as compared with LL vector and PBS.

e 100 PBS e r LL vector

F 80

s LL-CFA/I e

t 60 e

b 40

i D

20 % 0 0 5 0 5 0 5 0 5 1 1 2 2 3 3 4 4 Days post transfer

Figure 2-2 Continued.

)

l a

A t

f o

1 0 0

3 %

( 8 0

2 e

r 6 0 1

o c

4 0 0

i 2 0

i l

u 0 s s s ly n S k k k

I e e B e e e P e w w 3 2 W

)

B l

f 1 0 0

e 2 .0

o 3

% 2 7 5 c

( 1 .5

e 1 +

e o

5 0 0 g 1 .0

s 0 .5 v

i 2 5

A l

u 0 .0

s ) ) ) 0 8 7 7

n S ^ ^ ^

I h d B 0 0 0 S w 1 ig e o P 1 1 B x x x H M L 1 5 5 P I I ( ( . /I / / I I 1 / / ( A A A A A /I F F F F F A C C C C C - - - - F - C L L L L L - L L L L L L L

Figure 2-3. Dose optimization of LL-CFA/I in NOD mice. 4 wk-old female NOD mice were dosed with LL-CFA/I or PBS in varying doses and frequencies. A) Mice were dosed with 5x107 CFUs of LL-CFA/I ever wk, every 2 wks, or every 3 wks until 11 wks of age. Average insulitis levels are shown of a representative example of 3 experiments (n=12/group). B) Mice were dosed with 1x108, 5x107, or 1.57 CFUs of LL-CFA/I every 2 wks. Average insulitis scores are shown of a representative of 2 experiments (n=8/group). At termination of studies, insulin specific CD4+ and CD8+ T cells were examined from the PaLNs. C) Representative plots of InsB:9-23 specific CD4+ T cells and D) of InsB:15-23 specific CD8+ T cells of the PaLN of mice given different doses of LL-CFA/I. E) Summary graphs of insulin specific CD4+ and CD8+ T cells of the PaLN. Data were combined from 2 different experiments for n=8/group. F) FACS analysis of GAD206-220 specific CD4+ T cells of the PaLN (n=4/group). G) Insulitis and tetramer summary comparing optimized LL- CFA/I and LL vector therapy. H) Characterization of insulin specific CD4+ and CD8+ T cells of the PaLN. *p<0.05 for LL-CFA/I compared to all other groups, **p<0.005 as compared against all other groups, +p<0.05 as compared against PBS.

PBS High Medium Low

- InsB:9

CD4

PBS High Medium Low

- InsB:15

CD8

0 .5 0 .8

0 .4

+ + 8

4 0 .6 D

D 0 .3

C C

0 .4

f f o

o 0 .2

* + % % 0 .2 0 .1

0 .0 0 .0

S h d w S h d w B ig e o B ig e o P H M L P H M L

HEL GAD206-220 InsB9-23

Tetramer

Figure 2-3 Continued.

E x v iv o P e y e r's p a tc h e s C D 4

2 0 P B S

+ * L L v e c to r 4 1 5

D L L -C F A /I C

1 0

5 %

5 3 3 2 p x R D o C C F X C

Figure 2-4. Phenotyping Tregs in NOD mice. Four wk-old female NOD mice were orally dosed with 5x107 CFUs of LL-CFA/I, LL vector, or 200 μL of PBS. Additional doses were given every 2 weeks. At 11 wks of age mice were euthanized and CD4+ Treg phenotypes were examined in the A) spleen, PaLN, B) MLN, and PPs. C) and D) Summary of FACS analysis of Foxp3+CD4+ Tregs. E) Representative plots of Foxp3 and Tbet expression in pancreatic CD4+ T cells. F) Summary of Foxp3 and Tbet expression in pancreatic CD4+ T cells. G) Concentration of IL-10 found in cultures of cells from the spleen and MLNs stimulated with anti-CD3 and anti-CD28. H) Concentration of IFN-γ from stimulated cells of the spleen and MLNs. I) and J) Summary of FACS analysis of T effector-memory molecules, CD44 and CD62L of T cells of the spleen and MLN. *p<0.05 for LL-CFA/I compared to all other groups, **p<0.005 as compared against all other groups, +p<0.05 as compared against PBS.

E x v iv o S p le e n F o x p 3 E x v iv o P a L n F o x p 3

6 0 8 0

+ P B S

+ p

3 L L v e c to r

p x

x 6 0 o

4 0 o L L -C F A /I

+ 4

4 4 0

C f

2 0

o f

+ 2 0

* + % % 0 0 t 5 9 1 4 t t it 3 3 3 5 9 - 0  3 - -  e - 2 3 - e 2 3 ig R 0 N 1 F R b D A R b m 1 D D - D D L O T T i C F L G C T P T D C C I I X C C T R X T C C C C

PBS LL vector LL-CFA/I

Foxp3

Tbet Figure 2-4 Continued.

F )

) 4 0 +

4 8

D C

C 3 0

4 2 0

(

+ t 3 *

2 1 0

b o T 0

F 0 r I r I / / S o A S o A t B t B c F c F P e P e C C v - v - L L L L L L L L

S p le e n IF N - M L N IF N -

6 0 6 0

) )

L L

m m /

/ 4 0 4 0

g g

n n

( (

  -

- 2 0 2 0

N N

F F

I I

0 0 r I r I S / S / to A to A B c B c P F P F e C e C v - v - L L L L L L L L

S p le e n IL -1 0 M L N IL -1 0

2 5 1 0 .0

)

) l

2 0 L

7 .5

/ g

1 5 g

n (

( 5 .0

0 1 0

1 -

- 2 .5

L L

I 5 I

0 0 .0

r /I r I S o S o / B t A B t A P c F P c F e C e C v - v - L L L L L L L L

Figure 2-4 Continued.

S p le e n C D 4 S p le e n C D 8

8 0 8 0 P B S P B S

L L v e c to r L L v e c to r

6 0 6 0

8 4

L L -C F A /I L L -C F A /I

4 0 4 0

% % 2 0 2 0

0 0

4 + + 4 + + 4 L L 4 L L D 2 2 D 2 2 C 6 6 C 6 6 D D D D C C C C + + 4 4 4 4 D D C C

M L N C D 4 M L N C D 8

1 0 0 1 0 0 P B S P B S

8 0 L L v e c to r 8 0 L L v e c to r

8 D L L -C F A /I D L L -C F A /I

6 0 6 0

4 0 4 0

% % 2 0 2 0

0 0

4 + + 4 + + 4 L L 4 L L D 2 2 2 2 6 D 6 C 6 C 6 D D D D C C C C + + 4 4 4 4 D D C C

Figure 2-4 Continued.

PBS LL vector LL-CFA/I

Spleen

Lag

MLN

CD49b

S p le e n C D 4 S p le e n F o x p 3

1 0 0 1 0 0

+ P B S 4

8 0 D 8 0 L L v e c to r

C 4

+ L L -C F A /I D

6 0 3 6 0

f o

o 4 0 4 0

+ f %

2 0 o 2 0

0 % 0 t  t  5 9 3 - 0 1 4 5 9 - 0  1 4 2 3 p e 1 - 2 3 e 1 - - x b N - D A b N - F D A D D o T F L L D D T F L L C C I I P T C C I I G P T F C T C

Figure 2-5. LL-CFA/I does not induce Tr1 cells at 11 wks in NOD mice. 4 wk-old female NOD mice were orally dosed with 5x107 CFUs of LL-CFA/I, LL vector, or 200 μL of PBS. Additional doses were given every 2 wks. At 11 wks of age mice were euthanized. A) Representative plots of CD49b and Lag-3 expression on CD4+ T cells of the spleen and MLNs. Results of the combined data of 2 experiments (n=5/group). CD4+ Treg phenotypes were examined after stimulation with anti-CD3 and anti-CD28 in the B) spleen, C) MLNs, and D) PaLNs. Results are the combined data of 3 experiments of n=10/group. +p<0.05 for LL-CFA/I compared to PBS, #p<0.05 for LL-CFA/I compared to LL vector.

M L N C D 4 M L N F o x p 3

1 0 0 1 0 0

+ P B S 3

8 0 p 8 0 L L v e c to r

x 4

o L L -C F A /I F

D 6 0 6 0

f D

o 4 0 4 0

f %

2 0 o 2 0

0 % 0  t 5 9 - 0 7 1 3 5 9  0 7 1 2 3 1 1 - p e - - N - - b 2 3 N 1 1 D D D x D D - - D IF IL IL P o T F L L P C C F C C I I I

P a L N C D 4 P a L N F o x p 3

1 0 0 2 5

+ P B S 3

8 0 p 2 0 L L v e c to r

+ o

4 L L -C F A /I F

D 6 0 1 5

f D

o 4 0 1 0

% +

2 0 o 5

0 % 0

t 1   1 t 5 9 3 e - - 0 -4 -4 - 0 - e 2 3 p b N -1 N -1 b D D x D A A D o T P IF IL L L IF IL P T C C F T T C C

Fig. 2-5 Continued.

E x v iv o S p le e n C D 8 E x v iv o P a L N C D 8

2 0 1 5 P B S

L L v e c to r

8 1 5 +

8 1 0 L L -C F A /I

1 0 f

5 %

% 5

0 0

it 5 9 3 t 5 9 2 -1 3 4 e 2 3 2 ig R A 2 3 R b D D 1 D T L D D C T P C C C D X T C C X C C C C

E x v iv o M L N C D 8 E x v iv o p a tc h e s C D 8

2 5 1 0 P B S 2 0

+ 8 L L v e c to r

+ 8

8 L L -C F A /I D

1 5 D 6

f f

o 1 0

o 4

% 5 % 2

0 0 t t 2 1 5 9 4 i 3 3 t 2 - 2 3 e g - 5 3 D A b i R 2 e 1 D D L T T im C R b D P C C T T D C T X C C C C X C

Figure 2-6. LL-CFA/I suppresses inflammatory CD8+ T cell responses in NOD mice. 4 wk-old female NOD mice were orally dosed with 5x107 CFUs of LL-CFA/I, LL vector, or 200 μL of PBS. Additional doses were given every 2 wks. At 11 wks of age, mice were euthanized and CD8+ Treg phenotypes were examined in the A) spleen, PaLNs, B) MLNs, and PPs. C) and D) FACS analysis of stimulated splenocytes and lymphocytes from MLNs and PaLNs. Results are the combined data of 3 experiments of n=10/group. *p<0.05 for LL-CFA/I compared to all other groups, **p<0.005 as compared against all other groups.

S p le e n C D 8

1 0 0

8 0

+ 8

D 6 0

o 4 0

% * 2 0

0 t 5 9 2 3 4 -1 e 2 3 2 R A b D D 1 L D C P T C C D X T C C C

M L N C D 8 P a L N C D 8

8 0 1 0 0 P B S

8 0 L L v e c to r

6 0

8 8

L L -C F A /I D

D 6 0

f 4 0

4 0 % % 2 0 2 0

0 0  t t  5 9 2 - 0 7 1 5 9 - 0 1 2 3 2 1 1 - e 2 3 e 1 - N - - D b b N - D D D 1 F L L T D D T F L C C D I I I P C C I I P C

Fig. 2-6 Continued.

S p le n ic B c e lls M L N B c e lls

2 0 1 0 + + P B S P B S

L L v e c to r 8 L L v e c to r

1 5

9 9 1

1 L L -C F A /I L L -C F A /I D

D 6

C C

1 0

f f

o o

% % 5 2

0 0

5 d L 0 1 2  5 d L 0 1 2  D 1 1 L L - D 1 1 L L - S L F S L F C D O I D D C D O I D D C P P G C P P G IC T IC T

P a L N B c e lls

8 P B S

L L v e c to r

6 9

1 L L -C F A /I

D C

% 2

5 d L 0 1 2  D 1 1 L L - S L F C D O I D D C P P G IC T

Figure 2-7. LL-CFA/I promotes Bregs in the spleen. 4 wk-old female NOD mice (n=10/group) were orally dosed with 5x107 CFUs of LL-CFA/I, LL vector, or 200 μL of PBS. Additional doses were given every 2 wks. At 11 wks of age mice were euthanized. A) Breg phenotypes were examined by FACS analysis in the spleen, upper left, MLNs, upper right, and PaLNs, lower. +p<0.05 for LL-CFA/I compared to PBS.

E x v iv o s p le e n C D 4 E x v iv o S p le e n F o x p 3

8 0 5 0

+ P B S 3

p 4 0 L L v e c to r

6 0 x o

4 L L -C F A /I F

D 3 0

+ C

4 0

f o

D 2 0

2 0 f

1 0

0 % 0

  t   t 5 3 - 4 L 5 - 0 2 p - 4 2 e 2 1 - e x N F b N - F b D F D 6 T D F L T C o I G C D C I I G F T C T

E x v iv o M L N C D 4 E x v iv o M L N F o x p 3

8 0 8 0 P B S

+ L L v e c to r

6 0 3 6 0 4

p L L -C F A /I

o C

4 0 F

f 4 0

% 2 0 2 0 %

0 0  t 5   4 t 5 - 0  3 - 0 - L e 2 1 - e 2 p N -1 4 2 b N - F b D x F D 6 D F L T o IF IL G T I I G C C D C T F T C

Figure 2-8. LL-CFA/I maintains suppressive Tregs late in disease. 4 wk-old female NOD mice were orally dosed with 5x107 CFUs of LL-CFA/I, LL vector, or 200 μL of PBS. Additional doses were given every 2 wks. Mice were euthanized at 17 wks of age, 1 k after their final dose. A) Summary of CD4+ T cell, left, and Foxp3+CD4+ Treg phenotypes examined by FACS analysis in the sleen and B) MLNs. C) Summary of CD8+ T cells examined by FACS analysis in the spleen, left, and MLNs, right. D) Representative plots of IFN-γ production by splenic CD4+, upper, and CD8+, lower, T cells stimulated with anti-CD3 and anti-CD28. E) Total number of IFN-γ producing splenic CD4+, left, and CD8+, right, T cells after stimulation with anti-CD3 and anti-CD28. Lymphocytes from the PaLNs were collected and stimulated with anti-CD3 and anti-CD28. F) Representative plots of Foxp3+ Tregs of the PaLNs, upper, expressing IFN-γ, middle, and Tbet, lower. G) Summary of percentage of Tbet and IFN-γ expression in Foxp3+CD4+ Tregs of the PaLNs after stimulation with anti-CD3 and anti-CD28. Results are the combined data of 2 experiments of n=10/group. *p<0.05 for LL-CFA/I compared to all other groups, **p<0.005 as compared against all other groups.

E x v iv o s p le e n C D 8 E x v iv o M L N C D 8

1 0 0 5 0 P B S

8 0 4 0 L L v e c to r

8 D D L L -C F A /I

6 0 3 0

4 0 2 0

% % 2 0 1 0

0 0  t t 5 - 0 -  4 L e 5 - 0  4 L 2 1 4 2 2 1 - 4 2 e N - F 6 b N - F b D F L D T D D 6 I I G D IF IL G T C T C C C D C T C

D PBS LL vector LL-CFA/I

28.3±6.9 24.3±11.73 7.1±5.8

- CD4 IFN 45.9±7.2 40.5±13.5 20.65±6.5

CD8

l l

l 2 .5  1 0 5 1 .5  1 0 6

T 5

2  1 0

+ +

6 8 4 1  1 0

5 D

D 1 .5  1 0

C C

+ + 

 5 * - - 1  1 0

5 1 0 5 N

N  F

F I

I 4 **

5  1 0

t o

o 0 0

T T I I S r / S r / B to A B to A P c F P c F e C e C v - v - L L L L L L L L

Figure 2-8 Continued.

PBS LL vector LL-CFA/I

Foxp3

CD4

IFN

Tbet

CD4

)

p +

1 5 4 0 p 6 0

D +

3 0 +

f 1 0 4 0

C 2 0

f ( f *

+ 5 2 0

3 1 0

(



t -

F 0 0 0

N b

I F I I

S r / S r / T S r / B to A I B to A B to A P c F P c F P c F e C e C e C v - v - v - L L L L L L L L L L L L

Figure 2-8 Continued.

1 0 0

P B S

e e

r 8 0 L L v e c to r

s L L -C F A /I

e 6 0

t e

b 4 0 +

i D

2 0 %

0 1 4 1 6 1 8 2 0 2 2 2 4 2 6 2 8 3 0 A g e (w e e k s )

C D

4 0 8 +

+ 3 0 6

2 0 4

* % % 1 0 2

0 0

S r /I S r /I B to A B to A P c F P c F e C e C v - v - L L L L L L L L

Figure 2-9. Protection mediated by LL-CFA/I is stable out to 30 wks of age. 4 wk-old female NOD mice (n=10/group) were orally dosed with 5x107 CFUs of LL- CFA/I, LL vector, or 200 μL sterile PBS. Additional doses were given every 2 wks. A) Blood glucose levels of individual mice treated with PBS, LL vector, or LL-CFA/I. Values above the dotted lines are considered hyperglycemic. B) Summary of disease incidence of mice treated with PBS, LL vector, or LL- CFA/I. Lymphocytes were isolated from the pancreas and analyzed for expression of C) Tbet and D) Foxp3. +p<0.05 compared to PBS.

Table 2-1. List of antibodies used for flow cytometry in LL-CFA/I Treg studies. Antibody specificity Clone Source Dilution CD103 M290 BD 1/500 CD122 TM-b1 BD 1/500 CD19 EBio1D3 eBioscience 1/600 CD1d Ly-38 eBioscience 1/500 CD25 PC61.5 Biolegend 1/500 CD39 24DMS1 eBioscience 1/500 CD4 RM4-5 Biolegend 1/500 CD44 IM7 Biolegend 1/800 CD49B HMa2 BD 1/400 CD5 53-7.3 BD 1/500 CD62L MEL-14 BD 1/500 CD8α 53-6.7 eBioscience 1/500 CTLA-4 UC10-4F10-11 BD 1/500 CXCR3 CXCR3-173 eBioscience 1/500 Foxp3 FJK-16s eBioscience 1/250 Helios 22F6 eBioscience 1/250 ICOSL HK5.3 Biolegend 1/500 IFN-γ XMG1.2 BD 1/250 IL-10 JES5-16E3 eBioscience 1/250 Lag-3 C9B7W Biolegend 1/400 Neuropilin-1 3E12 Biolegend 1/500 PD-1 RMPI-30 eBioscience 1/500 PD-L1 10F.9g2 Biolegend 1/500 Tbet 4B10 Biolegend 1/250 TCR-β H57-597 Biolegend 1/500 TGF-β (Lap) TW7-16B4 Biolegend 1/400 TIGIT 1G9 Biolegend 1/400 TIM-3 B8.2C12 Biolegend 1/400 TNF-α MP6-XT22 Biolegend 1/250

CHAPTER 3 ESTABLISHMENT OF A REGULATORY MICROENVIRONMENT BY LACTOCOCCUS EXPRESSION COLONIZATION FACTOR ANTIGEN I

Background

T1D is a chronic autoimmune disease characterized by a patient’s inability to produce sufficient levels of insulin, and consequent inability to control blood glucose levels. This is due to an autoimmune attack on insulin-producing β cells in the islets of

Langerhans. In the NOD mouse, infiltration of the islets by inflammatory cells, termed insulitis, begins between 3 and 4 wks of age (106). APCs, including DCs and macrophages, are among the first cell types to infiltrate the islets of Langerhans (106).

This initial infiltrate is associated with an inflammatory cytokine signature including T1-

IFNs, TNF-α, and IL-1β (100, 105, 134, 141, 291). These findings demonstrate that DCs play an important role in the pathogenesis of T1D, and that tolerance towards islet- associated antigens is corrupted as early as 4 wks of age in the NOD mouse.

DCs are grouped into one of two large classes: plasmacytoid DCs (pDCs) and conventional DCs (cDCs) (292). In mice, pDCs are described as CD11cLow and Siglec-

H+, and are normally found in the blood and lymphoid organs (293). They are known for producing T1-IFNs in response to TLR engagement, which has been implicated in initiating T1D in the NOD mouse (100, 291, 293). However, the role pDCs play in the pathogenesis of T1D is unclear, as pDCs also produce the anti-inflammatory enzyme

IDO, which has been associated with pDC mediated protection from T1D (294, 295).

Together, these findings indicate that pDCs are preferentially inflammatory in the context of T1D, but can targeted as regulatory cells to protect against disease.

The second class of DCs, cDCs, can be further broken down into two subgroups;

CD11c+CD8α+CD11b- and CD11c+CD8α-CD11b+, which are commonly referred to as

CD8+ DC and CD11b+ DC, respectively (296). Under normal circumstances, T and B cells that recognize self-antigen are deleted or differentiated into regulatory cells as part of central tolerance (297). DCs play an integral role in this process, by presenting self- antigens to developing T cells (298). In the context of peripheral tolerance, CD8+ DCs promote Treg differentiation from naïve T cells (299, 300). However, NOD mice have reduced levels of CD8+ DCs, suggesting at least one mechanism of maintaining peripheral tolerance is defective in the context of T1D (301). This decrease in CD8+

DCs is associated with a concomitant rise in CD11b+ DCs, which have been shown to present self-antigen to autoreactive CD4+ T cells (295). CD11b+ DCs may also have a regulatory role, as studies using mAbs to target self-antigen to DCIR2+CD11b+ DCs has been shown to elicit tolerogenic T cell responses in NOD mice (302). DCs in NOD show increased expression of costimulatory molecules such as CD40, increasing their potential to prime inflammatory T cells (303). Collectively, these findings suggest that

DCs in the context of T1D preferentially stimulate inflammatory responses, but, like pDCs, are potential sources of regulatory function that can protect against T1D.

CFA/I fimbria is a virulence factor for ETEC, and has shown to be crucial for

ETEC’s ability to colonize the intestines of humans (304). CFA/I fimbriae were originally targeted as a vaccine candidate to protect against ETEC, however, it was found to suppress inflammatory cytokine production, suggesting it may act as an anti- inflammatory vaccine (206, 210). Consequently, it has been tested and found to treat and prevent mouse models of multiple sclerosis like disease as well as arthritis (211,

214). These studies utilized a Salmonella vector to deliver CFA/I fimbriae to the GALT.

In an effort to create an oral, food based therapy to treat autoimmune diseases, CFA/I

fimbriae were expressed on L. lactis lactis IL1403 (LL-CFA/I) and used to treat CIA

(228). More recently, LL-CFA/I has been shown to ameliorate T1D in NOD mice. Data from 17 wk old NOD mice showed that Foxp3+ Tregs gain expression of inflammatory mediators Tbet and IFN-γ, suggesting they are gaining inflammatory function. These data suggest that LL-CFA/I induces and maintains stable Tregs to protect against T1D.

Additionally, these findings are consistent with findings that Tregs become unable to suppress diabetogenic Teffs in NOD mice as T1D progresses (158, 305).

Previous studies with CFA/I fimbriae show that its mechanism of protection against autoimmune diseases is initiated through bystander suppression, but is ultimately dependent upon autoantigen-specific Tregs (306). However, it is unknown how this protection transitions from an antigen-independent mechanism to an antigen- specific mechanism. Studies with CFA/I fimbriae focus on the oral route of administration, implicating the GALT in the initial response towards CFA/I fimbriae.

Since DCs have been shown to sample luminal antigen and are crucial in determining whether a naïve T cell will differentiate into a Teff or Treg, it was hypothesized that protection mediated by LL-CFA/I is initiated by stimulating pDCs to produce IDO and increasing the prevalence of CD8 DCs in the MLNs, PPs, and PaLNs (147, 307).

Methods

NOD Mouse Husbandry

Female NOD/ShiLtJ mice (The Jackson Laboratory) were 3 or 5 wks of age upon arrival. Mice were housed 5/cage in SPF conditions with food and water available ad libitum. Mice were allowed to acclimate to the facility for at least 5 days prior to handling. After acclimation, mice were given an identifying mark by ear-punching. All procedures were documented in protocols approved by the University of Florida IACUC.

Growing L. lactis for Oral Administration

L. lactis lactis IL1403 was engineered to carry the pMSP3535H3 vector in its empty state or with a synthetic operon for CFA/I fimbriae as described previously (228).

M17-glucose was used as growing medium for all L. lactis strains. Sterile M17-glucose media was made by suspending 37.25 g of Difco M17 Broth (BD) and 5 g of glucose

(ThermoFisher) in 950 mL of deionized water. M17-glucose media were sterilized and allowed to cool to room temperature before use.

Starter cultures of LL-CFA/I and LL vector were started the day before dosing by adding 50 µL of LL-CFA/I or LL vector glycerol stock to a Falcon 14 mL Polystyrene

Round-Bottom Tube (Corning) with 6 mL of M17-glucose and 60 µg of erythromycin

(MilliporeSigma). Starter cultures were incubated overnight at 30°C with no shaking.

The following morning 2 mL of starter culture were added to 48 mL of pre-warmed M17- glucose media along with 500 µg of erythromycin (MilliporeSigma). Starting 1 hour after growth culture inoculation, OD was monitored using a BioMate spectrophotometer

(ThermoFisher). L. lactis mutants were allowed to grow to an OD of 0.19 to 0.21 before adding 25 μg of nisin (MillilporeSigma) to induce expression CFA/I fimbriae. Bacteria were incubated at 30°C for 4 hours after induction. After incubation, bacteria were collected by centrifugation and washed with DPBS to prepare for administration to mice.

Neutralization of stomach acid

were orally gavaged with 200 µL of the diluted sodium bicarbonate solution using a reusable AFN 20 gauge, 1.5’’ long, 2.25 mm-curved gavage needles (Cadence Science,

Inc.) 5 to 10 minutes before administration with bacteria.

Oral gavage of bacteria

Four hours after nisin induction, LL-CFA/I or LL vector were collected by centrifugation. Bacteria were washed twice with DPBS before being resuspended at the concentration required for the study. Concentration was adjusted such that mice would be orally gavaged with 100 – 200 µL of bacteria in DPBS. 10 minutes after neutralization of stomach acid with sodium bicarbonate LL-CFA/I, LL vector, or DPBS was loaded into a 1 mL syringe. Mice were orally gavaged with a reusable AFN 20 gauge, 1.5’’ long, 2.25 mm-curved gavage needles (Cadence Science, Inc.).

Tissue Collections

At specified study endpoints mice euthanized by carbon dioxide asphyxiation.

For necropsy, mice were pinned to a small dissection stage using 23 gauge needles.

Mice were sprayed with 70% ethanol, and sterile surgical instruments were used to harvest organs. Tissues were kept in IM until necropsy was complete. IM was prepared by adding 5 mL of HEPES solution (Genesee Scientific) and 5 mL of Penicillin-

Streptomycin solution (100x) (Genesee Scientific) to 500 mL of RPMI 1640 Medium

(Genesee Scientific).

Splenocyte isolation

Spleens were mechanically dissociated using a Tissuelyzer II (Qiagen) at 20 Hz for 1 minute, and then filtered through sterile nytex (Sefar Inc.). Cells were washed with

IM and resuspended in 1 mL of IM. 1 mL of 2X ACK lysis buffer was added to lyse red

100

blood cells. Cells were incubated with ACK lysis buffer at room temperature for 2 minutes. Cells were washed with IM and resuspended in CM.

Lymphocyte isolation

During necropsy, PaLNs, PPs, and MLNs were collected and placed into 2 mL

Safe-Lock Tubes (Eppendorf) tubes with 1 mL of IM and a sterile, 5 mm, stainless-steel bead. The tissue was first mechanically disrupted using a Tissuelyzer II set to 15 Hz for

1 minute. Cells were then filtered through sterile nytex (Sefar Inc.) and washed with IM and resuspended in CM.

Counting cells

DPBS. 20 µL of this mixture was added to a Cellometer Disposable Counting Chamber

(Nexcelom) and counted. For other tissues or blood the process was the same, except

20 µL of single cell suspensions were diluted in 20 µL of Trypan Blue-DPBS.

Cell Culture

BMDCs were generated from bone marrow of BALB/c mice or NOD mice, according to the experiment. During necropsy, the femur and tibia were isolated, cleaned of muscle tissue, and washed with 70% ethanol. After incubation bones were dried for 1 minute at RT and then added to a petri plate containing CM. At this point, a 3 mL syringe with a 27 gauge needle was loaded with CM. Then, one at a time, the ends of a bone were cut and the bone marrow was flushed into a 50 mL conical with CM using the prepared syringe and needle. The syringe was reloaded and the process

101

repeated as needed, flushing bone marrow into the same 50 mL conical. Using the same syringe and needle, bone marrow aggregates were broken up by passing in and out of the syringe. The sample was then filtered through a 70 µm Corning cell strainer

(MilliporeSigma). Samples were washed and supernatant was discarded and the sample was resuspended in 2 mL of CM and counted. The concentration of cells was adjusted to one million cells per mL using DC medium. DC medium was prepared by adding recombinant mouse GM-CSF (R&D Systems) to a working concentration of 20 ng/mL to CM lacking penicillin and streptomycin. One mL of cell suspension was added to each well of a 24 well plate and incubated at 37°C. On day 2 of culture, 1 mL of fresh

DC medium was added. On days 4 and 6, 1 mL of media was removed before adding 1 mL of fresh DC medium. Largest cell yields were found to be between days 6 and 9 of culture.

Infecting BMDCs

To determine the role of APCs in the initiation of LL-CFA/I mediated protection,

BMDCs were generated as described above. Resting BMDCs were collected by pipetting up and down. BMDCs were then washed and resuspended in DC media and counted. Concentration was adjusted to 1.0x106 cells/mL in DC medium and plated onto

24 well plates, 1 mL per well. LL-CFA/I and LL vector were grown and induced as described above. 4 hours after induction, LL-CFA/I and LL vector were collected by centrifugation and washed with DPBS. Bacteria were washed a total of 3 times before infection. Bacteria were then added to designated wells at BMDC to bacteria ratios indicated by the experiment. LL strains were allowed to infect BMDCs for 2 hours, after which, culture supernatants were carefully removed and plates were washed by gently adding 1 mL of DC media with 50 µg/mL of gentamicin (MilliporeSigma). This wash was

102

repeated once more and 1 mL of DC media with 2.5 µg/mL of gentamicin was added.

Plates were incubated overnight at 37°C. Supernatant were used for cytokine analysis via ELISA and BMDC phenotypes were observed by flow cytometry.

Co-culture of CD4+ T cells with APCs

To determine the role of APCs in the initiation of LL-CFA/I-mediated protection,

BMDCs that were previously infected with LL-CFA/I, LL vector, or not infected were co- cultured with naïve CD4+ T cells. BMDCs were generated and infected as described above. On day 1 mice were euthanized and spleens, MLN, and HNLNs were collected.

Tissue were processed as described above and single cell suspensions were obtained.

Cells from lymph nodes were combined into a single tube, washed, and resuspended in

1 mL of CD4+ buffer. CD4+ buffer was prepared by adding 9.6 g/L of PBS

(MilliporeSigma), 2% fetal calf serum (FCS), and 1 mM EDTA. Splenocytes from different mice were combined and washed in an identical manner. Naïve CD4+ T cells were defined as CD25-CD4+ T cells and were isolated through use of magnetic bead purification kits (Stemcell Technologies). CD25+CD4+ T cells were first removed using the EasySep Mouse CD25 Regulatory T cell Positive Selection Kit (Stemcell

Technologies). The fraction containing CD25-CD4+ T cells was washed and resuspended in CD4 buffer. The remaining CD4+ T cells were purified using the

EasySep Mouse CD4+ T Cell Isolation Kit (Stemcell Technologies). Purified CD4+ T cells were washed and resuspended in CM before counting. CD4+ T cells from spleens and lymph nodes were then combined and counted, and the cell concentration adjusted to 4x106 cells/mL using CM. BMDCs and CD25-CD4+ T cells were plated onto of 96 well, round bottom, tissue culture treated plates (MilliporeSigma) that had been coated in 1 µg/mL of anti-CD3 mAb (clone 17A2; Invitrogen), in ratios indicated by specific

103

experiments. Total number of cells in a well was 500,000 and total volume was 200 µL.

The volume of different wells was brought up to 200 µL in CM and anti-CD28 mAb

(clone 37.51, Invitrogen) was to a concentration of 2 µg/mL. Plates were incubated at

37°C for 48 hours, after which, culture supernatants were collected and stored at -20°C until cytokine analysis via ELISA was performed. Cells were immediately transferred to

FACS tubes, and phenotypes were analyzed by flow cytometry.

Flow Cytometry

To determine the phenotype of Tregs induced by pσ1 fusion proteins, cells were washed with PBS and stained for viability using a LIVE/DEAD Fixable Blue Dead Cell

Stain Kit, for UV excitation (ThermoFisher). Cells were then washed with FB and labeled with mAbs specific for CD11c, CD11b, DEC-205, DCIR2, CD8, CD19, TCR-β,

CD40, CD80, CD86, OX40L, CD103, TGF-β, and MHC class II. Then cells were washed and fixed with 1X fixation buffer from the True-Nuclear Transcription Factor

Buffer Set (BioLegend). Cells were then washed with 1X permeabilization buffer from the True-Nuclear Transcription Factor Buffer Set (BioLegend) and labeled with mAbs specific for IL-10, IL-1β, and IDO. Cells were labeled with different mAbs at dilutions according to Table 3-1.

Cytokine ELISA

Spleens and MLNs were aseptically removed at 11 or 24 wks of age from PBS-,

Burlington, MA, USA) in the presence of 5 µg/mL anti-CD3 (clone 17A2; Invitrogen) and

2.5 µg/mL anti-CD28 (clone 37.51; Invitrogen, Carlsbad, CA, USA) in a total volume of

104

200 µL. The supernatants were collected by centrifugation and stored at -80°C. Capture

ELISA was employed to quantify, on duplicate sets of samples, the levels of IL-10 and

TGF-β produced by BMDCs and lymphocytes.

For IL-10, 2 µg/mL rat anti-mouse IL-10 mAb (clone JES5-2A5; eBioscience, San

Diego, CA, USA) was used as capture antibody, and 1.5 µg/mL of biotinylated rat anti- mouse IL-10 mAb (clone SXC-1; BD) was used as detecting antibody.

For TGF-β, 2 µg/mL rat anti-mouse TGF-β mAb (clone A75-2; eBioscience, San

Diego, CA, USA) was used as capture antibody, and 5 µg/mL of biotinylated rat anti- mouse TGF-β mAb (clone A75-3; BD) was used as detecting antibody.

The color reaction was developed using horseradish peroxidase (HRP) conjugated goat anti-biotin Ab (Vector Laboratories, Burlingame, CA, USA) and ABTS peroxidase substrate (Moss, Inc., Pasadena, ME, USA). Cytokine concentrations were extrapolated from standard curves generated by recombinant murine cytokines IL-10 and TGF-β (R&D Systems).

Statistics

All presented data are the mean ± SEM Statistical significance was tested using

GraphPad Prism 7 (Prism). One-way ANOVA with Tukey’s multiple comparisons test were used to compare FACS data and cytokine production. All results were discerned to the 95% confidence interval.

Results

LL-CFA/I induces regulatory function in BMDCs

To test the hypothesis that protection mediated by LL-CFA/I is initiated through

DCs, in vitro infections of BMDCs were performed. BMDCs were generated from the bone marrow of BALB/c mice and were infected with LL-CFA/I at different ratios for 2

105

hours. After 2 hours, bacteria remaining in culture were washed out using gentamicin and BMDCs were incubated for 6 or 24 hours. After 24 hours of incubation, BMDCs infected at a 3:1 bacteria to DC ratio showed significant increase in IDO and TGF-β production compared to other infection ratios (Fig. 3-1A). To confirm these findings,

BMDCs were infected at a 3:1 bacteria to DC ratio with LL-CFA/I or LL vector for 2 hours. Bacteria were washed out as done previously and BMDCs were incubated for 24 hours. Infection with LL-CFA/I induced a 2-fold increase in IDO expression (p<0.005) as compared to LL vector (Fig. 3-1B). Additionally, LL-CFA/I-infected BMDCs produced significantly more TGF-β and IL-10 (p<0.05) as measured by cytokine ELISA (Fig. 3-

1C). These results suggest that LL-CFA/I directly induces regulatory function DCs and support the hypothesis that the protective effect of LL-CFA/I is initiated by DCs.

Kinetics of LL-CFA/I induced regulatory DCs and Tregs

Previous studies utilizing CFA/I fimbriae to treat EAE demonstrated that Foxp3+

Tregs are induced in the spleen, MLNs, and PPs as early as 1 wk after oral administration of Salmonella-CFA/I (212). Therefore, it was hypothesized that LL-CFA/I would stimulate IDO and TGF-β production in these tissues preceding Treg induction.

To test this hypothesis, 6 wk-old female NOD mice were orally dosed with 5x107 CFUs of LL-CFA/I, LL vector, or vehicle alone. On days 3, 7, 14, and 21 after treatment mice were euthanized from each treatment group and were examined for the presence of cDC, pDC, and Treg phenotypes in different tissues. Notably, LL-CFA/I induced a 3.7- fold increase in pDCs expressing TGF-β and IDO in the spleen (Fig. 3-2C and 3-2K) and a 3.2-fold increase in the PaLNs (Fig. 3-2H and 3-2J) 3 days after treatment. TGF-

β-producing IDO- pDCs were also detected in the spleens of treated mice (Fig. 3-2K).

TGF-β-producing CD8+ DCs and CD11b+ DCs were similarly detected in the spleens

106

(Fig.3-2A-B), but not the PaLNs (Fig. 3-2F-G) of treated mice. This phenotype was transient and no longer detectable by 1 wk post-treatment.

Since CD40 expression on cDCs of NOD mice is associated with T1D pathogenesis expression of costimulatory molecules CD40 and CD86 were also examined. Reductions in CD40 and CD86 were detectable in the MLNs beginning at 1 wk post-treatment (Fig. 3-2D), however, they became more pronounced and visible on

CD11b DCs at 3 wks post-treatment (Fig. 3-2E). Together, these data show that LL-

CFA/I induces a TGF-β and IDO response quickly after treatment and reductions in costimulatory molecules come later.

Foxp3+ Treg induction was then examined, following the same kinetic strategy as used with the DCs. Oral treatment with a single dose of LL-CFA/I significantly increased

Foxp3 expression (p<0.05) in the MLNs and PPs 1 wk after treatment (Fig. 3-3A). To determine if these Tregs being induced were due to expansion of tTregs or induction of new pTregs, Helios and neuropilin-1 (NRP-1) expression by Foxp3+CD4+ cells were examined. There were no significant changes in these molecules for tTregs (Fig. 3-3B-

C), suggesting that the increase in Tregs is due to induction of pTregs from naïve T cells.

Considered chronologically, these results show that IDO and TGF-β-producing pDCs are induced in the spleen (Fig. 3-2C) and the PaLNs (Fig. 3-2H) shortly after treatment with LL-CFA/I. Subsequently, Foxp3+ Tregs are induced in the mucosal inductive sites (Fig. 3-3A). This Treg induction is followed by reductions in costimulatory molecules at 2 and 3 wks post treatment (Fig. 3-2D-E). These findings suggest that IDO

107

and TGF-β are important for the induction of Tregs and that Tregs, in turn, control the inflammatory potential of DCs.

LL-CFA/I reduces inflammatory potential of cDCs

Previous work showed that LL-CFA/I reduced inflammatory mediators Tbet and

IFN-γ, beginning at 11 wks of age. Additionally, Treg activity, as marked by IL-10 production and Foxp3 expression were detected in the MLN and PP, respectively, at 11 wks of age. Since LL-CFA/I reduced expression of costimulatory molecules CD40 and

CD86 on cDCs in the MLNs it was hypothesized that Treg activity at mucosal inductive sites induced regulatory function in cDCs at this time-point, as defined by reduced expression of costimulatory markers CD40, CD80, CD86, and OX40L and increased prevalence of CD8 DCs. To test this hypothesis, 4 wk-old female NOD mice were orally dosed with 5x107 CFUs of LL-CFA/I, LL vector, or vehicle alone. Additional doses were given every 2 wks and mice were euthanized at 11 wks of age, 1 wk after their final dose. LL-CFA/I did not significantly alter the ratio of CD8 DCs to CD11b DCs in the spleen, MLNs, PaLNs, and PPs of treated mice (Fig. 3-4A). To test if LL-CFA/I affected the inflammatory potential of cDCs, expression of costimulatory molecules CD40, CD80,

CD86, and OX40L were examined. Treatment with LL-CFA/I modestly reduced OX40L expression (p<0.05) in the splenic CD11b DCs (Fig. 3-4B). Additionally, treatment significantly reduced expression of CD40 and CD86 on both CD8 DCs and CD11b DCs in the MLNs (Fig. 3-4C), suggesting that LL-CFA/I primarily affects DCs of the MLNs.

LL-CFA/I infected BMDCs do not induce Foxp3+CD4+ Tregs

DCs are crucial in determining the fate of differentiating T cells. Others have shown that IDO-producing DCs can induce differentiation of Foxp3+ Tregs from naïve T cells (308) In vitro infection of BMDCs with LL-CFA/I induced TGF-β and IDO production

108

(Fig. 3-2B-C). Therefore, it was hypothesized that LL-CFA/I-infected BMDCs would induce Foxp3+ Tregs from naïve CD4+ T cells. To that end, BMDCs were again generated from BALB/c mice and infected with LL-CFA/I or LL vector, as done previously. Naïve CD25-CD4+ T cells were isolated from the spleens, head and neck

LNs (HNLNs), and MLNs of BALB/c mice. CD25-CD4+ T cells and BMDCs were co- cultured at a 4:1 ratio for 48 hours, and FACS analysis was used to examine T cell and

DC phenotypes. The LL-CFA/I-infected DCs did not induce Foxp3 expression in naïve T cells (Fig. 3-5A). However, IL-10 concentration was significantly increased (p<0.0005) in cultures with LL-CFA/I infected BMDCs, suggesting that IL-10-producing Tregs are being induced (Fig. 3-5E). These CD4+ T cells co-cultured with LL-CFA/I infected

BMDCs had a high expression of CD44 and low expression of CD62L, suggesting they had gained a memory T cell phenotype, This, taken together with the observed increased in IL-10 production suggests these are activated Tregs (Fig. 3-5B-C).

Additionally, LL-CFA/I effectively suppressed IL-1β production in infected BMDCs, supporting the hypothesis that LL-CFA/I induced regulatory function in DCs (Fig. 3-5D).

Discussion

The exact pathogenesis and etiology of T1D is currently unknown, however, data from mice and humans suggests that it is heterogeneous and includes multiple, potentially compensatory, mechanisms as the disease progresses. Additionally, recent reports describing insulin neoantigens, hybrid insulin peptides, and defective ribosomal products demonstrate novel ways in which the disease can progress. Notably, these novel epitopes may provide a source of antigen lacking central tolerance, and thus provide a means for tolerance to be broken towards islet antigens (88, 89).

109

APCs play an important role in the pathogenesis of T1D. Tissue resident macrophages are present in healthy mice, however, they are thought to become pathogenic as the disease progresses as depleting them with mAb treatment blocks development of T1D (309). Others have observed that disease progression correlates with abundance of islet infiltrating DCs (310). cDCs and pDCs may contribute to the pathogenicity of macrophages through production of T1-IFNs (100, 291, 311).

Additionally, DCs have been shown to migrate to the islets early in the life of NOD mice, and subsequently move to the PaLN where they present self-antigen to autoreactive T cells (102). Diabetes-prone NOD mice exhibit numerous defects in DC phenotypes including a reduction in tolerogenic CD8 DCs. Others have detected heightened expression of NF-κB in resting NOD mice, suggesting that APCs are more primed for inflammation than in non-diabetes prone strains of mice (312, 313). Thus, affecting the number or phenotype of DCs in NOD mice can affect the progression to overt T1D

(314).

Oral tolerance strategies provide a powerful means of addressing inflammatory diseases such as T1D, through inducing anergy in pathogenic Teffs or inducing antigen- specific Tregs (144). However, oral tolerance strategies typically rely upon knowledge of the target or initiating antigen, which limits its efficacy in a heterogeneous disease such as T1D (219). This hypothesis is supported by results from human clinical trials where antigen monotherapies fail to prevent onset of disease in at risk patients (175, 177, 178,

180, 315). To overcome these hurdles, it was hypothesized that CFA/I fimbriae, which induce oral tolerance through bystander suppression, would prevent T1D in NOD mice.

Oral treatment with LL-CFA/I protects against T1D in the NOD mouse by suppressing

110

+ inflammatory TH1 responses. Notably, the data show Foxp3 Tregs and Bregs at 11 wks, stable Foxp3+Tbet-IFN-γ- Tregs in the PaLNs at 17 wks, and finally IFN-γ- and IL-

10-producing Tr1 cells at 24 wks. This reflects an evolving regulatory response that changes to meet that challenges of the different stages of T1D in NOD mice.

CFA/I fimbriae have previously been shown to protect mice against induced models of autoimmunity, including EAE and CIA (211, 212, 214). In these models, the mechanism of protection is dependent upon antigen-specific Tregs, but is shown to be initiated by bystander suppression (213). Bystander suppression functions through exposing a target antigen to the immune system while in the same environment as a secondary antigen that tolerance is enforced towards (219). DCs have been shown to be required for the induction of oral tolerance, and furthermore, are known to play a critical role in determining the fate of a T cell during differentiation (307, 316, 317). This is achieved through DCs providing different signals to T cells during activation: TCR engagement, costimulatory molecules such as CD80/CD86 and CD40, and cytokines

(318). In T1D, these mechanisms seemed poised to induce activated and inflammatory

T cell responses and targeting expression of costimulatory molecules, regulatory enzymes such as IDO, and cytokines production from DCs has been shown to ameliorate disease (294, 303, 319, 320). Thus, it was hypothesized that LL-CFA/I directly induces IL-10, TGF-β, and IDO production in DCs.

In vitro infection of BMDCs with LL-CFA/I or LL vector show that infection with

LL-CFA/I, but not LL vector, induces production of the regulatory cytokines TGF-β and

IL-10 (Fig. 3-2C) while suppressing production of the inflammatory cytokine IL-1β (Fig.

3-5D). Suppression of IL-1β after infection suggests that CFA/I fimbriae blocks activity

111

of NF-κB, which is notable since NF-κB has heightened expression in resting NOD mice

(312, 313, 321). Activation of the NF-κB pathway is associated with response to acute inflammation, therefore, expression in resting NOD mice is indicative of the ongoing, chronic inflammation present during disease. Additionally, heightened expression of NF-

κB is associated with inflammatory bowel disease, which supports NF-κB’s role as a marker for chronic inflammation (322, 323). However, NF-κB’s role in the pathogenesis of T1D is somewhat unclear, as deficiency in the NF-κB protein, c-Rel, prevents T1D in the NOD mouse model but will prevent streptozotocin-induced T1D (324). This was found to be due to c-Rel dependent Tregs. Interestingly, c-Rel has been shown to be important in the generation of pTregs in different tissues, suggesting that c-Rel may be important for Treg differentiation in the islets of Langerhans (325–328).

Infection with LL-CFA/I also stimulated production of the regulatory enzyme, IDO

(Fig. 3-2B). To study whether treatment with LL-CFA/I induced IDO in vivo, pDCs were examined shortly after dosing. The results show that LL-CFA/I induces IDO- and TGF-β- producing pDCs in the spleen (Fig. 3-3C) and PaLNs (Fig. 3-3H) shortly after treatment with LL-CFA/I. This is interesting, as the role pDCs play in the pathogenesis of T1D is unclear. Some reports have shown that pDCs play a role in the initiation of T1D by acting as a source of T1-IFNs in the islets (100, 291). However, IDO-producing pDCs have been shown to be protective in T1D (294, 320, 329). IDO is an important enzyme in immunoregulation, acting as the first, and rate limiting step in a tryptophan catabolism pathway and is produced by certain DCs and macrophages to suppress inflammation.

There are two aspects to IDO’s immunoregulatory function. The first entails suppressing the function of Teffs by limiting their access to tryptophan, which is required for the

112

function of activated T cells (330, 331). Additionally, one of the byproducts of this tryptophan catabolism pathway includes kynurenines. Kynurenines have been described as a ligand for the aryl hydrocarbon receptor, and play an important role in the metabolism of Tr1 cells (332, 333). The dual function of IDO provides a perfect candidate mechanism for the major findings in 11 and 17 wk-old NOD mice, where treatment with LL-CFA/I restrained inflammatory mediators Tbet and IFN-γ and maintained stable Foxp3+ Tregs in the PaLNs (Fig. 2-7F-G).

To determine the kinetics of LL-CFA/I induced bystander suppression pDCs, cDCs, and Foxp3+ Tregs were examined shortly after treatment. Results show an early and transient increase in pDCs producing both IDO and TGF-β (Fig. 3-3). Additionally, beginning at 1 wk post-treatment, CD8+ DCs showed reduced expression of the costimulatory molecule CD86 (Fig. 3-2D). This suppression of costimulatory molecules was further defined at 2 and 3 wks post treatment in CD11b+ DCs in the MLNs, but not the spleen or PaLNs (Fig. 3-2E). This was interesting when considered together with the kinetics of pTregs, which were only seen in the MLNs and PPs at 1 and 2 wks, respectively (Fig. 3-3A). This order of events suggests that pDCs are important for stimulating new pTregs and that these Tregs are reducing the amount or function of inflammatory DCs, as marked by CD86 and CD40.

In the NOD model of T1D, insulitis is well-established by 11 wks of age and is characterized by T cells, which are responsible for destroying β cells. Previously, treatment with LL-CFA/I was shown to suppress IFN-γ-producing CD8+ T cells in the spleen (Fig. 2-5C), and TH1 cells in the islets but that there was seemingly no increase in Tregs as marked by Foxp3 (Fig. 2-4F). It was hypothesized that regulatory DCs were

113

responsible for this restraint of IFN-γ and Tbet. Thus, the 11 wk time-point was used to examine regulatory DCs. LL-CFA/I therapy reduced expression of costimulatory molecules CD40 and CD86, principally in the MLNs (Fig. 3-1C). Heightened expression of costimulatory molecules, like heightened expression of NF-κB, is consistent with the chronic, low-grade inflammation characteristic of T1D. Additionally, blocking CD40 stimulation of T cells has been shown to be protective against T1D in the NOD mouse

(303).

Others have shown that regulatory DCs can stimulate differentiation of iTregs from naïve T cells (308). Since infection with LL-CFA/I induced regulatory function via

IDO, IL-10, and TGF-β in BMDCs it was hypothesized that these BMDCs would induce

Foxp3 expression in naïve T cells. However, this was not the case, as FACS analysis showed little Foxp3 expression, regardless of pre-treatment of BMDCs. However, T cells cultured with had significantly increased CD44 expression and significantly lower

CD62L expression, suggesting they are more activated than T cells from other cultures.

Culture supernatants revealed that co-cultures of these T cells and LL-CFA/I infected

BMDCs produced significantly more IL-10, suggesting that these are Foxp3-IL-10+ Tr1 cells.

114

IDO T G F -

+ I

3000 2 0 1:1 1 :1

* C 3:1 3 :1 H 1 5

2000 10:1 M 1 0 :1

+ 1

F 30:1 1 0 3 0 :1

1 M

1000 D

C 5

0 0 0 1 2 3 % 0 1 2 3 6 24 6 24 Time (Hours) Time (Hours)

2 0 I

Uninfected LL vector LL-CFA/I I **

1 5

C H

1 0

f o

% IDO 0

d r /I te to A c c F e e f v C n - i L L n L L U MHC C II

Figure 3-1. LL-CFA/I induces BMDCs to function as regulatory cells. BMDCs were generated from BALB/c bone marrow, stimulated with GM-CSF, and infected with LL-CFA/I or LL vector for 2 hours. Extracellular bacteria were washed away with media containing gentamicin. A) IDO and TGF-β expression induced by LL-CFA/I infected BMDCs was measured after 6 or 24 hours of culture. Dotted line represents expression levels in uninfected BMDCs. B) Representative FACS plots (left) of IDO expression in BMDCs infected with LL-CFA/I or LL vector at a 3:1 bacteria to BMDC ratio, and summary of observed percentages (right). C) Levels of TGF-β (left) and IL-10 (right) present in culture supernatants from BMDCs infected with LL-CFA/I or LL vector are shown. Results are the combined data of 2 experiments (n=6/group). *p<0.05 as compared against LL vector and PBS groups, **p<0.005 as compared against LL vector and PBS groups.

115

S p le e n c D C 1 C D 4 0 S p le e n c D C 1 C D 8 0 + 2 5 + 8 0

P B S P B S

c c

1 1

L L v e c to r L L v e c to r 1 2 0 1

6 0 D

L L -C F A /I D L L -C F A /I C

1 5 C

+ +

4 0

8 8

1 0

D D

C C

2 0 f

5 f

o o

% 0 % 0 0 1 2 3 0 1 2 3 W e e k W e e k

S p le e n c D C 1 C D 8 6 S p le e n c D C 1 T G F - + 6 0 + 8 P B S

c P B S

c # 1 L L v e c to r 1 1 L L v e c to r

1 6 D

4 0 L L -C F A /I D L L -C F A /I

+ +

8 D

2 0 D

% 0 % 0 0 1 2 3 0 1 2 3 W e e k W e e k

Figure 3-2. LL-CFA/I quickly induces IDO and TGF-β in the spleen and PaLNs of NOD mice. 6 wk-old female NOD mice were orally dosed with 5x107 CFUs of LL- CFA/I, LL vector, or 200 μL of PBS. Expression of CD40, CD80, CD86, and TGF-β examined by FACS. A) Summary of CD40, CD80, CD86, and TGF-β expression over time on splenic CD8+ DCs. B) Summary of CD40, CD80, CD86, and TGF-β expression over time on splenic CD11b+ DCs. C) Summary of IDO and TGF-β expression over time in splenic pDCS. D) Summary of CD40, CD80, CD86, and TGF-β expression over time on CD8+ DCs of the MLNs. E) Summary of CD40, CD80, CD86, and TGF-β expression over time on CD11b+ DCs of the MLNs. F) Summary of CD40, CD80, CD86, and TGF-β expression over time on CD8+ DCs of the PaLNs.G) Summary of CD40, CD80, CD86, and TGF-β expression over time on CD11b+ DCs of the PaLNs. H) Summary of IDO and TGF-β expression over time in pDCs of the PaLNs. I) Representative plots showing TGF-β and IDO expression on pDCs of the spleen, upper, and PaLNs, middle. Summary of observed percentages is also shown, lower. Total number of IDO-TGF-β+, left, and IDO+TGF-β+ pDCs observed in the J) PaLNs and K) spleen. Results are the combined data of 2 experiments (n=8/group). *p<0.05 as compared against LL vector and PBS groups, #p<0.05 as compared against the LL vector group, +p<0.05 as compared against the PBS group.

116

S p le e n c D C 2 C D 4 0 S p le e n c D C 2 C D 8 0

+ +

2 5 1 0 0 c

c P B S P B S

1 1

1 2 0 L L v e c to r 8 0 L L v e c to r

D C

C L L -C F A /I L L -C F A /I +

+ 1 5 6 0

b 1

1 1 0 4 0

D C

C 5 2 0

0 0 % % 0 1 2 3 0 1 2 3 W e e k W e e k

S p le e n c D C 2 C D 8 6 S p le e n c D C 2 T G F -

+ +

6 0 2 0 c

P B S c P B S

1 1

L L v e c to r 1 L L v e c to r D

D 1 5 C

4 0 L L -C F A /I C * L L -C F A /I

+ +

1 0

1 1

2 0 1 D

D 5

0 0 % 0 1 2 3 % 0 1 2 3 W e e k W e e k C

S p le e n p D C T G F - S p le e n p D C ID O

5 0 1 5

P B S P B S

+ + 4 0 L L v e c to r L L v e c to r

+ H *

- c

c L L -C F A /I 1 0 L L -C F A /I e

e 3 0

i S

S 2 0

f 5

1 0

% %

0 0 0 1 2 3 0 1 2 3 W e e k W e e k Figure 3-2 Continued.

117

M L N c D C 1 C D 4 0 M L N c D C 1 C D 8 0 + + 4 0 1 0 0

c P B S P B S

1 1

1 L L v e c to r L L v e c to r

1 8 0

3 0 D

L L -C F A /I D L L -C F A /I C

C 6 0

+ +

2 0

8 8

D 4 0

f 1 0

f 2 0

% % 0 0 0 1 2 3 0 1 2 3 W e e k W e e k

M L N c D C 1 C D 8 6 M L N c D C 1 T G F - + 5 0 + 6

c P B S

+ c P B S

1 1

1 L L v e c to r

4 0 1 L L v e c to r D

L L -C F A /I D 4 L L -C F A /I C

3 0 C

8 D

2 0 D

1 0 f

% 0 % 0 0 1 2 3 0 1 2 3 W e e k W e e k

M L N c D C 2 C D 4 0 M L N c D C 2 C D 8 0

+ + c

c 2 5 6 0 1

1 P B S P B S

1 1

2 0 + L L v e c to r L L v e c to r

D C

C L L -C F A /I 4 0 L L -C F A /I +

+ 1 5

b 1

1 + 1 1 1 0

2 0

0 0 % % 0 1 2 3 0 1 2 3 W e e k W e e k

M L N c D C 2 C D 8 6 M L N c D C 2 T G F -

+ c 4 0 c 8

1 P B S

* 1 P B S

1 1

L L v e c to r L L v e c to r D

3 0 D 6 C

L L -C F A /I C L L -C F A /I

+ b

2 0 b 4

D D

1 0 2

0 0 % 0 1 2 3 % 0 1 2 3 W e e k W e e k Figure 3-2 Continued.

118

P a L N c D C 1 C D 4 0 P a L N c D C 1 C D 8 0 +

+ 6 0 8 0 c

P B S c P B S

1 1 L L v e c to r 1 L L v e c to r

6 0 D

4 0 L L -C F A /I D L L -C F A /I

C C

+ +

4 0

8 D

2 0 D

f 2 0

% % 0 0 0 1 2 3 0 1 2 3 W e e k W e e k

P a L N c D C 1 C D 8 6 P a L N c D C 1 T G F - + 6 0 + 2 5

c P B S

1 1

1 L L v e c to r

1 2 0 L L v e c to r D

4 0 L L -C F A /I D L L -C F A /I C

C 1 5

8 D

D 1 0

2 0

f 5

% 0 % 0 0 1 2 3 0 1 2 3 W e e k W e e k G

P a L N c D C 2 C D 4 0 P a L N c D C 2 C D 8 0

+ + c

c 2 5 6 0 1

1 P B S P B S

1 1

2 0 L L v e c to r L L v e c to r

D D C

C L L -C F A /I 4 0 L L -C F A /I +

+ 1 5

b b

1 1 1

1 1 0 D

D 2 0

C C

f f

o o

0 0 % % 0 1 2 3 0 1 2 3 W e e k W e e k

P a L N c D C 2 C D 8 6 P a L N c D C 2 T G F -

+ c 5 0 c 1 5

1 P B S

1 1

4 0 L L v e c to r L L v e c to r

D C

L L -C F A /I C 1 0 L L -C F A /I +

3 0 +

1 1 2 0 1

D 5

1 0

0 0 % 0 1 2 3 % 0 1 2 3 W e e k W e e k Figure 3-2 Continued.

119

P a L N p D C T G F - P a L N p D C ID O

2 5 2 0 *

P B S P B S

+ + L L v e c to r L L v e c to r H

H 2 0 -

- 1 5 c

c L L -C F A /I L L -C F A /I

e l

l 1 5

g i

i 1 0 S

S 1 0

% %

0 0 0 1 2 3 0 1 2 3 W e e k W e e k

PBS LL vector LL-CFA/I

Spleen

TGF

PaLN

IDO

3 0

P B S

+ H

- L L v e c to r c

e 2 0 l L L -C F A /I

g *

f 1 0

- + O O D I ID +  + - -  F F G G T T

Figure 3-2 Continued.

120

- + + + P a L N ID O T G F - P a L N ID O T G F -

1 0 0 0 0 4 0 0 0

8 0 0 0 *

3 0 0 0

s l

6 0 0 0 l

l e

e 2 0 0 0 C 4 0 0 0 C

1 0 0 0 2 0 0 0

0 0

S r /I S r /I B to A B to A P c F P c F e C e C v - v - L L L L L L L L

- + + + S p le e n ID O T G F - S p le e n ID O T G F -

1 0 0 0 0 0 3 0 0 0 0 * 8 0 0 0 0 **

2 0 0 0 0

s l

l 6 0 0 0 0

e e

C 4 0 0 0 0 C 1 0 0 0 0 2 0 0 0 0

0 0

S r /I S r /I B to A B to A P c F P c F e C e C v - v - L L L L L L L L

Figure 3-2 Continued.

121

S p le e n F o x p 3 P a L N F o x p 3

2 0 2 0 P B S P B S

L L v e c to r L L v e c to r

1 5 1 5

4 4

L L -C F A /I L L -C F A /I

1 0 f 1 0

% % 5 5

0 0 0 1 2 3 0 1 2 3 W e e k W e e k

M L N F o x p 3 P e y e r's p a tc h e s F o x p 3

1 5 1 0 P B S + P B S

+ L L v e c to r 8 L L -C F A /I

4 4

1 0 L L -C F A /I D

D 6

% % 2

0 0 0 1 2 3 0 1 2 3 W e e k W e e k B

S p le e n F o x p 3 + H e lio s + M L N F o x p 3 + H e lio s +

4 0 8 0 P B S P B S

L L -C F A /I L L -C F A /I 3

3 3 0 6 0

o F

F 2 0 4 0

1 0 2 0

% %

0 0 0 1 2 3 0 1 2 3 W e e k W e e k

P a L N F o x p 3 + H e lio s + P e y e r's p a tc h e s F o x p 3 + H e lio s +

8 0 6 0 P B S P B S

L L -C F A /I L L -C F A /I 3

3 6 0 p

p 4 0

o F

F 4 0

2 0

% %

0 0 0 1 2 3 0 1 2 3 W e e k W e e k

Figure 3-3. LL-CFA/I promotes Foxp3+ Tregs in the MLNs and PPs. 6 wk-old female NOD mice were orally dosed with 5x107 CFUs of LL-CFA/I, LL vector, or 200 μL of PBS. A) Foxp3 expression over time was examined by FACS analysis in the spleen, MLNs, PaLNs, and PPs. B) Helios and C) NRP-1 expression was examined at different time-points by FACS analysis in the spleen, MLNs, PaLNs, and PPs. Results are the combined data of 2 experiments (n=8/group). +p<0.05 as compared against the PBS group.

122

Figure 3-3 Continued.

123

Spleen cDC MLN cDC +

80 c 80

PBS PBS

1 I

I LL vector LL vector

60 D 60 C

LL-CFA/I C LL-CFA/I

I I

M 40 40

20 M 20

0 0 % 1 2 1 2 C C C C cD cD cD cD

P a L N c D C P a tc h e s c D C

+ c

1 0 0 c 1 0 0

1 P B S P B S

1 1

8 0 L L v e c to r 1 8 0 L L -C F A /I

D C

L L -C F A /I C

+ +

I 6 0 6 0

C 4 0 4 0

2 0 f 2 0

0 % % 1 2 1 2 C C C C D D D D c c c c

S p le e n c D C 1 S p le e n c D C 2

+ c

+ 1 0 0 2 5 1

c P B S

1 1

1 8 0 2 0 L L v e c to r

D D

C L L -C F A /I +

C 6 0 1 5

8 1

D 4 0 1 0

C f

2 0 5 #

% 0 0 % 0 0 6 L 0 0 6 L 4 8 8 0 4 8 8 0 4 D D D 4 D D D x C C C C C C X O O

Figure 3-4. Treatment with LL-CFA/I reduces costimulatory molecules in MLNs. 4 wk- old female NOD mice were orally dosed with 5x107 CFUs of LL-CFA/I, LL vector, or 200 μL of PBS. Additional doses were given every 2 wks. Mice were euthanized at 11 weeks of age, 1 wk after their final dose. A) Summary of percentages of CD8+ and CD11b+ DCs in the spleen, upper left, MLNs, upper right, PaLNs, lower left, and PPs, lower right. B) Summary of expression of CD40, CD80, CD86, and OX40L on CD8+ DCs, left, and CD11b+ DCs, right in the B) spleen, C) MLNs, D) PaLNs, and E) PPs. Results are the combined data of 2 experiments (n=10/group). *p<0.05 as compared with PBS and LL vector groups, #p<0.05 as compared with LL vector, +p<0.05 as compared with PBS group. +++p<0.0005 as compared with PBS group.

124

M L N c D C 1 M L N c D C 2

+ c

+ 6 0 5 0 c

1 P B S

1 1

1 4 0 L L v e c to r

+++ D D

4 0 C + L L -C F A /I

C * + 3 0

+ #

1 D 1 2 0

2 0

f C

1 0 *

o % 0 0

0 0 6 L % 4 8 8 0 0 0 6 L 4 4 8 8 0 D D D D D D 4 C C C X C C C X O O

P a L N c D C 1 P a L N c D C 2

+ +

1 0 0 c 8 0 c

1 P B S

1 1

1 8 0 L L v e c to r

D 6 0 D

C L L -C F A /I C

6 0 +

b 8

1 4 0 1

D 4 0

C 2 0

2 0

% 0 0 % 0 0 6 L 0 0 6 L 4 8 8 0 4 8 8 0 D D D 4 4 X D D D C C C C C C X O O

P e y e r's p a tc h e s c D C 1 P e y e r's p a tc h e s c D C 2

4 0 1 0 0 P B S

8 0 L L -C F A /I 1

3 0 2

C D

D 6 0

2 0

f o

o 4 0

% % 1 0 2 0

0 0

0 6 0 6 8 8 8 8 D D D D C C C C

Figure 3-4 Continued.

125

Uninfected LL vector LL-CFA/I

Foxp3

CD4

Uninfected LL vector LL-CFA/I

CD44

CD62L

Figure 3-5. LL-CFA/I infected BMDCs induce IL-10 producing Foxp3- Tregs. BMDCs infected with LL-CFA/I or LL vector were co-cultured with CD25-CD4+ T cells at a 1:4 ratio in triplicate wells. A) Representative plots of Foxp3 expression in CD4+ T cells after co-culture. B) Representative plots of CD44, and CD62L expression on CD4+ T cells after co-culture. C) Summary of CD44 and CD62L expression on CD4+ T cells after co-culture. D) Summary of percentage IL-1β expression in BMDCs after co-culture. E) Concentration of IL-10 found in supernatants of co-cultures. *p<0.05 as compared against PBS and LL vector groups, +p<0.05 for LL-CFA/I vs LL vector, #p<0.05 for PBS vs LL vector.

126

T e ff M e m

5 0 * U n in fe c te d

4 0 L L v e c to r 4 L L -C F A /I

D 3 0

f o

2 0 * * % 1 0

+ + - 4 4 4 4 4 4 D D D C C C - + + L L L 2 2 2 6 6 6 D D D C C C

D E

T c e ll c o -c u ltu re w ith in fe c te d B M D C s

0 .8

) L

0 .6

g n

( 0 .4

0 1

- 0 .2

L I

0 .0

n r /I o to A ti c c F e C fe v - in L L L L o N

Figure 3-5 Continued.

127

Table 3-1. List of antibodies used for flow cytometry in LL-CFA/I DC studies. Antibody specificity Clone Source Dilution CD11b M1/70 Biolegend 1/600 CD11c N418 eBioscience 1/500 CD19 eBio1D3 eBioscience 1/600 CD40 3/23 Biolegend 1/500 CD8α 53-6.7 eBioscience 1/500 CD80 16-10A1 Biolegend 1/500 CD86 GL-1 eBioscience 1/500 DCIR2 33D1 BD 1/500 DEC-205 MG38 BD 1/500 ICOSL HK5.3 Biolegend 1/500 IDO eyedio Inivtrogen 1/250 IL-10 JES5-16E3 eBioscience 1/250 IL-1β NJTEN3 eBioscience 1/250 MHC II I-Ad Invitrogen 1/500 OX40L OX-86 eBioscience 1/500 PD-L1 10F.9g2 Biolegend 1/500 PD-L2 Ty25 BD 1/500 TCR-β H57-597 Biolegend 1/500 TGF-β TW7-16B4 Biolegend 1/400

128

CHAPTER 4 MUCOSAL TOLERANCE INDUCTION FACILITATED BY REOVIRUS PROTEIN SIGMA-1 AMELIORATES T1D IN NOD MICE

Background

In T1D, autoreactive T cells infiltrate the pancreas and destroy insulin-producing

β cells in the islets of Langerhans. The result is that a patient can no longer control blood glucose levels. The current treatment paradigm involves monitoring blood glucose levels individually and injecting corresponding amounts of exogenous insulin. Insulin replacement therapy is a safe and efficacious way of controlling blood glucose levels, allowing patients to live with what was once, 100 years ago, a deadly disease. However, even with insulin replacement therapy patients remain susceptible to a number of severe complications such kidney disease, nerve damage, blindness, and cardiovascular disease (216, 334). Additionally, it is now recognized that T1D raises likelihood of cardiovascular disease and the average lifespan of an individual with T1D is 10 years lower than the average. Thus, a robust therapy is needed that can halt the autoimmune attack on β cells, allowing for prevention or potentially reversal of established T1D. However, such a treatment must maintain the excellent safety standard that insulin replacement therapy has set.

Tolerance is an attractive means of achieving this goal. Defined as immunological unresponsiveness towards an antigen, tolerance can be induced mucosally, and historically has been shown to be very safe for humans and animals

(144, 200, 218). Tolerance induction occurs through one of two mechanisms; induction of Tregs or anergy in Teff cells, often dependent on the dose used (144). However, this strategy requires knowledge of the target or initiating antigen. Studies in NOD mice show that tolerization using InsB:9-23 and whole insulin have similar protective efficacy

129

when administered before 10 wks, however, whole insulin protects with greater efficacy than InsB:9-23 or GAD when given after 10 wks, suggesting that InsB:9-23 initiates T1D in NOD mice and the disease progresses through epitope spreading (190). More recent studies have shown that novel insulin epitopes are generated from defective ribosomal products of β cells and secreted into the blood, providing a potential means of breaking tolerance towards insulin and initiating T1D in the NOD mouse (89).

Oral tolerance strategies utilizing insulin or GAD have proven protective in NOD mice, suggesting therapy would be effective in diabetic patients (166, 168, 169, 171).

However, human clinical trials utilizing oral GAD or insulin have yet to reach their targeted outcome of preventing onset of T1D (176–178, 335). Two likely explanations for these outcomes are antigen selection and dosing efficiency. Translating dosing amounts from mice to humans is a challenging task. A recent human trial, called the

Immune Effects of Oral Insulin, has begun testing multiple dosing amounts. Marking the first clinical dose optimization study in humans for T1D.

M cells of the MALT are a specialized cell type that sample luminal antigen and facilitate antigen delivery for presentation to T and B cells (184, 307). Thus, M cells represent a potential target for antigen delivery to enhance induction of oral tolerance.

Reovirus pσ1 is an adhesin used to target M cells as a portal of entry into the host

(336). The Pascual lab found that pσ1, when fused to a heterologous protein, maintains specificity for M cell binding (146). Previously, it has been shown that genetically fusing the model antigen ovalbumin to pσ1 could induce tolerance in mice after a single dose

(145, 146). Additionally, the amount of protein required was shown to be 1000 times less than when using the native protein alone (146). Genetically fusing MOG peptide

130

29-146 to pσ1 have been shown to prevent or treat MOG-induced EAE through the induction of antigen-specific Tregs (186, 337).

Tregs generally classified as tTregs, which are generated in the thymus, or pTregs, which are differentiated in the periphery (237, 338). Classically described as

Foxp3 and CD25 expressing, Tregs are now appreciated as a plastic cell type, capable of expressing different markers such as CD39 or CD62L, and are capable of suppressing inflammation through production of a variety of cytokines and co-inhibitory markers (215, 339, 340). In previous studies utilizing pσ1, induced Tregs were found to produce IL-10 (186). Additionally, IL-10-producing Tregs have shown to protect NOD mice from T1D (121, 268). Thus, it was hypothesized that pσ1-induced Tregs would prevent the onset of T1D via antigen-specific IL-10-producing Tregs.

The studies described here examine when pσ1 fused to the B chain of insulin

(InsB-pσ1) or to full length GAD (GAD-pσ1) can prevent T1D in NOD mice when administered alone or in combination. The conferred protection is associated with IL-10- producing Foxp3+ Tregs. Interestingly, CD25 expression on these Tregs was found to be unique to the therapeutic used. Mice treated with InsB-pσ1 showed IL-10 production belonging to the Foxp3+CD25-CD4+ T cell subset. When GAD-pσ1 was introduced, either alone or in combination with InsB-pσ1, IL-10 was shown to be produced by the

Foxp3+CD25+ CD4+ T cells. These results suggest that targeting M cells for tolerance induction with insulin or GAD protects NOD mice from T1D.

Methods

Design, Construction, and Purification of Pσ1 Fusion Proteins

To obtain the InsB-pσ1 and GAD-pσ1 fusion proteins, sequences for InsB and

GAD were first hand-codon optimized for expression in yeast and synthetized by

131

GenScript. Pσ1 was recloned as previously described and respective plasmids were maintained the E. coli TOP10 vector (146). For InsB-pσ1, InsB and pσ1 were amplified from the relevant plasmid using appropriate primers. The 5’ primer for InsB encoded a

KpnI site along with an ATG initiation codon embedded into an optimal Kozak’s sequence. The 3’ primer for InsB and the 5’ primer for pσ1 included an AgeI site for framing into each other, while the 3’ primer for pσ1 provided an XhoI site. Polymerase chain reaction (PCR) products were gel purified to remove contamination caused by remaining TOP10 vector. The pYes2 vector (Invitrogen) was cut using KpnI and XhoI and a tripartite ligation was performed to join InsB, pσ1, and the vector. The resulting construct was transformed into E. coli and analyzed by restriction analysis and Sanger sequencing (GENEWIZ, South Plainfield, NJ, USA). The InsB-pσ1-pYes2 construct was then transformed into S. cerevisiae strain INVSc1 and transformants were analyzed for protein production by Western blot analysis using polyclonal rabbit anti-pσ1 (produced in-house).

For GAD-pσ1, GAD and pσ1 were amplified from the relevant plasmid using appropriate primers. The 5’ primer for GAD encoded a EcoRI site along with an ATG initiation codon embedded into an optimal Kozak’s sequence. The 3’ primer for GAD and the 5’ primer for pσ1 included a ScaI site for framing into each other, while the 3’ primer for pσ1 provided a KpnI site. PCR products were gel purified to remove contamination caused by remaining TOP10 vector. The pPicZα vector (Invitrogen) was cut using EcoRI and KpnI and a tripartite ligation was performed to join GAD, pσ1, and the vector. The resulting construct was transformed into E. coli and analyzed by restriction analysis and Sanger sequencing (GENEWIZ). The GAD-pσ1-pPicZα

132

construct was then transformed into P. pastoris strain X-33 and transformants were analyzed for protein production by Western blot analysis using polyclonal rabbit anti-pσ1

(produced in-house). Recombinant proteins were extracted from yeast cells by a bead- beater (Biospec Products, Bertlesville, OK) and purified on a Talon metal affinity resin

(BD) according to manufacturer’s instructions.

Quantifying and Analyzing Purified Protein

Purified proteins were analyzed to determine size, purity, and concentration. The

Pierce BCA Protein Assay kit (ThermoFisher) was used to measure protein concentration. Samples and standards were plated onto 96-well flat bottom plates and read on an Epoch Microplate Spectrophotometer (BioTek, Winooski, VT, USA). Gen5

2.0 (BioTek) was used to record data and data was analyzed using GraphPad (Prism).

To discern protein purity and size, gel electrophoresis and western blots were performed using 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels. Samples and protein controls were diluted in PBS so that 0.25 µg of protein would be loaded in each lane. Precision Plus Protein Kaleidoscope Pre-stained Protein Standards (Bio-Rad

Laboratories, Hercules, CA, USA) were used as molecular weight standards. Gels were either stained with Coomassie Brilliant Blue (ThermoFisher) or transferred to nitrocellulose blotting membrane (Pall Corporation, Pensacola, FL, USA). Gels stained with Coomassie Brilliant Blue were destained and purity was measured by densitometry. Protein quality and size were determined by detecting with rabbit anti-pσ1 polyclonal antibody (produced in-house).

NOD Mouse Husbandry

Female NOD/ShiLtJ mice (The Jackson Laboratory) were 3 or 5 wks of age upon arrival. Mice were housed 5/cage in SPF conditions with food and water available ad

133

libitum. Mice were allowed to acclimate to the facility for at least 5 days prior to handling. After acclimation, mice were given an identifying mark by ear-punching. All procedures were documented in protocols approved by the University of Florida IACUC.

P1 Treatments

Pσ1 fusion proteins were administered to NOD mice at the indicated time-points nasally or orally. For nasal dosing, solutions of InsB-pσ1, GAD-pσ1, insulin

(MilliporeSigma), or GAD (BioSynthesis, Lewisville, TX, USA) were prepared in PBS.

Protein solutions were administered with a micropipette dropwise into the anterior nares of the mice under isoflurane anesthesia. For oral dosing, proteins solutions were administered by gavage needle after neutralization of stomach acid.

Neutralization of stomach acid

Scientific). Mice were orally gavaged with 200 µL of the diluted sodium bicarbonate solution using a reusable AFN 20 gauge, 1.5’’ long, 2.25 mm-curved gavage needles

(Cadence Science) 5 to 10 minutes before administration with recombinant therapeutic.

Monitoring blood glucose levels

Starting at 10 wks of age, NOD mice were restrained using a stationary cone.

The tail was wiped with 70% before pricking the tail with the tip of a 23 gauge needle to draw blood. Blood was applied to an AlphaTRAK 2 (Abbott Animal Health) glucose test strip. A reading higher than 250 mg/dL was considered hyperglycemic and the mouse

134

was set to be screened again the following day. Diabetes was diagnosed after two consecutive blood glucose readings above 250 mg/dL or one reading at 500 mg/dL – the upper limit of the glucose test strip

Tissue Collections

All the animal experiments described in the present study were conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory

Animals of the National Institutes of Health. All animal studies were conducted under protocols approved by the University of Florida Institutional Animal Care and Use

Committee. All efforts were made to minimize suffering and ensure the highest ethical and humane standards.

Pancreata were collected using sterile forceps before either being fixed for staining with H&E or processed for isolation of lymphocytes. For H&E staining pancreata were spread evenly upon a thin sponge within a tissue cassette (University of

Florida Molecular Pathology Core; Gainesville, FL, USA). Tissue cassettes were incubated in 10% neutral buffered formalin (Leica) for at least 24 hours. Cassettes were then washed and stored in 70% ethanol. Processing, paraffin embedding, and cutting of slides were performed by the University of Florida Molecular Pathology Core. Two 5 µm sections were cut at least 150 µm away from each other to provide two distinct points of reference for histological analysis.

Spleens were harvested and placed into a sterile, 2.0 mL Safe-Lock Tube

(Eppendorf) with 1 mL of IM and a sterile, 5 mm, stainless-steel bead. Tissues were first disrupted by mechanical dissociation on a Tissuelyzer II (Qiagen). Cells were washed resuspended in 1 mL of IM. Red blood cells were lysed with ACK lysis buffer (produced in-house). Cells were washed and resuspended in CM before counting.

135

Lymphocyte isolation

PaLNs and MLNs were collected and placed into 2 mL Safe-Lock Tubes

(Eppendorf) tubes with 1 mL of IM and a sterile, 5 mm, stainless-steel bead. The tissue was first mechanically disrupted using a Tissuelyzer II set to 15 Hz for 1 minute. Cells were then filtered through sterile nytex (Sefar Inc.). Cells were washed and resuspended in CM before counting.

Counting Cells

Cells were counted on a Cellometer Auto T4 (Nexcelom). Single cell suspensions of cells from different tissues or peripheral blood were isolated as described above. A

Trypan Blue solution was prepared by diluting Trypan Blue Solution, 0.4%

(ThermoFisher) 1:1 in DPBS. For spleens, 10 µL of the cell suspension was added to 90

µL of Trypan Blue-DPBS. 20 µL of this mixture was added to a Cellometer Disposable

Counting Chamber (Nexcelom) and counted. For other tissues or blood the process was the same, except 20 µL of single cell suspensions were diluted in 20 µL of Trypan Blue-

DPBS.

Cell Culture and T Cell Stimulation

CD3 mAb (clone 17A2; Invitrogen). Anti-CD28 mAb (clone 37.51; Invitrogen) was then added to a final concentration of 2.5 µg/mL and samples incubated for 48 hours at

37°C. Stimulated cells meant for FACS analysis were treated with Brefeldin A

(Biovision), at a working concentration of 5 µg/mL, for the last 4 hours of culture.

136

Flow Cytometry

To determine the phenotype of Tregs induced by pσ1 fusion proteins, cells were washed with PBS and stained for viability using a LIVE/DEAD Fixable Blue Dead Cell

Stain Kit, for UV excitation (ThermoFisher). Cells were then washed with FB and labeled with mAbs specific for CD4, CD8α, TCR-β, CD19, CD25, CD39, and TGF-β.

Then cells were washed and fixed with 1X fixation buffer from the True-Nuclear

Transcription Factor Buffer Set (BioLegend). Cells were then washed with 1X permeabilization buffer from the True-Nuclear Transcription Factor Buffer Set

(BioLegend) and labeled with mAbs specific for Foxp3, IL-10, and IFN-γ. Cells were stained with conjugated mAbs at specific dilutions according to Table 4-1.

Cytokine ELISA

Splenocytes were isolated and prepared as described above. Cells were cultured at 5x106 cells/mL on 96 well, round bottom, tissue culture treated plates

(MilliporeSigma, Burlington, MA, USA) in the presence of 5 µg/mL anti-CD3 (clone

17A2; Invitrogen) and 2.5 µg/mL anti-CD28 (clone 37.51; Invitrogen, Carlsbad, CA,

USA) in a total volume of 200 µL. The supernatants were collected by centrifugation and stored at -80°C. Capture ELISA was employed to quantify, on duplicate sets of samples, the levels of IL-10 and TGF-β produced by BMDCs and lymphocytes.

For IFN-γ, 10 µg/mL rat anti-mouse IFN-γ mAb (clone R4-6A2, ThermoFisher) was used as the capture antibody, and 0.5 µg/mL of biotinylated rat anti-mouse IFN-γ mAb (clone XMG1.2; BD) was used as detecting antibody.

For TNF-α, 5 µg/mL rat anti-mouse TNF-α mAb (clone G281-2626, BD) was used as the capture antibody, and 1 µg/mL of biotinylated rat anti-mouse TNF-α mAb

(clone MP6-XT3, BD) was used as detecting antibody.

137

The color reaction was developed using HRP conjugated goat anti-biotin Ab

(Vector Laboratories, Burlingame, CA, USA) and ABTS peroxidase substrate (Moss,

Inc., Pasadena, ME, USA). Cytokine concentrations were extrapolated from standard curves generated by recombinant murine cytokines IL-10 and TGF-β (R&D Systems).

Statistical Analysis

All presented data are the mean ± SEM. Statistical significance was tested using

GraphPad Prism 7 (Prism). One-way ANOVA with Tukey’s multiple comparisons test were used to compare FACS data, cell counts, cytokine production, and insulitis scores.

Log-rank (Mantel-Cox) tests were used to compare incidence of T1D. All results were discerned to the 95% confidence interval.

Results

Development of Mucosally Targeted Tolerogens for T1D

Yeast production vectors provide support for proteins of eukaryotic origin and destination, such as correct protein folding and post-translational modifications (341).

Thus, InsB-pσ1 and GAD-pσ1 were designed for expression in yeast production vectors

Saccharomyces cerevisiae and Pichia pastoris, respectively. Sequences for InsB and

GAD were hand-codon optimized for expression in yeast and synthesized at GenScript

(GenScript, Piscataway, NJ, USA). Pσ1 was recloned, as previously described for genetic fusion (146). InsB and pσ1 were ligated into the Saccharomyces cerevisiae vector pYes2 (Invitrogen), analyzed by restriction analysis and Sanger sequencing, and subsequently transformed into Saccharomyces cerevisiae INVSc1 (Invitrogen). Western blots were used to analyze transformed S. cerevisiae by detecting protein production

(Fig. 4-1C).

138

GAD and pσ1 were ligated into the Pichia pastoris expression vector pPicZα

(Invitrogen), and similarly analyzed by restriction analysis and Sanger sequencing. pPicZα containing GAD-pσ1 was then transformed into P. pastoris X-33. Western blots were used to analyze transformed P. pichia by detecting protein production (Fig. 4-1D).

Recombinant proteins were extracted from yeast cells by a bead-beater (Biospec

Products, Bertlesville, OK) and purified on a Talon metal affinity resin (BD) according to manufacturer’s instructions.

Mucosal Pσ1 Therapy Ameliorates T1D

Previous studies with pσ1 fused to a MOG peptide demonstrated that a single, nasal dose could prevent EAE through the induction of Tregs (186). To test the hypothesis that pσ1 fused to T1D antigens could prevent disease onset, six week-old female NOD mice were nasally dosed with InsB-pσ1, GAD-pσ1, or a combination of these fusion proteins. Control mice were dosed with PBS, insulin, or GAD. Additional doses were given every 3 weeks and beginning at 15 weeks of age, mice were orally gavaged instead of nasally dosed. Mice treated with InsB-pσ1 or GAD-pσ1 were protected from T1D, with nearly 50% of the InsB-pσ1 treated mice (Fig.4-2A) and 70% of GAD-pσ1 treated mice (Fig. 4-2B) being diabetes free at the end of the study.

However, the combination therapy did not show significant improvement over the PBS treatment (Fig.4-2C).

At the end of the study, mice were euthanized and splenocytes were collected during necropsy. Splenic lymphocytes were stimulated in vitro with anti-CD3 and anti-

CD28 mAbs and T cell phenotypes were examined by flow cytometry. InsB-pσ1 dosed mice showed a greater than 2-fold increase (p<0.05) in CD25+ T cells compared to mice treated with PBS (Fig. 4-3B). Treatment with combination therapy induced a greater

139

than 2.5-fold increase in IL-10 producing Tregs as compared to PBS and GAD treatments (Fig. 4-3D). IL-10 production was limited to CD25+ expressing CD4+ T cells

(data not shown). Also, when GAD-pσ1 was introduced, either alone, or in combination with InsB-pσ1, CD25 expression was suppressed on stimulated CD4+ T cells (Fig. 4-

3D).

Furthermore, InsB-pσ1 modestly suppressed TNF-α production by CD4+ T cells while GAD-pσ1 significantly suppressed IFN-γ-producing CD4+ T cells. Combination therapy showed significant reductions in both TNF-α and IFN-γ-producing T cells.

Suggesting that combination therapy provided the most robust protection against inflammation.

Discussion

T1D is a chronic autoimmune disease becoming more common in developing nations. It is estimated that nearly 90,000 children are newly diagnosed each year (14).

Currently, the only available therapy is insulin replacement therapy. While insulin replacement allows patients to live with what was, 100 years ago, a deadly disease, patients remain susceptible to a variety of severe complications and have reduced life expectancy (12, 227, 334). As the incidence of this disease increases, so increases the healthcare burden and the need for a cure.

While the pathogenesis of T1D remains to be fully elucidated, it is thought to mediated by autoreactive T cells that infiltrate the pancreas and selectively destroy insulin-producing β cells (111, 342, 343). In support of this hypothesis, anti-CD3 mAb therapy has been shown to protect and treat NOD mice with T1D as well as slow progression of human disease (243, 255–257, 344). However, since anti-CD3 therapy was unable to prevent T1D it is possible that without complete ablation of autoreactive T

140

cells that prevention is impossible, suggesting that anti-CD3 therapy alone is insufficient to treat T1D without immunocompromising the patient.

Induction of tolerance towards antigens associated with T1D provides a safer means of achieving disease prevention. Tolerance is mediated through induction of anergy in Teffs or through induction of antigen-specific Tregs, and the mechanism is dependent upon the dose used (144). Tolerance studies targeting insulin or GAD have proven effective at protecting NOD mice from developing T1D (166, 168, 171). These initial findings have prompted multiple human clinical trials; however, trials featuring insulin or GAD-alum have not been able to prevent or treat diabetes in humans (174,

176–180, 345). These results indicate the major hurdles that must be overcome to adapt oral tolerance strategies to treat T1D. The first being antigen selection. The presence of islet associated autoantibodies is a sign of ongoing immune activity in the pancreas. However, multiple islet associated antibodies are required to serve as a signal that a patient will progress to overt T1D (90, 93, 193) Recent studies in humans show that patients with different risk of disease show differential expression of IFN-γ and IL-10, suggesting that, in humans, T1D may progress in a heterologous manner.

Additionally, islet associated antibodies do not appear in a set order, suggesting the etiology of T1D may be unique to the individual. These findings suggest therapies targeting a single antigen may not prove effective at treating T1D in all patients, or it may not effectively treat established T1D. The second hurdle to overcome is dosing efficiency. Recently, a human clinical trial – called the Immune Effects of Oral Insulin – has begun testing multiple doses, marking the first dose optimization studies in humans with T1D.

141

To overcome these hurdles, this report details the use of reovirus adhesin, pσ1 to enhance antigen delivery to the MALT and induce Tregs. Previously, pσ1 has been shown to induce tolerance towards a fused antigen with 1000-times less protein than traditional oral administration (146). Additionally, pσ1, when fused to MOG peptide, induces IL-10-producing Tregs to protect mice from EAE, demonstrating its efficacy in the context of autoimmunity (186). Thus, it was hypothesized that InsB-pσ1 and GAD- pσ1 fusion proteins would be able to protect NOD mice via induction of IL-10-producing

Tregs.

The data shows that mucosal administration of InsB-pσ1 or GAD-pσ1 fusion proteins protected 42% and 70% of treated animals, respectively. Additionally, pσ1 fusion proteins induced splenic IL-10-producing Tregs, suggesting treatment had a systemic effect. Since T1D is a T cell mediated autoimmune disease, IL-10 production frequently is found to correlate with protection (267, 268, 278). In humans, IL-10- producing Tregs are shown to correlate with patients having better glycemic control after diagnosis (288). This suggests that Treg induction after diagnosis may offer a therapeutic effect in helping patients control blood glucose levels.

Previous studies have shown that MOG-pσ1 could prevent or treat EAE through

+ + - + induction of IL-10-producing CD25 CD4 Tregs and IL-4-producing CD25 CD4 TH2 cells (186). Since Foxp3 does not correlate with the protective phenotypes seen in the present studies, an alternative is that these T cells more closely resemble the TH2 phenotype and potentially produce IL-4. Prior studies show that when IL-10-producing

CD25+CD4+ Treg function was blocked therapy was still able to protect against EAE, provided TGF-β was blocked, and effect was due to Tregs producing increased

142

amounts of IL-28 (185). IL-28 has been shown to prime tolerogenic DCs in vitro, and pσ1 based therapy has also been shown to induce regulatory DCs (337). However, regulatory DC induction by pσ1 was originally shown to precede Treg induction, suggesting that IL-28 production by Tregs may serve as an additional mechanism to enforce tolerance. IL-28’s role in T1D has not been studied, thus, future studies with

InsB-pσ1 and GAD-pσ1 will focus on the role regulatory DCs and IL-28 play in T1D.

Notably, the Treg phenotypes varied depending on the antigen targeted. Tregs induced by the combination therapy expressed high CD25 and IL-10. This is interesting as the IL-2 locus in NOD mice and in humans increases risk of developing T1D (32,

346, 347). Additionally, Treg function has been shown to decline as the disease progresses and this is thought to be caused by IL-2 deprivation (346). Tregs may also use high expression of CD25 to sequester IL-2 from Teffs, effectively inducing apoptosis in inflammatory T cells. Alternatively, it may reflect an additional mechanism those

Tregs use to suppress diabetogenic Teffs. Thus, inducing Tregs to both GAD and InsB, simultaneously, may act synergistically to induce IL-10-producing Tregs.

Mucosal administration of therapeutics has proven safe for both animals and humans. Additionally, tolerance induction by pσ1 is antigen specific, offering a means of suppressing an aberrant immune response without immunocompromising the host.

Thus, mucosal vaccination offers an active means of protecting or treating patients with

T1D.

143

A His-6 InsB Pσ1 Myc

B His-6 GAD Pσ1 Myc

C MW InsB-pσ1 Pσ1

100 kDa

50 kDa 52.59 kDa 51 kDa 37 kDa

D MW GAD-pσ1 Pσ1

150 kDa 100 kDa 114.72 kDa

50 kDa 51 kDa

Figure 4-1. Development of pσ1 fusion proteins for treatment of T1D. A) Schematic representation of InsB-pσ1 and B) GAD-pσ1: antigen and pσ1 (shaft and head), 6x histidine-tag, and Myc antigen-tag (components are not drawn to scale). C) Western blot of InsB-pσ1 and pσ1. D) Western blot of different elution fractions of GAD-pσ1 and purified pσ1. Proteins were detected with polyclonal rabbit anti-pσ1 antibody.

144

) l

d 1 0 0 e

/ 5 0 0 P B S

e g

r 8 0 m

4 0 0 F In s u lin

(

e In s B -p  1

e 6 0 t

s 3 0 0

e c b 4 0 *

u 2 0 0

2 0

d 1 0 0

% o l 0 0

B 1 0 1 2 1 4 1 6 1 8 2 0 2 2 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4 2 6 A g e (w e e k s ) A g e (W e e k s )

)

l d

/ 5 0 0 1 0 0 e

g P B S

e * r m 8 0

( 4 0 0 G A D c o n tro l

e s G A D -p  1 s 6 0

3 0 0 e

c b

u 4 0

l 2 0 0

1 0 0 2 0

% o

l 0 0

B 1 0 1 2 1 4 1 6 1 8 2 0 1 0 1 2 1 4 1 6 1 8 2 0 A g e (w e e k s ) A g e (w e e k s )

)

d / e 1 0 0

g 5 0 0

e P B S

m F ( 8 0

C o m b in a tio n

4 0 0

e s

3 0 0 t 6 0

c b

u 4 0 l

2 0 0 a

2 0

d 1 0 0

o %

o 0 l 0

B 1 0 1 2 1 4 1 6 1 8 2 0 1 0 1 2 1 4 1 6 1 8 2 0 A g e (W e e k s ) A g e (W e e k s )

Figure 4-2. Antigen-specific immunotherapy ameliorates T1D in NOD mice. Mice were nasally dosed with the indicated therapeutic at 6 wks of age. Additional doses were given every 3 wks. At 15 and 18 wks, mice were dosed orally. Blood glucose values of individual mice are shown (left) with the dotted line representing the limit for hyperglycemic values. Summary of disease incidence (right) of mice treated with A) PBS, insulin, or InsB-pσ1; B) PBS, GAD, or GAD-pσ1; and C) PBS or InsB-pσ1 in combination with GAD-pσ1. Dotted lines represent final disease incidence in mice given relevant pσ1 fusion protein. Data for InsB-pσ1 and PBS is a representation of 2 experiments, n=8/group. N=10/group for GAD-pσ1 and combination therapies and n=5/group for insulin and GAD control groups. *p<0.05 as compared against all other groups tested.

145

PBS Insulin InsB-pσ1

CD25

CD4

- IL

Foxp3

Figure 4-3. Pσ1 fusion proteins induce Tregs to protect NOD mice from T1D. Six week- old female NOD mice were nasally dosed with 50 μg of insulin, 50 μg InsB- pσ1, 100 μg of GAD, 100 μg of GAD-pσ1, 20 μL of DPBS, or a combination of GAD-pσ1 and InsB-pσ1. Additional doses were given every 3 wks. At 15 and 18 wks, mice were dosed orally. Splenocytes were recovered from mice treated with PBS, insulin, and InsB-pσ1, then stimulated with anti-CD3 and anti-CD28 to examine Treg phenotypes. A) Representative plots of CD25 expression on splenic CD4+ T cells, upper, and IL-10 and Foxp3 expression on the CD25 high population, lower, after stimulation. B) Total number of CD25+CD4+ T cells, upper left, IL-10+CD25-Foxp3+CD4+ T cells, upper right, IL-10+Foxp3+CD25+CD4+ T cells, lower left, and IL-10-Foxp3+CD25+CD4+ T cells, lower right. C) Splenocytes were recovered from mice treated with PBS, GAD, and GAD-pσ1, or combination therapy, then stimulated with anti-CD3 and anti-CD28 to examine Treg phenotypes. Representative plots of CD25 expression on splenic CD4+ T cells, upper, and IL-10 and Foxp3 expression on the CD25 high population, lower, after stimulation. D) Total number of CD25+CD4+ T cells, upper left, IL-10+CD25-Foxp3+CD4+ T cells, upper right, IL-10+Foxp3+CD25+CD4+ T cells, lower left, and IL-10-Foxp3+CD25+CD4+ T cells, lower right. E) Summary of percentages of TNF-α expression in splenic CD4+ T cells, left, and concentration of TNF-α in culture supernatant of splenocytes stimulated with anti-CD3 and anti-CD28. F) Summary of percentages of IFN-γ expression in splenic CD4+ T cells, left, and concentration of IFN-γ in culture supernatant of splenocytes stimulated with anti-CD3 and anti-CD28. Data for InsB-pσ1 and PBS is a representation of 2 experiments, n=8/group. N=10/group for GAD-pσ1 and Combination therapies and n=5/group for insulin and GAD control groups. *p<0.05 as compared against PBS group, ++p<0.005 as compared against PBS group, #p<0.05 as compared against GAD group, +++p<0.0005 as compared against PBS group.

146

Figure 4-3 Continued.

147

C D 2 5 H iC D 4 + IL -1 0 + F o x p 3 - C D 2 5 H i

8  1 0 0 6 1 5 0 0 0 0 +++ #

6  1 0 0 6

1 0 0 0 0 0

s s

l l l

l 0 6 e

e 4  1 0

C C + + 5 0 0 0 0 2  1 0 0 6

0 0

S D 1 n S D 1 n B  o B  o A p ti A p ti P G - a P G - a D in D in A b A b G m G m o o C C

IL -1 0 - F o x p 3 + C D 2 5 H i IL -1 0 + F o x p 3 + C D 2 5 H i

4 0 0 0 0 0 8 0 0 0 0

3 0 0 0 0 0 6 0 0 0 0

l e

e 2 0 0 0 0 0 4 0 0 0 0

C C 1 0 0 0 0 0 + + 2 0 0 0 0

0 0

S D 1 n S D 1 n B  o B  o A p ti A p ti P G - a P G - a D in D in A b A b G m G m o o C C

1 5 5

)

l e

c 4

m /

T 1 0 *

4 3

(



5 - 2

f ++ ++

N 1

% T 0 0 S 1 1 o B   b B D P s m S 1 1 o n A o B   b I G C B D P s m n A o I C G F

2 5 5 0

) e

2 0 l

c 4 0

/ T

1 5 g

4 3 0

n D ** (

1 0 C ** 

2 0

N o

F 1 0

I % 0 0 S 1 1 n   o B ti P -p -p S 1 1 n a   o B D in B ti s A b P -p -p In a G m B D in o s n A b C I G m o C Fig. 4-3 Continued.

148

Table 4-1. List of antibodies used in flow cytometry for pσ1 based therapies. Antibody specificity Clone Source Dilution CD19 EBio1D3 eBioscience 1/600 CD25 PC61.5 Biolegend 1/500 CD39 24DMS1 eBioscience 1/500 CD4 RM4-5 Biolegend 1/500 CD8α 53-6.7 eBioscience 1/500 Foxp3 FJK-16s eBioscience 1/250 IFN-γ XMG1.2 BD 1/250 IL-10 JES5-16E3 eBioscience 1/250 TCR-β H57-597 Biolegend 1/500 TGF-β (Lap) TW7-16B4 Biolegend 1/400 TNF-α MP6-XT22 Biolegend 1/250

149

CHAPTER 5 CONCLUSIONS AND FUTURE DIRECTIONS

Mucosal tolerance strategies to induce tolerance towards food antigens such as peanuts, eggs, and milk have shown promising results in human clinical trials (348–

350). A notable feature of these studies is the low rate of adverse reactions to therapy, showing the excellent safety profile of the mucosal route of administration. These findings suggest that mucosal tolerance strategies may be the gold standard for treating autoimmune diseases. This is due to their ability to correct aberrant immune responses without global immunosuppression and with an excellent safety profile. However, human trials have shown limited success with mucosal tolerance strategies in T1D

(175–179, 335, 345). These therapies have relied upon a single antigen, either insulin or GAD, to treat T1D. While there is a good deal of evidence suggesting important roles for insulin and GAD specific responses in the pathogenesis of T1D, the exact etiology is unknown (259). Antigen selection is complicated by the numerous antigens associated with disease pathogenesis; insulin, GAD, IA-2, chromogranin A, Znt8, IGRP, and heat shock protein (9, 221, 276, 289, 351). Autoantibodies towards 2 or more of these antigens, but not one, indicates high risk that a patient will go on to develop T1D (92).

Insulin is thought to be the initiating antigen, with others becoming relevant as a byproduct of epitope spreading (190). However, recent studies have described novel antigens, in the form of defective ribosomal products and fused-insulin peptides, able stimulate inflammatory responses (88, 89). These potentially represent different ways that T1D can be initiated. Together, these findings suggest that the pathogenesis of

T1D is heterologous and, thus, the first hurdle to overcome is antigen selection.

150

To overcome the first hurdle CFA/I fimbriae were utilized. Originally targeted as an antigen for a vaccine against ETEC, CFA/I fimbriae have been shown to prevent and treat animal models of autoimmune diseases such as EAE and CIA (211, 212, 214,

215). CFA/I mediates protection against autoinflammation through the induction of bystander tolerance (213). This mechanism allows CFA/I fimbriae to act in an antigen independent manner and provides an excellent alternative to mucosal tolerance through antigen-specific monotherapies. In an effort to provide a food based tolerogenic therapeutic, we expressed CFA/I fimbriae on the Gram-positive bacteria L. lactis (228).

L. lactis was selected as a vaccine vector because it is already used in food production for human consumption, even holding a generally recognized as safe rating from the

Food and Drug Administration (352, 353). Additionally, L. lactis does not colonize the intestines of mice or humans, suggesting that the resulting therapy would not cause additional complications to the host (277). L. lactis has been used as a vaccine vector previously, including as a shuttle for T1D antigens such as insulin and GAD (273, 274).

These studies suggest that L. lactis can effectively shuttle antigen through the hazardous environment of the mammalian gut as well as show that L. lactis does not provide protection against T1D on its own.

T1D is a disease characterized by insufficient insulin production brought on by a combination of the destruction of β cells and β cells becoming dysfunctional. The autoimmune attack on β cells is an ongoing process that starts as early as 3 weeks of age in the NOD mouse (106, 134). The islets are initially infiltrated by DCs and macrophages (105, 106, 309, 354). However, between 8 and 12 weeks of age, the immune infiltrate becomes dominated by CD4+ and CD8+ T cells (105, 106, 188).

151

Hyperglycemia is surest sign of β cell destruction or dysfunction, and is used to diagnose T1D in most studies. In the NOD mouse model, T1D is diagnosed anytime between 10 and 30 weeks of age, with most becoming hyperglycemic between 16 and

20 weeks of age. It was hypothesized that Tregs would be detectable at 11 weeks, when T cell infiltration was well established. However, CD4+ Tregs were not detected using conventional markers such as Foxp3, CD25, and CD39. There was an observed, significant, reduction in inflammatory mediators in the spleen and islets suggesting a regulatory element was present. Thus, non-CD4+ Tregs were considered: Bregs as marked by CD1d and CD5 as well as CD122+CD8+ Tregs were examined. Bregs expressing PD-L1 were detected, however, they were limited to the spleen at 11 weeks of age, suggesting that they were not mediating protection at the site of inflammation. A later disease time-point was then examined, first to see if the observed suppression of

Tbet and IFN-γ was stable, and second to examine Treg phenotypes just before onset of overt disease. We next examined T cell phenotypes at 17 weeks of. Foxp3+CD4+

Tregs from the PaLNs of PBS and LL vector treated mice expressed IFN-γ and Tbet to a high degree, suggesting that they had lost their suppressive function and had become ex-Tregs. However, Tregs from LL-CFA/I treated mice did not express these inflammatory mediators, suggesting that their suppressive function was stable and maintained at this late time-point. Additionally, suppression of Tbet and IFN-γ in splenocytes at 17 weeks was observed, much greater than at 11 weeks, showing the stability of LL-CFA/I based protection.

These data suggested a crucial role for Foxp3+CD4+ Tregs. To determine the role of Foxp3+ Tregs, future studies will utilize the NOD/ShiLtJ-Tg(Foxp3-

152

HBEGF/EGFP) mouse model. These mice express a fusion of diphtheria toxin receptor and enhanced green fluorescent protein (EGFP) under the promoter and enhancer regions of the Foxp3 gene. This allows for sorting of viable Foxp3+ Tregs using EGFP, thus enabling adoptive transfer studies targeting Foxp3+ Tregs. We hypothesize that these Tregs will effectively protect NOD.SCID mice from T1D caused by adoptive transfer of diabetogenic splenocytes.

The data show induction and maintenance of Foxp3+ Tregs by treatment with LL-

CFA/I as well as suppression of inflammatory mediators Tbet and IFN-γ in mixed cell cultures. Notably, treatment with LL-CFA/I elicited varied regulatory mechanisms at different disease time-points. This is important as reviews of different therapeutic interventions show efficacy is affected by the mouse’s age when dosing, suggesting that different pathogenic mechanisms are important at different disease time-points (266). At

11 wks, Foxp3+ Tregs PPs, IL-10-producing cells in the MLNs, and splenic Bregs all contribute to protection. Later in disease, at 17 wks of age, Foxp3+ Tregs that suppressed IFN-γ and Tbet responses and maintained protection. Finally, at 24 wks,

IFN-γ and IL-10-double producing Tr1 cells were responsible for protection. Collectively, these data indicate that LL-CFA/I induced bystander suppressing results in populations of regulatory cells uniquely equipped to suppress autoinflammation.

It is unknown exactly how bystander suppression induced by CFA/I fimbriae evolves into an antigen-specific regulatory response. Bystander suppression functions through exposing a target antigen to the immune system while in the same environment as a secondary antigen that tolerance is enforced towards (219). DCs have been shown to be required for the induction of oral tolerance, and furthermore, are known to play a

153

critical role in determining the fate of a T cell during differentiation (307, 316, 317). This is achieved through DCs providing different signals to T cells during activation: TCR engagement, costimulatory molecules such as CD80/CD86 and CD40, and cytokines

(318). In T1D, these mechanisms seemed poised to induce activated and inflammatory

These findings were confirmed in the NOD mouse as TGF-β- and IDO-producing pDCs were detected in the spleens and PaLNs of treated mice. Treatment kinetics showed that this regulatory pDC phenotype was no longer visible starting at 1 wk after dosing.

However, at this same time-point, treatment with LL-CFA/I lowered expression of costimulatory molecules CD86 and CD40 in the MLN. This phenotype became more pronounced by 3 wks post treatment, suggesting it was preceded by the observed induction of Tregs in the PPs and MLN. Collectively, these findings suggest that LL-

CFA/I induces bystander tolerance through pDCs, which in turn stimulate Tregs to further restrain inflammatory DCs in NOD mice. In support of this, LL-CFA/I infection of

BMDCs was shown to suppress IL-1β production, similar to observations with

Salmonella-CFA/I (209). Additionally, these BMDCs activate naïve CD4+ T cells to produce IL-10, suggesting that they can directly induce Tregs.

The data shows some discrepancy between pDCs being induced in the spleen and PaLNs and Tregs appearing in the MLNs and PPs. This is likely due to the time-

154

points we selected. We hypothesize that IDO producing pDCs are induced earlier in the lamina propria and migrate to sites of inflammation, such as the PaLNs in NOD mice.

Future studies will focus on DC phenotypes of the MLNs, PPs, and LP in the days just after treatment.

These data provide insight into how CFA/I fimbriae mediated protection shifts from bystander suppression to this autoantigen-specific response. Furthermore, knowing the cell types responsible for this early response will aid in determining the ligand CFA/I fimbriae interacts with. Others have suggested that the different subunits of

CFA/I have different binding specificities. The tip protein of the fimbriae, CfaE, is thought to bind to erythrocytes while the major fimbral subunit, CfaB, is believed to bind to glycosphingolipids (202, 203, 355). However, these findings do little to explain the observed bystander suppression found in autoimmune settings and suggest another ligand is being used, potentially by DCs of the GALT (211, 213, 215).

Together these findings suggest that LL-CFA/I induces pDCs that produce IDO and TGF-β that create a regulatory microenvironment that preferentially stimulates

Tregs. These Tregs, in turn, control and suppress inflammatory DCs through reducing expression of costimulatory molecules, CD40 and CD86. This combination of Tregs, pDCs, and suppression of inflammatory DCs restrains diabetogenic Teffs in the PaLNs and islets of Langerhans, protecting NOD mice from T1D.

Together, the data from different disease time-points – early (4-6 wks), disease onset (11 wks), overt disease (17 wks), and late stage (24-30 wks) – show LL-CFA/I induces a robust regulatory response to protect against T1D. Within days of oral treatment, LL-CFA/I induces IDO+TGF-β+ pDCs in the spleen and PaLNs. Data from in

155

vitro studies demonstrate that LL-CFA/I infected BMDCs induce IL-10 production from naïve CD25-CD4+ T cells, suggesting that pDCs are inducing regulatory Tr1 cells in the

PaLNs and systemically. This pDC response is quickly followed by an induction of

Foxp3+CD4+ Tregs in the PPs and MLNs, which is still visible at 11 wks of age (Fig. 5-

1A, upper right). These Tregs are thought to play a role in controlling the inflammatory potential of cDCs at the mucosal inductive sites, starting within 2 wks of treatment and stable at least to 11 wks (Fig. 5-1B). These Tregs and regulatory cDCs are associated with suppression of IFN-γ-producing CD8+ T cells and Tbet+CD4+ T cells in the spleens and pancreas (Fig. 5-1A, lower right). By 17 wks, Tregs are present in the PaLNs with suppression of Tbet and IFN-γ (Fig. 5-1A, left side). Additionally, this systemic suppression of pathogenic TH1 responses is stable in the spleens of treated mice (Fig.

5-1A, lower left). At the late stage of disease, Tregs have gained expression of IL-10 and IFN-γ in the spleen and continue to protect against T1D through suppressing

Tbet+CD4+ T cells (Fig. 5-1A, upper left). These findings show that LL-CFA/I induces

Tregs that gain functions to protect against the evolving pathogenesis of T1D.

Our complimentary approach utilized reovirus pσ1 which is used by the virus to target M cells of the PPs (336). Pσ1 has been shown to provide an efficient means of antigen delivery to M cells when antigen is genetically fused to it, lowering the amount of protein required for tolerization 1000x over (146). Studies with mucosal administration of InsB-pσ1 and GAD-pσ1 demonstrate that these fusion proteins are capable of protecting NOD mice from T1D. Additionally, in the splenic IL-10-producing Tregs were detected. Interestingly, we detected different phenotypes depending on the therapy used. Combination therapy induced IL-10+CD25HighCD4+ Tregs while InsB-pσ1 induced

156

CD25HighCD4+ Tregs with suppressive activity independent of IL-10. These results suggest combination therapy provides the most robust protection against T1D and that

IL-10 is crucial to protection mediated by pσ1 fusion proteins. Given the differential expression of CD25, sequestration of IL-2 may be important to suppressing GAD specific inflammation but not crucial to suppressing insulin specific responses. These results suggest inflammatory responses to different T1D antigens may be unique and overlapping and thus requires more broadly functioning regulatory action to protect against disease. These findings are notable as studies in humans have shown that IAAs specific for insulin and GAD first appear at different ages; IAAs for insulin appear around 2 years of age and IAAs specific for GAD appear later, between 3 and 5 years of age. This variation in disease kinetics suggests that the initiation of T1D is a heterogeneous process that may involve different pathogenic mechanisms.

157

Figure 5-1. Summary of protection mediated by LL-CFA/I over time. A) At 11 wks, LL- CFA/I induces IL-10-producing cells in the MLNs, Foxp3+CD4+ Tregs in the PPs, and PD-L1+ Bregs in the spleen. IFN-γ+CD8+ T cells are suppressed in the spleen. Tbet+CD4+ T cells are suppressed in the pancreas. At 17 wks, IFN- γ-producing CD4+ and CD8+ T cells are suppressed in the spleen, and Foxp3+Tbet-IFN-γ-CD4+ Tregs are maintained in the PaLNs. Finally, at 24 and 30 wks, Foxp3+IL-10+IFN-γ+ Tr1 cells are induced in the spleen and Tbet+CD4+ T cells are suppressed in the pancreas. B) Summary of regulatory responses induced by LL-CFA/I to protect the NOD mouse from T1D at various stages of disease.

158

+ + - - + + Initial dose of LL- Splenic PD-L1 Foxp3 Tbet IFN-γ IL-10 IFN-γ Tr1 CFA/I + Bregs CD4 Tregs in cells in the spleen PaLN at 24 wks. + Foxp3 Tregs + Foxp3 Tregs in induced in MLNs PPs. IL-10 IFN-γ producing + + Tbet CD4 T cells and Peyer’s producing cells in + + CD4 and CD8 T suppressed in patches 1-2 wks MLN cells suppressed after treatment. pancreas at 30 in the spleen wks. + + IDO TGF-β Suppression of pDCs induced in Tbet and IFN-γ responses in spleen and spleen and

PaLNs pancreas. Beta cell mass/function cell Beta Suppression of CD40 and CD86 on cDCs

Early (4 weeks) Disease onset Overt disease Established or late stage (11 weeks) (17 weeks) (24 weeks)

Age of NOD mouse

Figure 5-1 Continued.

159

LIST OF REFERENCES

1. Giacco, F., and M. Brownlee. 2010. Oxidative stress and diabetic complications. Circ Res 107: 1058–1070.

2. Paneni, F., J. A. Beckman, M. A. Creager, and F. Cosentino. 2013. Diabetes and vascular disease: pathophysiology, clinical consequences, and medical therapy: part I. Eur Heart J 34: 2436–2443.

3. Dobson, M., and J. Fothergill. 1776. Experiments and Observations on the Urine in a Diabetes,.

4. 2017. Principles of diabetes mellitus, (L. Poretsky, ed). Springer International Publishing, Cham.

5. Rosenfeld, L. 2002. Insulin: discovery and controversy. Clin Chem 48: 2270–2288.

6. Lawrence, R. D. 1951. Types of human diabetes. Br Med J 1: 373–375.

7. In’t Veld, P. 2011. Insulitis in human type 1 diabetes: The quest for an elusive lesion. Islets 3: 131–138.

8. Bottazzo, G. F., A. Florin-Christensen, and D. Doniach. 1974. Islet-cell antibodies in diabetes mellitus with autoimmune polyendocrine deficiencies. The Lancet 2: 1279– 1283.

9. Bonifacio, E., V. Lampasona, S. Genovese, M. Ferrari, and E. Bosi. 1995. Identification of protein tyrosine phosphatase-like IA2 (islet cell antigen 512) as the insulin-dependent diabetes-related 37/40K autoantigen and a target of islet-cell antibodies. J Immunol 155: 5419–5426.

10. Baekkeskov, S., H. J. Aanstoot, S. Christgau, A. Reetz, M. Solimena, M. Cascalho, F. Folli, H. Richter-Olesen, and P. De Camilli. 1990. Identification of the 64K autoantigen in insulin-dependent diabetes as the GABA-synthesizing enzyme glutamic acid decarboxylase. Nature 347: 151–156.

11. Palmer, J. P., C. M. Asplin, P. Clemons, K. Lyen, O. Tatpati, P. K. Raghu, and T. L. Paquette. 1983. Insulin antibodies in insulin-dependent diabetics before insulin treatment. Science 222: 1337–1339.

12. Miller, R. G., A. M. Secrest, R. K. Sharma, T. J. Songer, and T. J. Orchard. 2012. Improvements in the life expectancy of type 1 diabetes: the Pittsburgh Epidemiology of Diabetes Complications study cohort. Diabetes 61: 2987–2992.

13. Katsarou, A., S. Gudbjörnsdottir, A. Rawshani, D. Dabelea, E. Bonifacio, B. J. Anderson, L. M. Jacobsen, D. A. Schatz, and Å. Lernmark. 2017. Type 1 diabetes mellitus. Nat. Rev. Dis. Primers 3: 17016.

160

14. Diaz-Valencia, P. A., P. Bougnères, and A.-J. Valleron. 2015. Global epidemiology of type 1 diabetes in young adults and adults: a systematic review. BMC Public Health 15: 255.

15. Rawshani, A., M. Landin-Olsson, A.-M. Svensson, L. Nyström, H. J. Arnqvist, J. Bolinder, and S. Gudbjörnsdottir. 2014. The incidence of diabetes among 0-34 year olds in Sweden: new data and better methods. Diabetologia 57: 1375–1381.

16. Soltesz, G., C. C. Patterson, G. Dahlquist, and EURODIAB Study Group. 2007. Worldwide childhood type 1 diabetes incidence--what can we learn from epidemiology? Pediatr Diabetes 8 Suppl 6: 6–14.

17. Atkinson, M. A., G. S. Eisenbarth, and A. W. Michels. 2014. Type 1 diabetes. The Lancet 383: 69–82.

18. Podar, T., A. Solntsev, M. Karvonen, Z. Padaiga, G. Brigis, B. Urbonaite, M. Viik- Kajander, A. Reunanen, and J. Tuomilehto. 2001. Increasing incidence of childhood- onset type I diabetes in 3 Baltic countries and Finland 1983-1998. Diabetologia 44 Suppl 3: B17–20.

19. Singal, D. P., and M. A. Blajchman. 1973. Histocompatibility (HL-A) antigens, lymphocytotoxic antibodies and tissue antibodies in patients with diabetes mellitus. Diabetes 22: 429–432.

20. Nerup, J., P. Platz, O. O. Andersen, M. Christy, J. Lyngsoe, J. E. Poulsen, L. P. Ryder, L. S. Nielsen, M. Thomsen, and A. Svejgaard. 1974. HL-A antigens and diabetes mellitus. The Lancet 2: 864–866.

21. Thomsen, M., P. Platz, O. O. Andersen, M. Christy, J. Lyngsoe, J. Nerup, K. Rasmussen, L. P. Ryder, L. S. Nielsen, and A. Svejgaard. 1975. MLC typing in juvenile diabetes mellitus and idiopathic addison’s disease. Immunol Rev 22: 125–147.

22. Noble, J. A., A. M. Valdes, M. Cook, W. Klitz, G. Thomson, and H. A. Erlich. 1996. The role of HLA class II genes in insulin-dependent diabetes mellitus: molecular analysis of 180 Caucasian, multiplex families. Am J Hum Genet 59: 1134–1148.

23. Pugliese, A., R. Gianani, R. Moromisato, Z. L. Awdeh, C. A. Alper, H. A. Erlich, R. A. Jackson, and G. S. Eisenbarth. 1995. HLA-DQB1*0602 is associated with dominant protection from diabetes even among islet cell antibody-positive first-degree relatives of patients with IDDM. Diabetes 44: 608–613.

24. Rewers, M., T. L. Bugawan, J. M. Norris, A. Blair, B. Beaty, M. Hoffman, R. S. McDuffie, R. F. Hamman, G. Klingensmith, G. S. Eisenbarth, and H. A. Erlich. 1996. Newborn screening for HLA markers associated with IDDM: diabetes autoimmunity study in the young (DAISY). Diabetologia 39: 807–812.

161

25. Vehik, K., R. F. Hamman, D. Lezotte, J. M. Norris, G. J. Klingensmith, M. Rewers, and D. Dabelea. 2008. Trends in high-risk HLA susceptibility genes among Colorado youth with type 1 diabetes. Diabetes Care 31: 1392–1396.

26. Delli, A. J., B. Lindblad, A. Carlsson, G. Forsander, S.-A. Ivarsson, J. Ludvigsson, C. Marcus, A. Lernmark, and Better Diabetes Diagnosis (BDD) Study Group. 2010. Type 1 diabetes patients born to immigrants to Sweden increase their native diabetes risk and differ from Swedish patients in HLA types and islet autoantibodies. Pediatr Diabetes 11: 513–520.

27. Bell, G. I., S. Horita, and J. H. Karam. 1984. A polymorphic locus near the human insulin gene is associated with insulin-dependent diabetes mellitus. Diabetes 33: 176– 183.

28. Nisticò, L., R. Buzzetti, L. E. Pritchard, B. Van der Auwera, C. Giovannini, E. Bosi, M. T. Larrad, M. S. Rios, C. C. Chow, C. S. Cockram, K. Jacobs, C. Mijovic, S. C. Bain, A. H. Barnett, C. L. Vandewalle, F. Schuit, F. K. Gorus, R. Tosi, P. Pozzilli, and J. A. Todd. 1996. The CTLA-4 gene region of chromosome 2q33 is linked to, and associated with, type 1 diabetes. Belgian Diabetes Registry. Hum Mol Genet 5: 1075–1080.

29. Bottini, N., L. Musumeci, A. Alonso, S. Rahmouni, K. Nika, M. Rostamkhani, J. MacMurray, G. F. Meloni, P. Lucarelli, M. Pellecchia, G. S. Eisenbarth, D. Comings, and T. Mustelin. 2004. A functional variant of lymphoid tyrosine phosphatase is associated with type I diabetes. Nat Genet 36: 337–338.

30. Todd, J. A., N. M. Walker, J. D. Cooper, D. J. Smyth, K. Downes, V. Plagnol, R. Bailey, S. Nejentsev, S. F. Field, F. Payne, C. E. Lowe, J. S. Szeszko, J. P. Hafler, L. Zeitels, J. H. M. Yang, A. Vella, S. Nutland, H. E. Stevens, H. Schuilenburg, G. Coleman, M. Maisuria, W. Meadows, L. J. Smink, B. Healy, O. S. Burren, A. A. C. Lam, N. R. Ovington, J. Allen, E. Adlem, H.-T. Leung, C. Wallace, J. M. M. Howson, C. Guja, C. Ionescu-Tîrgovişte, Genetics of Type 1 Diabetes in Finland, M. J. Simmonds, J. M. Heward, S. C. L. Gough, Wellcome Trust Case Control Consortium, D. B. Dunger, L. S. Wicker, and D. G. Clayton. 2007. Robust associations of four new chromosome regions from genome-wide analyses of type 1 diabetes. Nat Genet 39: 857–864.

31. Chentoufi, A. A., and C. Polychronakos. 2002. Insulin expression levels in the thymus modulate insulin-specific autoreactive T-cell tolerance: the mechanism by which the IDDM2 locus may predispose to diabetes. Diabetes 51: 1383–1390.

32. Hyttinen, V., J. Kaprio, L. Kinnunen, M. Koskenvuo, and J. Tuomilehto. 2003. Genetic liability of type 1 diabetes and the onset age among 22,650 young Finnish twin pairs: a nationwide follow-up study. Diabetes 52: 1052–1055.

33. Metcalfe, K. A., G. A. Hitman, R. E. Rowe, M. Hawa, X. Huang, T. Stewart, and R. D. Leslie. 2001. Concordance for type 1 diabetes in identical twins is affected by insulin genotype. Diabetes Care 24: 838–842.

162

34. Redondo, M. J., J. Jeffrey, P. R. Fain, G. S. Eisenbarth, and T. Orban. 2008. Concordance for islet autoimmunity among monozygotic twins. N Engl J Med 359: 2849–2850.

35. Söderström, U., J. Aman, and A. Hjern. 2012. Being born in Sweden increases the risk for type 1 diabetes - a study of migration of children to Sweden as a natural experiment. Acta Paediatr 101: 73–77.

36. Oilinki, T., T. Otonkoski, J. Ilonen, M. Knip, and P. J. Miettinen. 2012. Prevalence and characteristics of diabetes among Somali children and adolescents living in Helsinki, Finland. Pediatr Diabetes 13: 176–180.

37. Bach, J.-F. 2002. The effect of infections on susceptibility to autoimmune and allergic diseases. N Engl J Med 347: 911–920.

38. Bach, J.-F., and L. Chatenoud. 2012. The hygiene hypothesis: an explanation for the increased frequency of insulin-dependent diabetes. Cold Spring Harb Perspect Med 2: a007799.

39. Okada, H., C. Kuhn, H. Feillet, and J. F. Bach. 2010. The “hygiene hypothesis” for autoimmune and allergic diseases: an update. Clin Exp Immunol 160: 1–9.

40. Cardwell, C. R., D. J. Carson, and C. C. Patterson. 2008. No association between routinely recorded infections in early life and subsequent risk of childhood-onset Type 1 diabetes: a matched case-control study using the UK General Practice Research Database. Diabet Med 25: 261–267.

41. Gamble, D. R., M. L. Kinsley, M. G. FitzGerald, R. Bolton, and K. W. Taylor. 1969. Viral antibodies in diabetes mellitus. Br Med J 3: 627–630.

42. Yoon, J. W., M. Austin, T. Onodera, and A. L. Notkins. 1979. Isolation of a virus from the pancreas of a child with diabetic ketoacidosis. N Engl J Med 300: 1173–1179.

43. Schulte, B. M., J. Bakkers, K. H. W. Lanke, W. J. G. Melchers, C. Westerlaken, W. Allebes, H.-J. Aanstoot, G. J. Bruining, G. J. Adema, F. J. M. Van Kuppeveld, and J. M. D. Galama. 2010. Detection of enterovirus RNA in peripheral blood mononuclear cells of type 1 diabetic patients beyond the stage of acute infection. Viral Immunol 23: 99– 104.

44. Simonen-Tikka, M. L., M. Pflueger, P. Klemola, C. Savolainen-Kopra, T. Smura, S. Hummel, S. Kaijalainen, K. Nuutila, O. Natri, M. Roivainen, and A. G. Ziegler. 2011. Human enterovirus infections in children at increased risk for type 1 diabetes: the Babydiet study. Diabetologia 54: 2995–3002.

163

45. Tracy, S., K. M. Drescher, N. M. Chapman, K. S. Kim, S. D. Carson, S. Pirruccello, P. H. Lane, J. R. Romero, and J. S. Leser. 2002. Toward testing the hypothesis that group B coxsackieviruses (CVB) trigger insulin-dependent diabetes: inoculating nonobese diabetic mice with CVB markedly lowers diabetes incidence. J Virol 76: 12097–12111.

46. Larsson, P. G., T. Lakshmikanth, E. Svedin, C. King, and M. Flodström-Tullberg. 2013. Previous maternal infection protects offspring from enterovirus infection and prevents experimental diabetes development in mice. Diabetologia 56: 867–874.

47. Webb, A. R., L. Kline, and M. F. Holick. 1988. Influence of season and latitude on the cutaneous synthesis of vitamin D3: exposure to winter sunlight in Boston and Edmonton will not promote vitamin D3 synthesis in human skin. J Clin Endocrinol Metab 67: 373–378.

48. Holick, M. F., T. C. Chen, Z. Lu, and E. Sauter. 2007. Vitamin D and skin physiology: a D-lightful story. J Bone Miner Res 22 Suppl 2: V28–33.

49. Moltchanova, E. V., N. Schreier, N. Lammi, and M. Karvonen. 2009. Seasonal variation of diagnosis of Type 1 diabetes mellitus in children worldwide. Diabet Med 26: 673–678.

50. Hayes, C. E., S. L. Hubler, J. R. Moore, L. E. Barta, C. E. Praska, and F. E. Nashold. 2015. Vitamin D actions on CD4(+) T cells in autoimmune disease. Front Immunol 6: 100.

51. Dong, J.-Y., W.-G. Zhang, J. J. Chen, Z.-L. Zhang, S.-F. Han, and L.-Q. Qin. 2013. Vitamin D intake and risk of type 1 diabetes: a meta-analysis of observational studies. Nutrients 5: 3551–3562.

52. Miettinen, M. E., L. Reinert, L. Kinnunen, V. Harjutsalo, P. Koskela, H. M. Surcel, C. Lamberg-Allardt, and J. Tuomilehto. 2012. Serum 25-hydroxyvitamin D level during early pregnancy and type 1 diabetes risk in the offspring. Diabetologia 55: 1291–1294.

53. Sørensen, I. M., G. Joner, P. A. Jenum, A. Eskild, P. A. Torjesen, and L. C. Stene. 2012. Maternal serum levels of 25-hydroxy-vitamin D during pregnancy and risk of type 1 diabetes in the offspring. Diabetes 61: 175–178.

54. Pozzilli, P., A. Signore, A. J. K. Williams, and P. E. Beales. 1993. NOD mouse colonies around the world- recent facts and figures. Immunol Today 14: 193–196.

55. Alam, C., E. Bittoun, D. Bhagwat, S. Valkonen, A. Saari, U. Jaakkola, E. Eerola, P. Huovinen, and A. Hänninen. 2011. Effects of a germ-free environment on gut immune regulation and diabetes progression in non-obese diabetic (NOD) mice. Diabetologia 54: 1398–1406.

56. King, C., and N. Sarvetnick. 2011. The incidence of type-1 diabetes in NOD mice is modulated by restricted flora not germ-free conditions. PLoS ONE 6: e17049.

164

57. Giongo, A., K. A. Gano, D. B. Crabb, N. Mukherjee, L. L. Novelo, G. Casella, J. C. Drew, J. Ilonen, M. Knip, H. Hyöty, R. Veijola, T. Simell, O. Simell, J. Neu, C. H. Wasserfall, D. Schatz, M. A. Atkinson, and E. W. Triplett. 2011. Toward defining the autoimmune microbiome for type 1 diabetes. ISME J 5: 82–91.

58. Brown, C. T., A. G. Davis-Richardson, A. Giongo, K. A. Gano, D. B. Crabb, N. Mukherjee, G. Casella, J. C. Drew, J. Ilonen, M. Knip, H. Hyöty, R. Veijola, T. Simell, O. Simell, J. Neu, C. H. Wasserfall, D. Schatz, M. A. Atkinson, and E. W. Triplett. 2011. Gut microbiome metagenomics analysis suggests a functional model for the development of autoimmunity for type 1 diabetes. PLoS ONE 6: e25792.

59. Wolf, K. J., J. G. Daft, S. M. Tanner, R. Hartmann, E. Khafipour, and R. G. Lorenz. 2014. Consumption of acidic water alters the gut microbiome and decreases the risk of diabetes in NOD mice. J Histochem Cytochem 62: 237–250.

60. Mizuno, M., D. Noto, N. Kaga, A. Chiba, and S. Miyake. 2017. The dual role of short fatty acid chains in the pathogenesis of autoimmune disease models. PLoS ONE 12: e0173032.

61. Mariño, E., J. L. Richards, K. H. McLeod, D. Stanley, Y. A. Yap, J. Knight, C. McKenzie, J. Kranich, A. C. Oliveira, F. J. Rossello, B. Krishnamurthy, C. M. Nefzger, L. Macia, A. Thorburn, A. G. Baxter, G. Morahan, L. H. Wong, J. M. Polo, R. J. Moore, T. J. Lockett, J. M. Clarke, D. L. Topping, L. C. Harrison, and C. R. Mackay. 2017. Gut microbial metabolites limit the frequency of autoimmune T cells and protect against type 1 diabetes. Nat Immunol 18: 552–562.

62. Santisteban, M. M., Y. Qi, J. Zubcevic, S. Kim, T. Yang, V. Shenoy, C. T. Cole- Jeffrey, G. O. Lobaton, D. C. Stewart, A. Rubiano, C. S. Simmons, F. Garcia-Pereira, R. D. Johnson, C. J. Pepine, and M. K. Raizada. 2017. Hypertension-Linked Pathophysiological Alterations in the Gut. Circ Res 120: 312–323.

63. De Goffau, M. C., K. Luopajärvi, M. Knip, J. Ilonen, T. Ruohtula, T. Härkönen, L. Orivuori, S. Hakala, G. W. Welling, H. J. Harmsen, and O. Vaarala. 2013. Fecal microbiota composition differs between children with β-cell autoimmunity and those without. Diabetes 62: 1238–1244.

64. Anderson, M. S., and J. A. Bluestone. 2005. The NOD mouse: a model of immune dysregulation. Annu Rev Immunol 23: 447–485.

65. Pearson, J. A., F. S. Wong, and L. Wen. 2016. The importance of the Non Obese Diabetic (NOD) mouse model in autoimmune diabetes. J Autoimmun 66: 76–88.

66. Driver, J. P., D. V. Serreze, and Y.-G. Chen. 2011. Mouse models for the study of autoimmune type 1 diabetes: a NOD to similarities and differences to human disease. Semin Immunopathol 33: 67–87.

67. Eisenbarth, G. S. 1986. Type I diabetes mellitus. A chronic autoimmune disease. N Engl J Med 314: 1360–1368.

165

68. Dotta, F., S. Censini, A. G. S. van Halteren, L. Marselli, M. Masini, S. Dionisi, F. Mosca, U. Boggi, A. O. Muda, S. Del Prato, J. F. Elliott, A. Covacci, R. Rappuoli, B. O. Roep, and P. Marchetti. 2007. Coxsackie B4 virus infection of beta cells and natural killer cell insulitis in recent-onset type 1 diabetic patients. Proc Natl Acad Sci U S A 104: 5115–5120.

69. Elshebani, A., A. Olsson, J. Westman, T. Tuvemo, O. Korsgren, and G. Frisk. 2007. Effects on isolated human pancreatic islet cells after infection with strains of enterovirus isolated at clinical presentation of type 1 diabetes. Virus Res 124: 193–203.

70. Laitinen, O. H., H. Honkanen, O. Pakkanen, S. Oikarinen, M. M. Hankaniemi, H. Huhtala, T. Ruokoranta, V. Lecouturier, P. André, R. Harju, S. M. Virtanen, J. Lehtonen, J. W. Almond, T. Simell, O. Simell, J. Ilonen, R. Veijola, M. Knip, and H. Hyöty. 2014. Coxsackievirus B1 is associated with induction of β-cell autoimmunity that portends type 1 diabetes. Diabetes 63: 446–455.

71. Lin, H.-C., C.-H. Wang, F.-J. Tsai, K.-P. Hwang, W. Chen, C.-C. Lin, and T.-C. Li. 2015. Enterovirus infection is associated with an increased risk of childhood type 1 diabetes in Taiwan: a nationwide population-based cohort study. Diabetologia 58: 79– 86.

72. Oikarinen, S., S. Tauriainen, D. Hober, B. Lucas, A. Vazeou, A. Sioofy-Khojine, E. Bozas, P. Muir, H. Honkanen, J. Ilonen, M. Knip, P. Keskinen, M.-T. Saha, H. Huhtala, G. Stanway, C. Bartsocas, J. Ludvigsson, K. Taylor, H. Hyöty, and VirDiab Study Group. 2014. Virus antibody survey in different European populations indicates risk association between coxsackievirus B1 and type 1 diabetes. Diabetes 63: 655–662.

73. Horwitz, M. S., L. M. Bradley, J. Harbertson, T. Krahl, J. Lee, and N. Sarvetnick. 1998. Diabetes induced by Coxsackie virus: initiation by bystander damage and not molecular mimicry. Nat Med 4: 781–785.

74. Serreze, D. V., E. W. Ottendorfer, T. M. Ellis, C. J. Gauntt, and M. A. Atkinson. 2000. Acceleration of type 1 diabetes by a coxsackievirus infection requires a preexisting critical mass of autoreactive T-cells in pancreatic islets. Diabetes 49: 708– 711.

75. Bottazzo, G. F. 1986. Lawrence lecture. Death of a beta cell: homicide or suicide? Diabet Med 3: 119–130.

76. Atkinson, M. A., J. A. Bluestone, G. S. Eisenbarth, M. Hebrok, K. C. Herold, D. Accili, M. Pietropaolo, P. R. Arvan, M. Von Herrath, D. S. Markel, and C. J. Rhodes. 2011. How does type 1 diabetes develop?: the notion of homicide or β-cell suicide revisited. Diabetes 60: 1370–1379.

77. Wachlin, G., P. Augstein, D. Schröder, B. Kuttler, I. Klöting, P. Heinke, and S. Schmidt. 2003. IL-1beta, IFN-gamma and TNF-alpha increase vulnerability of pancreatic beta cells to autoimmune destruction. J Autoimmun 20: 303–312.

166

78. Cnop, M., N. Welsh, J.-C. Jonas, A. Jörns, S. Lenzen, and D. L. Eizirik. 2005. Mechanisms of pancreatic beta-cell death in type 1 and type 2 diabetes: many differences, few similarities. Diabetes 54 Suppl 2: S97–107.

79. Thomas, H. E., J. L. Parker, R. D. Schreiber, and T. W. Kay. 1998. IFN-gamma action on pancreatic beta cells causes class I MHC upregulation but not diabetes. J Clin Invest 102: 1249–1257.

80. Zhao, Y., N. A. Scott, H. S. Quah, B. Krishnamurthy, F. Bond, T. Loudovaris, S. I. Mannering, T. W. H. Kay, and H. E. Thomas. 2015. Mouse pancreatic beta cells express MHC class II and stimulate CD4(+) T cells to proliferate. Eur J Immunol 45: 2494–2503.

81. Schuit, F. C., P. A. In’t Veld, and D. G. Pipeleers. 1988. Glucose stimulates proinsulin biosynthesis by a dose-dependent recruitment of pancreatic beta cells. Proc Natl Acad Sci U S A 85: 3865–3869.

82. Liu, M., I. Hodish, C. J. Rhodes, and P. Arvan. 2007. Proinsulin maturation, misfolding, and proteotoxicity. Proc Natl Acad Sci U S A 104: 15841–15846.

83. De Jong, V. M., J. R. F. Abreu, A. A. Verrijn Stuart, A. R. van der Slik, K. Verhaeghen, M. A. Engelse, B. Blom, F. J. T. Staal, F. K. Gorus, and B. O. Roep. 2013. Alternative splicing and differential expression of the islet autoantigen IGRP between pancreas and thymus contributes to immunogenicity of pancreatic islets but not diabetogenicity in humans. Diabetologia 56: 2651–2658.

84. Sollid, L. M., and B. Jabri. 2011. Celiac disease and transglutaminase 2: a model for posttranslational modification of antigens and HLA association in the pathogenesis of autoimmune disorders. Curr Opin Immunol 23: 732–738.

85. Mannering, S. I., L. C. Harrison, N. A. Williamson, J. S. Morris, D. J. Thearle, K. P. Jensen, T. W. H. Kay, J. Rossjohn, B. A. Falk, G. T. Nepom, and A. W. Purcell. 2005. The insulin A-chain epitope recognized by human T cells is posttranslationally modified. J Exp Med 202: 1191–1197.

86. McGinty, J. W., I.-T. Chow, C. Greenbaum, J. Odegard, W. W. Kwok, and E. A. James. 2014. Recognition of posttranslationally modified GAD65 epitopes in subjects with type 1 diabetes. Diabetes 63: 3033–3040.

87. Strollo, R., C. Vinci, M. H. Arshad, D. Perrett, C. Tiberti, F. Chiarelli, N. Napoli, P. Pozzilli, and A. Nissim. 2015. Antibodies to post-translationally modified insulin in type 1 diabetes. Diabetologia 58: 2851–2860.

88. Delong, T., T. A. Wiles, R. L. Baker, B. Bradley, G. Barbour, R. Reisdorph, M. Armstrong, R. L. Powell, N. Reisdorph, N. Kumar, C. M. Elso, M. DeNicola, R. Bottino, A. C. Powers, D. M. Harlan, S. C. Kent, S. I. Mannering, and K. Haskins. 2016. Pathogenic CD4 T cells in type 1 diabetes recognize epitopes formed by peptide fusion. Science 351: 711–714.

167

89. Wan, X., B. H. Zinselmeyer, P. N. Zakharov, A. N. Vomund, R. Taniguchi, L. Santambrogio, M. S. Anderson, C. F. Lichti, and E. R. Unanue. 2018. Pancreatic islets communicate with lymphoid tissues via exocytosis of insulin peptides. Nature 560: 107– 111.

90. Bingley, P. J., E. Bonifacio, A. J. Williams, S. Genovese, G. F. Bottazzo, and E. A. Gale. 1997. Prediction of IDDM in the general population: strategies based on combinations of autoantibody markers. Diabetes 46: 1701–1710.

91. Bingley, P. J. 2010. Clinical applications of diabetes antibody testing. J Clin Endocrinol Metab 95: 25–33.

92. Bingley, P. J., D. C. Boulware, J. P. Krischer, and Type 1 Diabetes TrialNet Study Group. 2016. The implications of autoantibodies to a single islet antigen in relatives with normal glucose tolerance: development of other autoantibodies and progression to type 1 diabetes. Diabetologia 59: 542–549.

93. Knip, M., S. Korhonen, P. Kulmala, R. Veijola, A. Reunanen, O. T. Raitakari, J. Viikari, and H. K. Akerblom. 2010. Prediction of type 1 diabetes in the general population. Diabetes Care 33: 1206–1212.

94. Ziegler, A. G., M. Rewers, O. Simell, T. Simell, J. Lempainen, A. Steck, C. Winkler, J. Ilonen, R. Veijola, M. Knip, E. Bonifacio, and G. S. Eisenbarth. 2013. Seroconversion to multiple islet autoantibodies and risk of progression to diabetes in children. JAMA 309: 2473–2479.

95. Wong, F. S., L. Wen, M. Tang, M. Ramanathan, I. Visintin, J. Daugherty, L. G. Hannum, C. A. Janeway, and M. J. Shlomchik. 2004. Investigation of the role of B-cells in type 1 diabetes in the NOD mouse. Diabetes 53: 2581–2587.

96. Xiu, Y., C. P. Wong, J.-D. Bouaziz, Y. Hamaguchi, Y. Wang, S. M. Pop, R. M. Tisch, and T. F. Tedder. 2008. B lymphocyte depletion by CD20 monoclonal antibody prevents diabetes in nonobese diabetic mice despite isotype-specific differences in Fc gamma R effector functions. J Immunol 180: 2863–2875.

97. Hu, C., D. Rodriguez-Pinto, W. Du, A. Ahuja, O. Henegariu, F. S. Wong, M. J. Shlomchik, and L. Wen. 2007. Treatment with CD20-specific antibody prevents and reverses autoimmune diabetes in mice. J Clin Invest 117: 3857–3867.

98. Jansen, A., F. Homo-Delarche, H. Hooijkaas, P. J. Leenen, M. Dardenne, and H. A. Drexhage. 1994. Immunohistochemical characterization of monocytes-macrophages and dendritic cells involved in the initiation of the insulitis and beta-cell destruction in NOD mice. Diabetes 43: 667–675.

99. Bouma, G., J. M. C. Coppens, S. Mourits, T. Nikolic, S. Sozzani, H. A. Drexhage, and M. A. Versnel. 2005. Evidence for an enhanced adhesion of DC to fibronectin and a role of CCL19 and CCL21 in the accumulation of DC around the pre-diabetic islets in NOD mice. Eur J Immunol 35: 2386–2396.

168

100. Diana, J., Y. Simoni, L. Furio, L. Beaudoin, B. Agerberth, F. Barrat, and A. Lehuen. 2013. Crosstalk between neutrophils, B-1a cells and plasmacytoid dendritic cells initiates autoimmune diabetes. Nat Med 19: 65–73.

101. Höglund, P., J. Mintern, C. Waltzinger, W. Heath, C. Benoist, and D. Mathis. 1999. Initiation of autoimmune diabetes by developmentally regulated presentation of islet cell antigens in the pancreatic lymph nodes. J Exp Med 189: 331–339.

102. Calderon, B., J. A. Carrero, M. J. Miller, and E. R. Unanue. 2011. Cellular and molecular events in the localization of diabetogenic T cells to islets of Langerhans. Proc Natl Acad Sci U S A 108: 1561–1566.

103. Nikolic, T., S. B. Geutskens, N. van Rooijen, H. A. Drexhage, and P. J. M. Leenen. 2005. Dendritic cells and macrophages are essential for the retention of lymphocytes in (peri)-insulitis of the nonobese diabetic mouse: a phagocyte depletion study. Lab Invest 85: 487–501.

104. Steer, S. A., A. L. Scarim, K. T. Chambers, and J. A. Corbett. 2006. Interleukin-1 stimulates beta-cell necrosis and release of the immunological adjuvant HMGB1. PLoS Med 3: e17.

105. Jörns, A., T. Arndt, A. Meyer zu Vilsendorf, J. Klempnauer, D. Wedekind, H.-J. Hedrich, L. Marselli, P. Marchetti, N. Harada, Y. Nakaya, G.-S. Wang, F. W. Scott, C. Gysemans, C. Mathieu, and S. Lenzen. 2014. Islet infiltration, cytokine expression and beta cell death in the NOD mouse, BB rat, Komeda rat, LEW.1AR1-iddm rat and humans with type 1 diabetes. Diabetologia 57: 512–521.

106. Magnuson, A. M., G. M. Thurber, R. H. Kohler, R. Weissleder, D. Mathis, and C. Benoist. 2015. Population dynamics of islet-infiltrating cells in autoimmune diabetes. Proc Natl Acad Sci U S A 112: 1511–1516.

107. Christianson, S. W., L. D. Shultz, and E. H. Leiter. 1993. Adoptive transfer of diabetes into immunodeficient NOD-scid/scid mice. Relative contributions of CD4+ and CD8+ T-cells from diabetic versus prediabetic NOD.NON-Thy-1a donors. Diabetes 42: 44–55.

108. Wicker, L. S., E. H. Leiter, J. A. Todd, R. J. Renjilian, E. Peterson, P. A. Fischer, P. L. Podolin, M. Zijlstra, R. Jaenisch, and L. B. Peterson. 1994. Beta 2-microglobulin- deficient NOD mice do not develop insulitis or diabetes. Diabetes 43: 500–504.

109. Verdaguer, J., D. Schmidt, A. Amrani, B. Anderson, N. Averill, and P. Santamaria. 1997. Spontaneous autoimmune diabetes in monoclonal T cell nonobese diabetic mice. J Exp Med 186: 1663–1676.

110. Matsumoto, M., H. Yagi, K. Kunimoto, J. Kawaguchi, S. Makino, and M. Harada. 1993. Transfer of autoimmune diabetes from diabetic NOD mice to NOD athymic nude mice: the roles of T cell subsets in the pathogenesis. Cell Immunol 148: 189–197.

169

111. Coppieters, K. T., F. Dotta, N. Amirian, P. D. Campbell, T. W. H. Kay, M. A. Atkinson, B. O. Roep, and M. G. von Herrath. 2012. Demonstration of islet-autoreactive CD8 T cells in insulitic lesions from recent onset and long-term type 1 diabetes patients. J Exp Med 209: 51–60.

112. Peterson, J. D., and K. Haskins. 1996. Transfer of diabetes in the NOD-scid mouse by CD4 T-cell clones. Differential requirement for CD8 T-cells. Diabetes 45: 328–336.

113. Yagi, H., M. Matsumoto, K. Kunimoto, J. Kawaguchi, S. Makino, and M. Harada. 1992. Analysis of the roles of CD4+ and CD8+ T cells in autoimmune diabetes of NOD mice using transfer to NOD athymic nude mice. Eur J Immunol 22: 2387–2393.

114. Katz, J. D., C. Benoist, and D. Mathis. 1995. T helper cell subsets in insulin- dependent diabetes. Science 268: 1185–1188.

115. Rapoport, M. J., A. Jaramillo, D. Zipris, A. H. Lazarus, D. V. Serreze, E. H. Leiter, P. Cyopick, J. S. Danska, and T. L. Delovitch. 1993. Interleukin 4 reverses T cell proliferative unresponsiveness and prevents the onset of diabetes in nonobese diabetic mice. J Exp Med 178: 87–99.

116. Savinov, A. Y., F. S. Wong, and A. V. Chervonsky. 2001. IFN-gamma affects homing of diabetogenic T cells. J Immunol 167: 6637–6643.

117. Yi, Z., L. Li, A. Garland, Q. He, H. Wang, J. D. Katz, R. Tisch, and B. Wang. 2012. IFN-γ receptor deficiency prevents diabetes induction by diabetogenic CD4+, but not CD8+, T cells. Eur J Immunol 42: 2010–2018.

118. Zhang, J., Z. Huang, R. Sun, Z. Tian, and H. Wei. 2012. IFN-γ induced by IL-12 administration prevents diabetes by inhibiting pathogenic IL-17 production in NOD mice. J Autoimmun 38: 20–28.

119. Sobel, D. O., J. Han, J. Williams, J.-W. Yoon, H.-S. Jun, and B. Ahvazi. 2002. Gamma interferon paradoxically inhibits the development of diabetes in the NOD mouse. J Autoimmun 19: 129–137.

120. Satoh, J., H. Seino, T. Abo, S. Tanaka, S. Shintani, S. Ohta, K. Tamura, T. Sawai, T. Nobunaga, and T. Oteki. 1989. Recombinant human tumor necrosis factor alpha suppresses autoimmune diabetes in nonobese diabetic mice. J Clin Invest 84: 1345– 1348.

121. Fousteri, G., A. Dave, A. Bot, T. Juntti, S. Omid, and M. von Herrath. 2010. Subcutaneous insulin B:9-23/IFA immunisation induces Tregs that control late-stage prediabetes in NOD mice through IL-10 and IFNgamma. Diabetologia 53: 1958–1970.

122. Emamaullee, J. A., J. Davis, S. Merani, C. Toso, J. F. Elliott, A. Thiesen, and A. M. J. Shapiro. 2009. Inhibition of Th17 cells regulates autoimmune diabetes in NOD mice. Diabetes 58: 1302–1311.

170

123. Jain, R., D. M. Tartar, R. K. Gregg, R. D. Divekar, J. J. Bell, H.-H. Lee, P. Yu, J. S. Ellis, C. M. Hoeman, C. L. Franklin, and H. Zaghouani. 2008. Innocuous IFNgamma induced by adjuvant-free antigen restores normoglycemia in NOD mice through inhibition of IL-17 production. J Exp Med 205: 207–218.

124. Martin-Orozco, N., Y. Chung, S. H. Chang, Y.-H. Wang, and C. Dong. 2009. Th17 cells promote pancreatic inflammation but only induce diabetes efficiently in lymphopenic hosts after conversion into Th1 cells. Eur J Immunol 39: 216–224.

125. Bending, D., H. De la Peña, M. Veldhoen, J. M. Phillips, C. Uyttenhove, B. Stockinger, and A. Cooke. 2009. Highly purified Th17 cells from BDC2.5NOD mice convert into Th1-like cells in NOD/SCID recipient mice. J Clin Invest 119: 565–572.

126. Lau, K., P. Benitez, A. Ardissone, T. D. Wilson, E. L. Collins, G. Lorca, N. Li, D. Sankar, C. Wasserfall, J. Neu, M. A. Atkinson, D. Shatz, E. W. Triplett, and J. Larkin. 2011. Inhibition of type 1 diabetes correlated to a Lactobacillus johnsonii N6.2-mediated Th17 bias. J Immunol 186: 3538–3546.

127. Kuriya, G., T. Uchida, S. Akazawa, M. Kobayashi, K. Nakamura, T. Satoh, I. Horie, E. Kawasaki, H. Yamasaki, L. Yu, Y. Iwakura, H. Sasaki, Y. Nagayama, A. Kawakami, and N. Abiru. 2013. Double deficiency in IL-17 and IFN-γ signalling significantly suppresses the development of diabetes in the NOD mouse. Diabetologia 56: 1773– 1780.

128. Katz, J., C. Benoist, and D. Mathis. 1993. Major histocompatibility complex class I molecules are required for the development of insulitis in non-obese diabetic mice. Eur J Immunol 23: 3358–3360.

129. Trivedi, P., K. L. Graham, B. Krishnamurthy, S. Fynch, R. M. Slattery, T. W. H. Kay, and H. E. Thomas. 2016. Perforin facilitates beta cell killing and regulates autoreactive CD8+ T-cell responses to antigen in mouse models of type 1 diabetes. Immunol Cell Biol 94: 334–341.

130. Dudek, N. L., H. E. Thomas, L. Mariana, R. M. Sutherland, J. Allison, E. Estella, E. Angstetra, J. A. Trapani, P. Santamaria, A. M. Lew, and T. W. H. Kay. 2006. Cytotoxic T-cells from T-cell receptor transgenic NOD8.3 mice destroy beta-cells via the perforin and Fas pathways. Diabetes 55: 2412–2418.

131. Kägi, D., B. Odermatt, P. Seiler, R. M. Zinkernagel, T. W. Mak, and H. Hengartner. 1997. Reduced incidence and delayed onset of diabetes in perforin-deficient nonobese diabetic mice. J Exp Med 186: 989–997.

132. Mollah, Z. U., K. L. Graham, B. Krishnamurthy, P. Trivedi, T. C. Brodnicki, J. A. Trapani, T. W. Kay, and H. E. Thomas. 2012. Granzyme B is dispensable in the development of diabetes in non-obese diabetic mice. PLoS ONE 7: e40357.

171

133. Kobayashi, M., C. Kaneko-Koike, N. Abiru, T. Uchida, S. Akazawa, K. Nakamura, G. Kuriya, T. Satoh, H. Ida, E. Kawasaki, H. Yamasaki, Y. Nagayama, H. Sasaki, and A. Kawakami. 2013. Genetic deletion of granzyme B does not confer resistance to the development of spontaneous diabetes in non-obese diabetic mice. Clin Exp Immunol 173: 411–418.

134. Carrero, J. A., B. Calderon, F. Towfic, M. N. Artyomov, and E. R. Unanue. 2013. Defining the transcriptional and cellular landscape of type 1 diabetes in the NOD mouse. PLoS ONE 8: e59701.

135. Ewel, C. H., D. O. Sobel, B. J. Zeligs, and J. A. Bellanti. 1992. Poly I:C accelerates development of diabetes mellitus in diabetes-prone BB rat. Diabetes 41: 1016–1021.

136. Sobel, D. O., C. H. Ewel, B. Zeligs, V. Abbassi, J. Rossio, and J. A. Bellanti. 1994. Poly I:C induction of alpha-interferon in the diabetes-prone BB and normal Wistar rats. Dose-response relationships. Diabetes 43: 518–522.

137. Welzen-Coppens, J. M. C., C. G. van Helden-Meeuwsen, P. J. M. Leenen, H. A. Drexhage, and M. A. Versnel. 2013. The kinetics of plasmacytoid dendritic cell accumulation in the pancreas of the NOD mouse during the early phases of insulitis. PLoS ONE 8: e55071.

138. Beaudoin, L., J. Diana, L. Ghazarian, Y. Simoni, C. Boitard, and A. Lehuen. 2014. Plasmacytoid dendritic cells license regulatory T cells, upon iNKT-cell stimulation, to prevent autoimmune diabetes. Eur J Immunol 44: 1454–1466.

139. Alba, A., M. C. Puertas, J. Carrillo, R. Planas, R. Ampudia, X. Pastor, F. Bosch, R. Pujol-Borrell, J. Verdaguer, and M. Vives-Pi. 2004. IFN beta accelerates autoimmune type 1 diabetes in nonobese diabetic mice and breaks the tolerance to beta cells in nondiabetes-prone mice. J Immunol 173: 6667–6675.

140. Vassileva, G., S.-C. Chen, M. Zeng, S. Abbondanzo, K. Jensen, D. Gorman, B. M. Baroudy, Y. Jiang, N. Murgolo, and S. A. Lira. 2003. Expression of a novel murine type I IFN in the pancreatic islets induces diabetes in mice. J Immunol 170: 5748–5755.

141. Stewart, T. A., B. Hultgren, X. Huang, S. Pitts-Meek, J. Hully, and N. J. MacLachlan. 1993. Induction of type I diabetes by interferon-alpha in transgenic mice. Science 260: 1942–1946.

142. Pelegrin, M., J. C. Devedjian, C. Costa, J. Visa, G. Solanes, A. Pujol, G. Asins, A. Valera, and F. Bosch. 1998. Evidence from transgenic mice that interferon-beta may be involved in the onset of diabetes mellitus. J Biol Chem 273: 12332–12340.

143. Brandtzaeg, P. 1998. Development and basic mechanisms of human gut immunity. Nutr Rev 56: S5–18.

144. Weiner, H. L., A. P. da Cunha, F. Quintana, and H. Wu. 2011. Oral tolerance. Immunol Rev 241: 241–259.

172

145. Suzuki, H., S. Sekine, K. Kataoka, D. W. Pascual, M. Maddaloni, R. Kobayashi, K. Fujihashi, H. Kozono, J. R. McGhee, and K. Fujihashi. 2008. Ovalbumin-protein sigma 1 M-cell targeting facilitates oral tolerance with reduction of antigen-specific CD4+ T cells. Gastroenterology 135: 917–925.

146. Rynda, A., M. Maddaloni, D. Mierzejewska, J. Ochoa-Repáraz, T. Maslanka, K. Crist, C. Riccardi, B. Barszczewska, K. Fujihashi, J. R. McGhee, and D. W. Pascual. 2008. Low-dose tolerance is mediated by the microfold cell ligand, reovirus protein sigma1. J Immunol 180: 5187–5200.

147. Lelouard, H., M. Fallet, B. de Bovis, S. Méresse, and J.-P. Gorvel. 2012. Peyer’s patch dendritic cells sample antigens by extending dendrites through M cell-specific transcellular pores. Gastroenterology 142: 592–601.e3.

148. Rescigno, M., M. Urbano, B. Valzasina, M. Francolini, G. Rotta, R. Bonasio, F. Granucci, J. P. Kraehenbuhl, and P. Ricciardi-Castagnoli. 2001. Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nat Immunol 2: 361–367.

149. Asano, M., M. Toda, N. Sakaguchi, and S. Sakaguchi. 1996. Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J Exp Med 184: 387–396.

150. Sakaguchi, S., N. Sakaguchi, M. Asano, M. Itoh, and M. Toda. 1995. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 155: 1151–1164.

151. Himmel, M. E., K. G. MacDonald, R. V. Garcia, T. S. Steiner, and M. K. Levings. 2013. Helios+ and Helios- cells coexist within the natural FOXP3+ T regulatory cell subset in humans. J Immunol 190: 2001–2008.

152. Singh, K., M. Hjort, L. Thorvaldson, and S. Sandler. 2015. Concomitant analysis of Helios and Neuropilin-1 as a marker to detect thymic derived regulatory T cells in naïve mice. Sci Rep 5: 7767.

153. Yadav, M., C. Louvet, D. Davini, J. M. Gardner, M. Martinez-Llordella, S. Bailey- Bucktrout, B. A. Anthony, F. M. Sverdrup, R. Head, D. J. Kuster, P. Ruminski, D. Weiss, D. Von Schack, and J. A. Bluestone. 2012. Neuropilin-1 distinguishes natural and inducible regulatory T cells among regulatory T cell subsets in vivo. J Exp Med 209: 1713–22, S1.

154. Chen, W., W. Jin, N. Hardegen, K.-J. Lei, L. Li, N. Marinos, G. McGrady, and S. M. Wahl. 2003. Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3. J Exp Med 198: 1875–1886.

173

155. Lindley, S., C. M. Dayan, A. Bishop, B. O. Roep, M. Peakman, and T. I. M. Tree. 2005. Defective suppressor function in CD4(+)CD25(+) T-cells from patients with type 1 diabetes. Diabetes 54: 92–99.

156. Brusko, T. M., C. H. Wasserfall, M. J. Clare-Salzler, D. A. Schatz, and M. A. Atkinson. 2005. Functional defects and the influence of age on the frequency of CD4+ CD25+ T-cells in type 1 diabetes. Diabetes 54: 1407–1414.

157. Schneider, A., M. Rieck, S. Sanda, C. Pihoker, C. Greenbaum, and J. H. Buckner. 2008. The effector T cells of diabetic subjects are resistant to regulation via CD4+ FOXP3+ regulatory T cells. J Immunol 181: 7350–7355.

158. Lawson, J. M., J. Tremble, C. Dayan, H. Beyan, R. D. G. Leslie, M. Peakman, and T. I. M. Tree. 2008. Increased resistance to CD4+CD25hi regulatory T cell-mediated suppression in patients with type 1 diabetes. Clin Exp Immunol 154: 353–359.

159. D’Alise, A. M., V. Auyeung, M. Feuerer, J. Nishio, J. Fontenot, C. Benoist, and D. Mathis. 2008. The defect in T-cell regulation in NOD mice is an effect on the T-cell effectors. Proc Natl Acad Sci U S A 105: 19857–19862.

160. Yang, J. H. M., A. J. Cutler, R. C. Ferreira, J. L. Reading, N. J. Cooper, C. Wallace, P. Clarke, D. J. Smyth, C. S. Boyce, G.-J. Gao, J. A. Todd, L. S. Wicker, and T. I. M. Tree. 2015. Natural Variation in Interleukin-2 Sensitivity Influences Regulatory T-Cell Frequency and Function in Individuals With Long-standing Type 1 Diabetes. Diabetes 64: 3891–3902.

161. Long, S. A., K. Cerosaletti, P. L. Bollyky, M. Tatum, H. Shilling, S. Zhang, Z.-Y. Zhang, C. Pihoker, S. Sanda, C. Greenbaum, and J. H. Buckner. 2010. Defects in IL-2R signaling contribute to diminished maintenance of FOXP3 expression in CD4(+)CD25(+) regulatory T-cells of type 1 diabetic subjects. Diabetes 59: 407–415.

162. Kleijwegt, F. S., S. Laban, G. Duinkerken, A. M. Joosten, B. P. C. Koeleman, T. Nikolic, and B. O. Roep. 2011. Transfer of regulatory properties from tolerogenic to proinflammatory dendritic cells via induced autoreactive regulatory T cells. J Immunol 187: 6357–6364.

163. Long, S. A., K. Cerosaletti, J. Y. Wan, J. C. Ho, M. Tatum, S. Wei, H. G. Shilling, and J. H. Buckner. 2011. An autoimmune-associated variant in PTPN2 reveals an impairment of IL-2R signaling in CD4(+) T cells. Genes Immun 12: 116–125.

164. Marwaha, A. K., S. Q. Crome, C. Panagiotopoulos, K. B. Berg, H. Qin, Q. Ouyang, L. Xu, J. J. Priatel, M. K. Levings, and R. Tan. 2010. Cutting edge: Increased IL-17- secreting T cells in children with new-onset type 1 diabetes. J Immunol 185: 3814– 3818.

174

165. McClymont, S. A., A. L. Putnam, M. R. Lee, J. H. Esensten, W. Liu, M. A. Hulme, U. Hoffmüller, U. Baron, S. Olek, J. A. Bluestone, and T. M. Brusko. 2011. Plasticity of human regulatory T cells in healthy subjects and patients with type 1 diabetes. J Immunol 186: 3918–3926.

166. Zhang, Z. J., L. Davidson, G. Eisenbarth, and H. L. Weiner. 1991. Suppression of diabetes in nonobese diabetic mice by oral administration of porcine insulin. Proc Natl Acad Sci U S A 88: 10252–10256.

167. Kobayashi, M., N. Abiru, T. Arakawa, K. Fukushima, H. Zhou, E. Kawasaki, H. Yamasaki, E. Liu, D. Miao, F. S. Wong, G. S. Eisenbarth, and K. Eguchi. 2007. Altered B:9-23 insulin, when administered intranasally with cholera toxin adjuvant, suppresses the expression of insulin autoantibodies and prevents diabetes. J Immunol 179: 2082– 2088.

168. Tian, J., M. A. Atkinson, M. Clare-Salzler, A. Herschenfeld, T. Forsthuber, P. V. Lehmann, and D. L. Kaufman. 1996. Nasal administration of glutamate decarboxylase (GAD65) peptides induces Th2 responses and prevents murine insulin-dependent diabetes. J Exp Med 183: 1561–1567.

169. Liu, B., Y. Yue, Y. Yang, and Y. Jin. 2016. Oral Administration of Silkworm- Produced GAD65 and Insulin Bi-Autoantigens against Type 1 Diabetes. PLoS ONE 11: e0147260.

170. Bergerot, I., N. Fabien, V. Maguer, and C. Thivolet. 1994. Oral administration of human insulin to NOD mice generates CD4+ T cells that suppress adoptive transfer of diabetes. J Autoimmun 7: 655–663.

171. Ramiya, V. K., X. Z. Shang, C. H. Wasserfall, and N. K. Maclaren. 1997. Effect of oral and intravenous insulin and glutamic acid decarboxylase in NOD mice. Autoimmunity 26: 139–151.

172. Ploix, C., I. Bergerot, A. Durand, C. Czerkinsky, J. Holmgren, and C. Thivolet. 1999. Oral administration of cholera toxin B-insulin conjugates protects NOD mice from autoimmune diabetes by inducing CD4+ regulatory T-cells. Diabetes 48: 2150–2156.

173. Ma, S., Y. Huang, Z. Yin, R. Menassa, J. E. Brandle, and A. M. Jevnikar. 2004. Induction of oral tolerance to prevent diabetes with transgenic plants requires glutamic acid decarboxylase (GAD) and IL-4. Proc Natl Acad Sci U S A 101: 5680–5685.

174. Diabetes Prevention Trial--Type 1 Diabetes Study Group. 2002. Effects of insulin in relatives of patients with type 1 diabetes mellitus. N Engl J Med 346: 1685–1691.

175. Skyler, J. S., J. P. Krischer, J. Wolfsdorf, C. Cowie, J. P. Palmer, C. Greenbaum, D. Cuthbertson, L. E. Rafkin-Mervis, H. P. Chase, and E. Leschek. 2005. Effects of oral insulin in relatives of patients with type 1 diabetes: The Diabetes Prevention Trial--Type 1. Diabetes Care 28: 1068–1076.

175

176. Writing Committee for the Type 1 Diabetes TrialNet Oral Insulin Study Group, J. P. Krischer, D. A. Schatz, B. Bundy, J. S. Skyler, and C. J. Greenbaum. 2017. Effect of oral insulin on prevention of diabetes in relatives of patients with type 1 diabetes: A randomized clinical trial. JAMA 318: 1891–1902.

177. Bonifacio, E., A.-G. Ziegler, G. Klingensmith, E. Schober, P. J. Bingley, M. Rottenkolber, A. Theil, A. Eugster, R. Puff, C. Peplow, F. Buettner, K. Lange, J. Hasford, P. Achenbach, and Pre-POINT Study Group. 2015. Effects of high-dose oral insulin on immune responses in children at high risk for type 1 diabetes: the Pre-POINT randomized clinical trial. JAMA 313: 1541–1549.

178. Ludvigsson, J., M. Faresjö, M. Hjorth, S. Axelsson, M. Chéramy, M. Pihl, O. Vaarala, G. Forsander, S. Ivarsson, C. Johansson, A. Lindh, N.-O. Nilsson, J. Aman, E. Ortqvist, P. Zerhouni, and R. Casas. 2008. GAD treatment and insulin secretion in recent-onset type 1 diabetes. N Engl J Med 359: 1909–1920.

179. Ludvigsson, J., D. Krisky, R. Casas, T. Battelino, L. Castaño, J. Greening, O. Kordonouri, T. Otonkoski, P. Pozzilli, J.-J. Robert, H. J. Veeze, J. Palmer, U. Samuelsson, H. Elding Larsson, J. Åman, G. Kärdell, J. Neiderud Helsingborg, G. Lundström, E. Albinsson, A. Carlsson, M. Nordvall, H. Fors, C.-G. Arvidsson, S. Edvardson, R. Hanås, K. Larsson, B. Rathsman, H. Forsgren, H. Desaix, G. Forsander, N.-Ö. Nilsson, C.-G. Åkesson, P. Keskinen, R. Veijola, T. Talvitie, K. Raile, T. Kapellen, W. Burger, A. Neu, I. Engelsberger, B. Heidtmann, S. Bechtold, D. Leslie, F. Chiarelli, A. Cicognani, G. Chiumello, F. Cerutti, G. V. Zuccotti, A. Gomez Gila, I. Rica, R. Barrio, M. Clemente, M. J. López Garcia, M. Rodriguez, I. Gonzalez, J. P. Lopez, M. Oyarzabal, H. M. Reeser, R. Nuboer, P. Stouthart, N. Bratina, N. Bratanic, M. de Kerdanet, J. Weill, N. Ser, P. Barat, A. M. Bertrand, J.-C. Carel, R. Reynaud, R. Coutant, and S. Baron. 2012. GAD65 antigen therapy in recently diagnosed type 1 diabetes mellitus. N Engl J Med 366: 433–442.

180. Ludvigsson, J., M. Chéramy, S. Axelsson, M. Pihl, L. Akerman, R. Casas, and Clinical GAD-Study Group in Sweden. 2014. GAD-treatment of children and adolescents with recent-onset type 1 diabetes preserves residual insulin secretion after 30 months. Diabetes Metab Res Rev 30: 405–414.

181. Mowat, A. M. 2003. Anatomical basis of tolerance and immunity to intestinal antigens. Nat Rev Immunol 3: 331–341.

182. Fujihashi, K., T. Dohi, P. D. Rennert, M. Yamamoto, T. Koga, H. Kiyono, and J. R. McGhee. 2001. Peyer’s patches are required for oral tolerance to proteins. Proc Natl Acad Sci U S A 98: 3310–3315.

183. Mowat, A. M., A. M. Donachie, L. A. Parker, N. C. Robson, H. Beacock-Sharp, L. J. McIntyre, O. Millington, and F. Chirdo. 2003. The role of dendritic cells in regulating mucosal immunity and tolerance. Novartis Found Symp 252: 291–302; discussion 302– 5.

176

184. Weiner, H. L. 2000. Oral tolerance, an active immunologic process mediated by multiple mechanisms. J Clin Invest 106: 935–937.

185. Rynda, A., M. Maddaloni, J. Ochoa-Repáraz, G. Callis, and D. W. Pascual. 2010. IL-28 supplants requirement for T(reg) cells in protein sigma1-mediated protection against murine experimental autoimmune encephalomyelitis (EAE). PLoS ONE 5: e8720.

186. Rynda-Apple, A., E. Huarte, M. Maddaloni, G. Callis, J. A. Skyberg, and D. W. Pascual. 2011. Active immunization using a single dose immunotherapeutic abates established EAE via IL-10 and regulatory T cells. Eur J Immunol 41: 313–323.

187. Nakayama, M., N. Abiru, H. Moriyama, N. Babaya, E. Liu, D. Miao, L. Yu, D. R. Wegmann, J. C. Hutton, J. F. Elliott, and G. S. Eisenbarth. 2005. Prime role for an insulin epitope in the development of type 1 diabetes in NOD mice. Nature 435: 220– 223.

188. Wegmann, D. R., M. Norbury-Glaser, and D. Daniel. 1994. Insulin-specific T cells are a predominant component of islet infiltrates in pre-diabetic NOD mice. Eur J Immunol 24: 1853–1857.

189. Daniel, D., R. G. Gill, N. Schloot, and D. Wegmann. 1995. Epitope specificity, cytokine production profile and diabetogenic activity of insulin-specific T cell clones isolated from NOD mice. Eur J Immunol 25: 1056–1062.

190. Prasad, S., A. P. Kohm, J. S. McMahon, X. Luo, and S. D. Miller. 2012. Pathogenesis of NOD diabetes is initiated by reactivity to the insulin B chain 9-23 epitope and involves functional epitope spreading. J Autoimmun 39: 347–353.

191. Yang, J., N. A. Danke, D. Berger, S. Reichstetter, H. Reijonen, C. Greenbaum, C. Pihoker, E. A. James, and W. W. Kwok. 2006. Islet-specific glucose-6-phosphatase catalytic subunit-related protein-reactive CD4+ T cells in human subjects. J Immunol 176: 2781–2789.

192. Leslie, R. D., M. A. Atkinson, and A. L. Notkins. 1999. Autoantigens IA-2 and GAD in Type I (insulin-dependent) diabetes. Diabetologia 42: 3–14.

193. Verge, C. F., R. Gianani, E. Kawasaki, L. Yu, M. Pietropaolo, R. A. Jackson, H. P. Chase, and G. S. Eisenbarth. 1996. Prediction of type I diabetes in first-degree relatives using a combination of insulin, GAD, and ICA512bdc/IA-2 autoantibodies. Diabetes 45: 926–933.

194. Evans, D. J., and D. G. Evans. 1990. Colonization factor antigens of human pathogens. Curr Top Microbiol Immunol 151: 129–145.

177

195. Edelman, R., R. G. Russell, G. Losonsky, B. D. Tall, C. O. Tacket, M. M. Levine, and D. H. Lewis. 1993. Immunization of rabbits with enterotoxigenic E. coli colonization factor antigen (CFA/I) encapsulated in biodegradable microspheres of poly (lactide-co- glycolide). Vaccine 11: 155–158.

196. Evans, D. G., D. Y. Graham, and D. J. Evans. 1984. Administration of purified colonization factor antigens (CFA/I, CFA/II) of enterotoxigenic Escherichia coli to volunteers. Response to challenge with virulent enterotoxigenic Escherichia coli. Gastroenterology 87: 934–940.

197. Schmidt, M., E. P. Kelly, L. Y. Tseng, and E. C. Boedeker. 1985. Towards an oral Escherichia-coli pilus vaccine for travelers diarrhea: susceptibility of purified colonization factor antigen II to proteolytic digestion. Gastroenterology 88: 1575.

198. Lin, I. Y. C., T. T. H. Van, and P. M. Smooker. 2015. Live-Attenuated Bacterial Vectors: Tools for Vaccine and Therapeutic Agent Delivery. Vaccines (Basel) 3: 940– 972.

199. Galen, J. E., M. F. Pasetti, S. Tennant, P. Ruiz-Olvera, M. B. Sztein, and M. M. Levine. 2009. Salmonella enterica serovar Typhi live vector vaccines finally come of age. Immunol Cell Biol 87: 400–412.

200. Mestecky, J., H. Nguyen, C. Czerkinsky, and H. Kiyono. 2008. Oral immunization: an update. Curr Opin Gastroenterol 24: 713–719.

201. Anantha, R. P., A. L. McVeigh, L. H. Lee, M. K. Agnew, F. J. Cassels, D. A. Scott, T. S. Whittam, and S. J. Savarino. 2004. Evolutionary and functional relationships of colonization factor antigen i and other class 5 adhesive fimbriae of enterotoxigenic Escherichia coli. Infect Immun 72: 7190–7201.

202. Li, Y.-F., S. Poole, K. Nishio, K. Jang, F. Rasulova, A. McVeigh, S. J. Savarino, D. Xia, and E. Bullitt. 2009. Structure of CFA/I fimbriae from enterotoxigenic Escherichia coli. Proc Natl Acad Sci U S A 106: 10793–10798.

203. Jansson, L., J. Tobias, M. Lebens, A.-M. Svennerholm, and S. Teneberg. 2006. The major subunit, CfaB, of colonization factor antigen i from enterotoxigenic Escherichia coli is a glycosphingolipid binding protein. Infect Immun 74: 3488–3497.

204. Yang, X., Z. Suo, T. Thornburg, K. Holderness, L. Cao, T. Lim, N. Walters, L. Kellerman, L. Loetterle, R. Avci, and D. W. Pascual. 2012. Expression of Escherichia coli virulence usher protein attenuates wild-type Salmonella. Virulence 3: 29–42.

205. Wu, S., D. W. Pascual, J. L. VanCott, J. R. McGhee, D. R. Maneval, M. M. Levine, and D. M. Hone. 1995. Immune responses to novel Escherichia coli and Salmonella typhimurium vectors that express colonization factor antigen I (CFA/I) of enterotoxigenic E. coli in the absence of the CFA/I positive regulator cfaR. Infect Immun 63: 4933–4938.

178

206. Pascual, D. W., D. M. Hone, S. Hall, F. W. van Ginkel, M. Yamamoto, N. Walters, K. Fujihashi, R. J. Powell, S. Wu, J. L. Vancott, H. Kiyono, and J. R. McGhee. 1999. Expression of recombinant enterotoxigenic Escherichia coli colonization factor antigen I by Salmonella typhimurium elicits a biphasic T helper cell response. Infect Immun 67: 6249–6256.

207. Nauciel, C., and F. Espinasse-Maes. 1992. Role of gamma interferon and tumor necrosis factor alpha in resistance to Salmonella typhimurium infection. Infect Immun 60: 450–454.

208. Mastroeni, P., B. Villarreal-Ramos, and C. E. Hormaeche. 1992. Role of T cells, TNF alpha and IFN gamma in recall of immunity to oral challenge with virulent salmonellae in mice vaccinated with live attenuated aro- Salmonella vaccines. Microb Pathog 13: 477–491.

209. Walters, N., T. Trunkle, M. Sura, and D. W. Pascual. 2005. Enhanced immunoglobulin A response and protection against Salmonella enterica serovar typhimurium in the absence of the substance P receptor. Infect Immun 73: 317–324.

210. Pascual, D. W., T. Trunkle, and J. Sura. 2002. Fimbriated Salmonella enterica serovar typhimurium abates initial inflammatory responses by macrophages. Infect Immun 70: 4273–4281.

211. Jun, S., W. Gilmore, G. Callis, A. Rynda, A. Haddad, and D. W. Pascual. 2005. A live diarrheal vaccine imprints a Th2 cell bias and acts as an anti-inflammatory vaccine. J Immunol 175: 6733–6740.

212. Ochoa-Repáraz, J., C. Riccardi, A. Rynda, S. Jun, G. Callis, and D. W. Pascual. 2007. Regulatory T cell vaccination without autoantigen protects against experimental autoimmune encephalomyelitis. J Immunol 178: 1791–1799.

213. Jun, S., J. Ochoa-Repáraz, D. Zlotkowska, T. Hoyt, and D. W. Pascual. 2012. Bystander-mediated stimulation of proteolipid protein-specific regulatory T (Treg) cells confers protection against experimental autoimmune encephalomyelitis (EAE) via TGF- β. J Neuroimmunol 245: 39–47.

214. Kochetkova, I., T. Trunkle, G. Callis, and D. W. Pascual. 2008. Vaccination without autoantigen protects against collagen II-induced arthritis via immune deviation and regulatory T cells. J Immunol 181: 2741–2752.

215. Kochetkova, I., T. Thornburg, G. Callis, and D. W. Pascual. 2011. Segregated regulatory CD39+CD4+ T cell function: TGF-β-producing Foxp3- and IL-10-producing Foxp3+ cells are interdependent for protection against collagen-induced arthritis. J Immunol 187: 4654–4666.

216. Huxley, R. R., S. A. E. Peters, G. D. Mishra, and M. Woodward. 2015. Risk of all- cause mortality and vascular events in women versus men with type 1 diabetes: a systematic review and meta-analysis. Lancet Diabetes Endocrinol. 3: 198–206.

179

217. Lind, M., A.-M. Svensson, M. Kosiborod, S. Gudbjörnsdottir, A. Pivodic, H. Wedel, S. Dahlqvist, M. Clements, and A. Rosengren. 2014. Glycemic control and excess mortality in type 1 diabetes. N Engl J Med 371: 1972–1982.

218. Ilan, Y., E. Zigmond, G. Lalazar, A. Dembinsky, A. Ben Ya’acov, N. Hemed, I. Kasis, E. Axelrod, L. Zolotarov, A. Klein, M. El Haj, R. Gandhi, C. Baecher-Allan, H. Wu, G. Murugaiyan, P. Kivisakk, M. F. Farez, F. J. Quintana, S. J. Khoury, and H. L. Weiner. 2010. Oral administration of OKT3 monoclonal antibody to human subjects induces a dose-dependent immunologic effect in T cells and dendritic cells. J Clin Immunol 30: 167–177.

219. Wang, X., A. Sherman, G. Liao, K. W. Leong, H. Daniell, C. Terhorst, and R. W. Herzog. 2013. Mechanism of oral tolerance induction to therapeutic proteins. Adv Drug Deliv Rev 65: 759–773.

220. Pihoker, C., L. K. Gilliam, C. S. Hampe, and A. Lernmark. 2005. Autoantibodies in diabetes. Diabetes 54 Suppl 2: S52–61.

221. Zhang, L., and G. S. Eisenbarth. 2011. Prediction and prevention of Type 1 diabetes mellitus. J Diabetes 3: 48–57.

222. Ilonen, J., A. Hammais, A.-P. Laine, J. Lempainen, O. Vaarala, R. Veijola, O. Simell, and M. Knip. 2013. Patterns of β-cell autoantibody appearance and genetic associations during the first years of life. Diabetes 62: 3636–3640.

223. Phillips, J. M., N. M. Parish, T. Raine, C. Bland, Y. Sawyer, H. De La Peña, and A. Cooke. 2009. Type 1 diabetes development requires both CD4+ and CD8+ T cells and can be reversed by non-depleting antibodies targeting both T cell populations. Rev Diabet Stud 6: 97–103.

224. Ludvigsson, J., and L. D. I. I. S. Group. 2009. The role of immunomodulation therapy in autoimmune diabetes. J Diabetes Sci Technol 3: 320–30.

225. Serreze, D. V., C. M. Post, H. D. Chapman, E. A. Johnson, B. Lu, and P. B. Rothman. 2000. Interferon-gamma receptor signaling is dispensable in the development of autoimmune type 1 diabetes in NOD mice. Diabetes 49: 2007–2011.

226. Joseph, J., S. Bittner, F. M. P. Kaiser, H. Wiendl, and S. Kissler. 2012. IL-17 silencing does not protect nonobese diabetic mice from autoimmune diabetes. J Immunol 188: 216–221.

227. GBD Diarrhoeal Diseases Collaborators. 2017. Estimates of global, regional, and national morbidity, mortality, and aetiologies of diarrhoeal diseases: a systematic analysis for the Global Burden of Disease Study 2015. Lancet Infect Dis 17: 909–948.

228. Maddaloni, M., I. Kochetkova, S. Jun, G. Callis, T. Thornburg, and D. W. Pascual. 2015. Milk-based nutraceutical for treating autoimmune arthritis via the stimulation of IL- 10- and TGF-β-producing CD39+ regulatory T cells. PLoS ONE 10: e0117825.

180

229. Zmuda, E. J., C. A. Powell, and T. Hai. 2011. A method for murine islet isolation and subcapsular kidney transplantation. J Vis Exp .

230. Ishigame, H., L. A. Zenewicz, S. Sanjabi, P. Licona-Limón, M. Nakayama, W. J. Leonard, and R. A. Flavell. 2013. Excessive Th1 responses due to the absence of TGF- β signaling cause autoimmune diabetes and dysregulated Treg cell homeostasis. Proc Natl Acad Sci U S A 110: 6961–6966.

231. Rohane, P. W., A. Shimada, D. T. Kim, C. T. Edwards, B. Charlton, L. D. Shultz, and C. G. Fathman. 1995. Islet-infiltrating lymphocytes from prediabetic NOD mice rapidly transfer diabetes to NOD-scid/scid mice. Diabetes 44: 550–554.

232. Gregori, S., N. Giarratana, S. Smiroldo, and L. Adorini. 2003. Dynamics of pathogenic and suppressor T cells in autoimmune diabetes development. J Immunol 171: 4040–4047.

233. Baecher-Allan, C., J. A. Brown, G. J. Freeman, and D. A. Hafler. 2001. CD4+CD25high regulatory cells in human peripheral blood. J Immunol 167: 1245–1253.

234. Hombach, A. A., D. Kofler, A. Hombach, G. Rappl, and H. Abken. 2007. Effective proliferation of human regulatory T cells requires a strong costimulatory CD28 signal that cannot be substituted by IL-2. J Immunol 179: 7924–7931.

235. Wong, F. S., L. K. Siew, G. Scott, I. J. Thomas, S. Chapman, C. Viret, and L. Wen. 2009. Activation of insulin-reactive CD8 T-cells for development of autoimmune diabetes. Diabetes 58: 1156–1164.

236. Wegmann, D. R., R. G. Gill, M. Norbury-Glaser, N. Schloot, and D. Daniel. 1994. Analysis of the spontaneous T cell response to insulin in NOD mice. J Autoimmun 7: 833–843.

237. Sakaguchi, S., M. Ono, R. Setoguchi, H. Yagi, S. Hori, Z. Fehervari, J. Shimizu, T. Takahashi, and T. Nomura. 2006. Foxp3+ CD25+ CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease. Immunol Rev 212: 8–27.

238. Zhao, C., Z. Wang, M. W. Robertson, and J. D. Davies. 2008. Cachexia in the nonobese diabetic mouse is associated with CD4+ T-cell lymphopenia. Immunology 125: 48–58.

239. De Matos-Neto, E. M., J. D. C. C. Lima, W. O. de Pereira, R. G. Figuerêdo, D. M. D. R. Riccardi, K. Radloff, R. X. das Neves, R. G. Camargo, L. F. Maximiano, F. Tokeshi, J. P. Otoch, R. Goldszmid, N. O. S. Câmara, G. Trinchieri, P. S. M. de Alcântara, and M. Seelaender. 2015. Systemic Inflammation in Cachexia - Is Tumor Cytokine Expression Profile the Culprit? Front Immunol 6: 629.

240. Koch, M. A., G. Tucker-Heard, N. R. Perdue, J. R. Killebrew, K. B. Urdahl, and D. J. Campbell. 2009. The transcription factor T-bet controls regulatory T cell homeostasis and function during type 1 inflammation. Nat Immunol 10: 595–602.

181

241. Yang, B. H., S. Hagemann, P. Mamareli, U. Lauer, U. Hoffmann, M. Beckstette, L. Föhse, I. Prinz, J. Pezoldt, S. Suerbaum, T. Sparwasser, A. Hamann, S. Floess, J. Huehn, and M. Lochner. 2016. Foxp3(+) T cells expressing RORγt represent a stable regulatory T-cell effector lineage with enhanced suppressive capacity during intestinal inflammation. Mucosal Immunol 9: 444–457.

242. Zhao, H., C. Bo, Y. Kang, and H. Li. 2017. What else can CD39 tell us? Front Immunol 8: 727.

243. Lasch, S., P. Müller, M. Bayer, J. M. Pfeilschifter, A. D. Luster, E. Hintermann, and U. Christen. 2015. Anti-CD3/Anti-CXCL10 Antibody Combination Therapy Induces a Persistent Remission of Type 1 Diabetes in Two Mouse Models. Diabetes 64: 4198– 4211.

244. Frigerio, S., T. Junt, B. Lu, C. Gerard, U. Zumsteg, G. A. Holländer, and L. Piali. 2002. β cells are responsible for CXCR3-mediated T-cell infiltration in insulitis. Nat Med 8: 1414–1420.

245. Zeng, H., R. Zhang, B. Jin, and L. Chen. 2015. Type 1 regulatory T cells: a new mechanism of peripheral immune tolerance. Cell Mol Immunol 12: 566–571.

246. Gagliani, N., C. F. Magnani, S. Huber, M. E. Gianolini, M. Pala, P. Licona-Limon, B. Guo, D. R. Herbert, A. Bulfone, F. Trentini, C. Di Serio, R. Bacchetta, M. Andreani, L. Brockmann, S. Gregori, R. A. Flavell, and M.-G. Roncarolo. 2013. Coexpression of CD49b and LAG-3 identifies human and mouse T regulatory type 1 cells. Nat Med 19: 739–746.

247. Li, S., Q. Xie, Y. Zeng, C. Zou, X. Liu, S. Wu, H. Deng, Y. Xu, X. C. Li, and Z. Dai. 2014. A naturally occurring CD8(+)CD122(+) T-cell subset as a memory-like Treg family. Cell Mol Immunol 11: 326–331.

248. Liu, J., D. Chen, G. D. Nie, and Z. Dai. 2015. CD8(+)CD122(+) T-Cells: A Newly Emerging Regulator with Central Memory Cell Phenotypes. Front Immunol 6: 494.

249. Kleffel, S., A. Vergani, S. Tezza, M. Ben Nasr, M. A. Niewczas, S. Wong, R. Bassi, F. D’Addio, T. Schatton, R. Abdi, M. Atkinson, M. H. Sayegh, L. Wen, C. H. Wasserfall, K. C. O’Connor, and P. Fiorina. 2015. Interleukin-10+ regulatory B cells arise within antigen-experienced CD40+ B cells to maintain tolerance to islet autoantigens. Diabetes 64: 158–171.

250. Dai, H., N. Wan, S. Zhang, Y. Moore, F. Wan, and Z. Dai. 2010. Cutting edge: programmed death-1 defines CD8+CD122+ T cells as regulatory versus memory T cells. J Immunol 185: 803–807.

182

251. Spanier, J. A., N. L. Sahli, J. C. Wilson, T. Martinov, T. Dileepan, A. L. Burrack, E. B. Finger, B. R. Blazar, A. W. Michels, A. Moran, M. K. Jenkins, and B. T. Fife. 2017. Increased Effector Memory Insulin-Specific CD4+ T Cells Correlate With Insulin Autoantibodies in Patients With Recent-Onset Type 1 Diabetes. Diabetes 66: 3051– 3060.

252. Campbell-Thompson, M., A. Fu, J. S. Kaddis, C. Wasserfall, D. A. Schatz, A. Pugliese, and M. A. Atkinson. 2016. Insulitis and β-Cell Mass in the Natural History of Type 1 Diabetes. Diabetes 65: 719–731.

253. Cerosaletti, K., F. Barahmand-Pour-Whitman, J. Yang, H. A. DeBerg, M. J. Dufort, S. A. Murray, E. Israelsson, C. Speake, V. H. Gersuk, J. A. Eddy, H. Reijonen, C. J. Greenbaum, W. W. Kwok, E. Wambre, M. Prlic, R. Gottardo, G. T. Nepom, and P. S. Linsley. 2017. Single-Cell RNA Sequencing Reveals Expanded Clones of Islet Antigen- Reactive CD4+ T Cells in Peripheral Blood of Subjects with Type 1 Diabetes. J Immunol 199: 323–335.

254. Seay, H. R., E. Yusko, S. J. Rothweiler, L. Zhang, A. L. Posgai, M. Campbell- Thompson, M. Vignali, R. O. Emerson, J. S. Kaddis, D. Ko, M. Nakayama, M. J. Smith, J. C. Cambier, A. Pugliese, M. A. Atkinson, H. S. Robins, and T. M. Brusko. 2016. Tissue distribution and clonal diversity of the T and B cell repertoire in type 1 diabetes. JCI Insight 1: e88242.

255. Hagopian, W., R. J. Ferry, N. Sherry, D. Carlin, E. Bonvini, S. Johnson, K. E. Stein, S. Koenig, A. G. Daifotis, K. C. Herold, J. Ludvigsson, and Protégé Trial Investigators. 2013. Teplizumab preserves C-peptide in recent-onset type 1 diabetes: two-year results from the randomized, placebo-controlled Protégé trial. Diabetes 62: 3901–3908.

256. Daifotis, A. G., S. Koenig, L. Chatenoud, and K. C. Herold. 2013. Anti-CD3 clinical trials in type 1 diabetes mellitus. Clin Immunol 149: 268–278.

257. Mottram, P. L., L. J. Murray-Segal, W. Han, J. Maguire, and A. N. Stein-Oakley. 2002. Remission and pancreas isograft survival in recent onset diabetic NOD mice after treatment with low-dose anti-CD3 monoclonal antibodies. Transpl Immunol 10: 63–72.

258. Yamagata, K., H. Nakajima, K. Tomita, N. Itoh, J. Miyagawa, T. Hamaguchi, M. Namba, S. Tamura, S. Kawata, N. Kono, M. Kuwajima, T. Noguchi, T. Hanafusa, and Y. Matsuzawa. 1996. Dominant TCR α-chain clonotypes and interferon-γ are expressed in the pancreas of patients with recent-onset insulin-dependent diabetes mellitus. Diabetes Res Clin Pract 34: 37–46.

259. Babon, J. A. B., M. E. DeNicola, D. M. Blodgett, I. Crèvecoeur, T. S. Buttrick, R. Maehr, R. Bottino, A. Naji, J. Kaddis, W. Elyaman, E. A. James, R. Haliyur, M. Brissova, L. Overbergh, C. Mathieu, T. Delong, K. Haskins, A. Pugliese, M. Campbell-Thompson, C. Mathews, M. A. Atkinson, A. C. Powers, D. M. Harlan, and S. C. Kent. 2016. Analysis of self-antigen specificity of islet-infiltrating T cells from human donors with type 1 diabetes. Nat Med 22: 1482–1487.

183

260. Trembleau, S., G. Penna, S. Gregori, H. D. Chapman, D. V. Serreze, J. Magram, and L. Adorini. 1999. Pancreas-infiltrating Th1 cells and diabetes develop in IL-12- deficient nonobese diabetic mice. J Immunol 163: 2960–2968.

261. Driver, J. P., J. J. Racine, C. Ye, D. J. Lamont, B. N. Newby, C. M. Leeth, H. D. Chapman, T. M. Brusko, Y.-G. Chen, C. E. Mathews, and D. V. Serreze. 2017. Interferon-γ Limits Diabetogenic CD8+ T-Cell Effector Responses in Type 1 Diabetes. Diabetes 66: 710–721.

262. Utsugi, T., J. W. Yoon, B. J. Park, M. Imamura, N. Averill, S. Kawazu, and P. Santamaria. 1996. Major histocompatibility complex class I-restricted infiltration and destruction of pancreatic islets by NOD mouse-derived beta-cell cytotoxic CD8+ T-cell clones in vivo. Diabetes 45: 1121–1131.

263. Nagata, M., P. Santamaria, T. Kawamura, T. Utsugi, and J. W. Yoon. 1994. Evidence for the role of CD8+ cytotoxic T cells in the destruction of pancreatic beta-cells in nonobese diabetic mice. J Immunol 152: 2042–2050.

264. Collier, J. J., S. J. Burke, M. E. Eisenhauer, D. Lu, R. C. Sapp, C. J. Frydman, and S. R. Campagna. 2011. Pancreatic β-cell death in response to pro-inflammatory cytokines is distinct from genuine apoptosis. PLoS ONE 6: e22485.

265. Arif, S., P. Leete, V. Nguyen, K. Marks, N. M. Nor, M. Estorninho, D. Kronenberg- Versteeg, P. J. Bingley, J. A. Todd, C. Guy, D. B. Dunger, J. Powrie, A. Willcox, A. K. Foulis, S. J. Richardson, E. de Rinaldis, N. G. Morgan, A. Lorenc, and M. Peakman. 2014. Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes. Diabetes 63: 3835–3845.

266. Shoda, L. K. M., D. L. Young, S. Ramanujan, C. C. Whiting, M. A. Atkinson, J. A. Bluestone, G. S. Eisenbarth, D. Mathis, A. A. Rossini, S. E. Campbell, R. Kahn, and H. T. C. Kreuwel. 2005. A comprehensive review of interventions in the NOD mouse and implications for translation. Immunity 23: 115–126.

267. Goudy, K. S., B. R. Burkhardt, C. Wasserfall, S. Song, M. L. Campbell-Thompson, T. Brusko, M. A. Powers, M. J. Clare-Salzler, E. S. Sobel, T. M. Ellis, T. R. Flotte, and M. A. Atkinson. 2003. Systemic overexpression of IL-10 induces CD4+CD25+ cell populations in vivo and ameliorates type 1 diabetes in nonobese diabetic mice in a dose-dependent fashion. J Immunol 171: 2270–2278.

268. Yang, Z., M. Chen, R. Wu, L. B. Fialkow, J. S. Bromberg, M. McDuffie, A. Naji, and J. L. Nadler. 2002. Suppression of autoimmune diabetes by viral IL-10 gene transfer. J Immunol 168: 6479–6485.

269. Yang, X. D., R. Tisch, S. M. Singer, Z. A. Cao, R. S. Liblau, R. D. Schreiber, and H. O. McDevitt. 1994. Effect of tumor necrosis factor alpha on insulin-dependent diabetes mellitus in NOD mice. I. The early development of autoimmunity and the diabetogenic process. J Exp Med 180: 995–1004.

184

270. Li, C.-R., M. F. Deiro, E. Godebu, and L. M. Bradley. 2011. IL-7 uniquely maintains FoxP3(+) adaptive Treg cells that reverse diabetes in NOD mice via integrin-β7- dependent localization. J Autoimmun 37: 217–227.

271. Grinberg-Bleyer, Y., A. Baeyens, S. You, R. Elhage, G. Fourcade, S. Gregoire, N. Cagnard, W. Carpentier, Q. Tang, J. Bluestone, L. Chatenoud, D. Klatzmann, B. L. Salomon, and E. Piaggio. 2010. IL-2 reverses established type 1 diabetes in NOD mice by a local effect on pancreatic regulatory T cells. J Exp Med 207: 1871–1878.

272. Schuster, C., F. Jonas, F. Zhao, and S. Kissler. 2018. Peripherally induced regulatory T cells contribute to the control of autoimmune diabetes in the NOD mouse model. Eur J Immunol 48: 1211–1216.

273. Robert, S., C. Gysemans, T. Takiishi, H. Korf, I. Spagnuolo, G. Sebastiani, K. Van Huynegem, L. Steidler, S. Caluwaerts, P. Demetter, C. H. Wasserfall, M. A. Atkinson, F. Dotta, P. Rottiers, T. L. Van Belle, and C. Mathieu. 2014. Oral delivery of glutamic acid decarboxylase (GAD)-65 and IL10 by Lactococcus lactis reverses diabetes in recent- onset NOD mice. Diabetes 63: 2876–2887.

274. Takiishi, T., D. P. Cook, H. Korf, G. Sebastiani, F. Mancarella, J. P. M. C. M. Cunha, C. Wasserfall, N. Casares, J. J. Lasarte, L. Steidler, P. Rottiers, F. Dotta, C. Gysemans, and C. Mathieu. 2017. Reversal of Diabetes in NOD Mice by Clinical-Grade Proinsulin and IL-10-Secreting Lactococcus lactis in Combination With Low-Dose Anti- CD3 Depends on the Induction of Foxp3-Positive T Cells. Diabetes 66: 448–459.

275. Takiishi, T., H. Korf, T. L. Van Belle, S. Robert, F. A. Grieco, S. Caluwaerts, L. Galleri, I. Spagnuolo, L. Steidler, K. Van Huynegem, P. Demetter, C. Wasserfall, M. A. Atkinson, F. Dotta, P. Rottiers, C. Gysemans, and C. Mathieu. 2012. Reversal of autoimmune diabetes by restoration of antigen-specific tolerance using genetically modified Lactococcus lactis in mice. J Clin Invest 122: 1717–1725.

276. Liu, K.-F., X.-R. Liu, G.-L. Li, S.-P. Lu, L. Jin, and J. Wu. 2016. Oral administration of Lactococcus lactis-expressing heat shock protein 65 and tandemly repeated IA2P2 prevents type 1 diabetes in NOD mice. Immunol Lett 174: 28–36.

277. Daniel, C., S. Poiret, V. Dennin, D. Boutillier, and B. Pot. 2013. Bioluminescence imaging study of spatial and temporal persistence of Lactobacillus plantarum and Lactococcus lactis in living mice. Appl Environ Microbiol 79: 1086–1094.

278. You, S., C. Chen, W.-H. Lee, T. Brusko, M. Atkinson, and C.-P. Liu. 2004. Presence of diabetes-inhibiting, glutamic acid decarboxylase-specific, IL-10-dependent, regulatory T cells in naive nonobese diabetic mice. J Immunol 173: 6777–6785.

279. Kassiotis, G., D. Gray, Z. Kiafard, J. Zwirner, and B. Stockinger. 2006. Functional specialization of memory Th cells revealed by expression of integrin CD49b. J Immunol 177: 968–975.

185

280. White, A. M., and D. C. Wraith. 2016. Tr1-Like T Cells - An Enigmatic Regulatory T Cell Lineage. Front Immunol 7: 355.

281. El Mir, S., and F. Triebel. 2000. A soluble lymphocyte activation gene-3 molecule used as a vaccine adjuvant elicits greater humoral and cellular immune responses to both particulate and soluble antigens. J Immunol 164: 5583–5589.

282. Pot, C., H. Jin, A. Awasthi, S. M. Liu, C.-Y. Lai, R. Madan, A. H. Sharpe, C. L. Karp, S.-C. Miaw, I.-C. Ho, and V. K. Kuchroo. 2009. Cutting edge: IL-27 induces the transcription factor c-Maf, cytokine IL-21, and the costimulatory receptor ICOS that coordinately act together to promote differentiation of IL-10-producing Tr1 cells. J Immunol 183: 797–801.

283. Apetoh, L., F. J. Quintana, C. Pot, N. Joller, S. Xiao, D. Kumar, E. J. Burns, D. H. Sherr, H. L. Weiner, and V. K. Kuchroo. 2010. The aryl hydrocarbon receptor interacts with c-Maf to promote the differentiation of type 1 regulatory T cells induced by IL-27. Nat Immunol 11: 854–861.

284. Kastelein, R. A., C. A. Hunter, and D. J. Cua. 2007. Discovery and biology of IL-23 and IL-27: related but functionally distinct regulators of inflammation. Annu Rev Immunol 25: 221–242.

285. Ben Nasr, M., S. Tezza, F. D’Addio, C. Mameli, V. Usuelli, A. Maestroni, D. Corradi, S. Belletti, L. Albarello, G. Becchi, G. P. Fadini, C. Schuetz, J. Markmann, C. Wasserfall, L. Zon, G. V. Zuccotti, and P. Fiorina. 2017. PD-L1 genetic overexpression or pharmacological restoration in hematopoietic stem and progenitor cells reverses autoimmune diabetes. Sci Transl Med 9.

286. Visperas, A., and D. A. A. Vignali. 2016. Are regulatory T cells defective in type 1 diabetes and can we fix them? J Immunol 197: 3762–3770.

287. Yu, H., N. Gagliani, H. Ishigame, S. Huber, S. Zhu, E. Esplugues, K. C. Herold, L. Wen, and R. A. Flavell. 2017. Intestinal type 1 regulatory T cells migrate to periphery to suppress diabetogenic T cells and prevent diabetes development. Proc Natl Acad Sci U S A 114: 10443–10448.

288. Sanda, S., B. O. Roep, and M. von Herrath. 2008. Islet antigen specific IL-10+ immune responses but not CD4+CD25+FoxP3+ cells at diagnosis predict glycemic control in type 1 diabetes. Clin Immunol 127: 138–143.

289. Chujo, D., T.-S. Nguyen, E. Foucat, D. Blankenship, J. Banchereau, G. T. Nepom, D. Chaussabel, and H. Ueno. 2015. Adult-onset type 1 diabetes patients display decreased IGRP-specific Tr1 cells in blood. Clin Immunol 161: 270–277.

290. Arif, S., T. I. Tree, T. P. Astill, J. M. Tremble, A. J. Bishop, C. M. Dayan, B. O. Roep, and M. Peakman. 2004. Autoreactive T cell responses show proinflammatory polarization in diabetes but a regulatory phenotype in health. J Clin Invest 113: 451– 463.

186

291. Newby, B. N., and C. E. Mathews. 2017. Type I interferon is a catastrophic feature of the diabetic islet microenvironment. Front Endocrinol (Lausanne) 8: 232.

292. Merad, M., P. Sathe, J. Helft, J. Miller, and A. Mortha. 2013. The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting. Annu Rev Immunol 31: 563–604.

293. Reizis, B., A. Bunin, H. S. Ghosh, K. L. Lewis, and V. Sisirak. 2011. Plasmacytoid dendritic cells: recent progress and open questions. Annu Rev Immunol 29: 163–183.

294. Fallarino, F., C. Volpi, T. Zelante, C. Vacca, M. Calvitti, M. C. Fioretti, P. Puccetti, L. Romani, and U. Grohmann. 2009. IDO mediates TLR9-driven protection from experimental autoimmune diabetes. J Immunol 183: 6303–6312.

295. Saxena, V., J. K. Ondr, A. F. Magnusen, D. H. Munn, and J. D. Katz. 2007. The countervailing actions of myeloid and plasmacytoid dendritic cells control autoimmune diabetes in the nonobese diabetic mouse. J Immunol 179: 5041–5053.

296. Morel, P. A. 2013. Dendritic cell subsets in type 1 diabetes: friend or foe? Front Immunol 4: 415.

297. Millar, D. G., and P. S. Ohashi. 2016. Central tolerance: what you see is what you don’t get! Nat Immunol 17: 115–116.

298. Proietto, A. I., S. van Dommelen, P. Zhou, A. Rizzitelli, A. D’Amico, R. J. Steptoe, S. H. Naik, M. H. Lahoud, Y. Liu, P. Zheng, K. Shortman, and L. Wu. 2008. Dendritic cells in the thymus contribute to T-regulatory cell induction. Proc Natl Acad Sci U S A 105: 19869–19874.

299. Yamazaki, S., D. Dudziak, G. F. Heidkamp, C. Fiorese, A. J. Bonito, K. Inaba, M. C. Nussenzweig, and R. M. Steinman. 2008. CD8+ CD205+ splenic dendritic cells are specialized to induce Foxp3+ regulatory T cells. J Immunol 181: 6923–6933.

300. Yamazaki, S., A. J. Bonito, R. Spisek, M. Dhodapkar, K. Inaba, and R. M. Steinman. 2007. Dendritic cells are specialized accessory cells along with TGF- for the differentiation of Foxp3+ CD4+ regulatory T cells from peripheral Foxp3 precursors. Blood 110: 4293–4302.

301. Vasquez, A. C., M. Feili-Hariri, R. J. Tan, and P. A. Morel. 2004. Qualitative and quantitative abnormalities in splenic dendritic cell populations in NOD mice. Clin Exp Immunol 135: 209–218.

302. Price, J. D., C. Hotta-Iwamura, Y. Zhao, N. M. Beauchamp, and K. V. Tarbell. 2015. DCIR2+ cDC2 DCs and Zbtb32 Restore CD4+ T-Cell Tolerance and Inhibit Diabetes. Diabetes 64: 3521–3531.

187

303. Price, J. D., N. M. Beauchamp, G. Rahir, Y. Zhao, C. C. Rieger, A. W. Lau-Kilby, and K. V. Tarbell. 2014. CD8+ dendritic cell-mediated tolerance of autoreactive CD4+ T cells is deficient in NOD mice and can be corrected by blocking CD40L. J Leukoc Biol 95: 325–336.

304. Boedeker, E. C. 2005. Vaccines for enterotoxigenic Escherichia coli: current status. Curr Opin Gastroenterol 21: 15–19.

305. Mellanby, R. J., D. Thomas, J. M. Phillips, and A. Cooke. 2007. Diabetes in nonobese diabetic mice is not associated with quantitative changes in CD4+ CD25+ Foxp3+ regulatory T cells. Immunology 121: 15–28.

306. Pascual, D. W., X. Yang, K. Holderness, S. Jun, M. Maddaloni, and I. Kochetkova. 2014. Regulatory T-cell vaccination independent of auto-antigen. Exp Mol Med 46: e82.

307. Fleeton, M., N. Contractor, F. Leon, J. He, D. Wetzel, T. Dermody, A. Iwasaki, and B. Kelsall. 2004. Involvement of dendritic cell subsets in the induction of oral tolerance and immunity. Ann N Y Acad Sci 1029: 60–65.

308. Forteza, M. J., K. A. Polyzos, R. Baumgartner, B. E. Suur, M. Mussbacher, D. K. Johansson, A. Hermansson, G. K. Hansson, and D. F. J. Ketelhuth. 2018. Activation of the Regulatory T-Cell/Indoleamine 2,3-Dioxygenase Axis Reduces Vascular Inflammation and Atherosclerosis in Hyperlipidemic Mice. Front Immunol 9: 950.

309. Carrero, J. A., D. P. McCarthy, S. T. Ferris, X. Wan, H. Hu, B. H. Zinselmeyer, A. N. Vomund, and E. R. Unanue. 2017. Resident macrophages of pancreatic islets have a seminal role in the initiation of autoimmune diabetes of NOD mice. Proc Natl Acad Sci U S A 114: E10418–E10427.

310. Thornley, T. B., K. A. Agarwal, P. Kyriazis, L. Ma, V. Chipashvili, J. E. Aker, S. Korniotis, E. Csizmadia, T. B. Strom, and M. Koulmanda. 2016. Contrasting roles of islet resident immunoregulatory macrophages and dendritic cells in experimental autoimmune type 1 diabetes. PLoS ONE 11: e0150792.

311. Newby, B. N., T. M. Brusko, B. Zou, M. A. Atkinson, M. Clare-Salzler, and C. E. Mathews. 2017. Type 1 interferons potentiate human CD8(+) T cell cytotoxicity through a STAT4 and granzyme B dependent pathway. Diabetes .

312. Weaver, D. J., B. Poligone, T. Bui, U. M. Abdel-Motal, A. S. Baldwin, and R. Tisch. 2001. Dendritic cells from nonobese diabetic mice exhibit a defect in NF-kappa B regulation due to a hyperactive I kappa B kinase. J Immunol 167: 1461–1468.

313. Liu, J., and D. Beller. 2002. Aberrant production of IL-12 by macrophages from several autoimmune-prone mouse strains is characterized by intrinsic and unique patterns of NF-kappa B expression and binding to the IL-12 p40 promoter. J Immunol 169: 581–586.

188

314. Billiard, F., C. Lobry, G. Darrasse-Jèze, J. Waite, X. Liu, H. Mouquet, A. DaNave, M. Tait, J. Idoyaga, M. Leboeuf, C. A. Kyratsous, J. Burton, J. Kalter, A. Klinakis, W. Zhang, G. Thurston, M. Merad, R. M. Steinman, A. J. Murphy, G. D. Yancopoulos, I. Aifantis, and D. Skokos. 2012. Dll4-Notch signaling in Flt3-independent dendritic cell development and autoimmunity in mice. J Exp Med 209: 1011–1028.

315. Michels, A. W., and P. A. Gottlieb. 2018. Learning from past failures of oral insulin trials. Diabetes 67: 1211–1215.

316. Legge, K. L., R. K. Gregg, R. Maldonado-Lopez, L. Li, J. C. Caprio, M. Moser, and H. Zaghouani. 2002. On the role of dendritic cells in peripheral T cell tolerance and modulation of autoimmunity. J Exp Med 196: 217–227.

317. Audiger, C., M. J. Rahman, T. J. Yun, K. V. Tarbell, and S. Lesage. 2017. The importance of dendritic cells in maintaining immune tolerance. J Immunol 198: 2223– 2231.

318. Sallusto, F., and A. Lanzavecchia. 2002. The instructive role of dendritic cells on T- cell responses. Arthritis Res 4 Suppl 3: S127–32.

319. Szypowska, A., A. Stelmaszczyk-Emmel, U. Demkow, and W. Luczyński. 2014. High expression of OX40 (CD134) and 4-1BB (CD137) molecules on CD4(+)CD25(high) cells in children with type 1 diabetes. Adv Med Sci 59: 39–43.

320. Pallotta, M. T., C. Orabona, R. Bianchi, C. Vacca, F. Fallarino, M. L. Belladonna, C. Volpi, G. Mondanelli, M. Gargaro, M. Allegrucci, V. N. Talesa, P. Puccetti, and U. Grohmann. 2014. Forced IDO1 expression in dendritic cells restores immunoregulatory signalling in autoimmune diabetes. J Cell Mol Med 18: 2082–2091.

321. Irvin, A. E., G. Jhala, Y. Zhao, T. S. Blackwell, B. Krishnamurthy, H. E. Thomas, and T. W. H. Kay. 2018. NF-κB is weakly activated in the NOD mouse model of type 1 diabetes. Sci Rep 8: 4217.

322. Schreiber, S., S. Nikolaus, and J. Hampe. 1998. Activation of nuclear factor kappa B inflammatory bowel disease. Gut 42: 477–484.

323. Rogler, G., K. Brand, D. Vogl, S. Page, R. Hofmeister, T. Andus, R. Knuechel, P. A. Baeuerle, J. Schölmerich, and V. Gross. 1998. Nuclear factor kappaB is activated in macrophages and epithelial cells of inflamed intestinal mucosa. Gastroenterology 115: 357–369.

324. Ramakrishnan, P., M. A. Yui, J. A. Tomalka, D. Majumdar, R. Parameswaran, and D. Baltimore. 2016. Deficiency of Nuclear Factor-κB c-Rel Accelerates the Development of Autoimmune Diabetes in NOD Mice. Diabetes 65: 2367–2379.

325. Wang, T., W. Shi, T. Fan, X. Wan, Y. H. Chen, and Q. Ruan. 2016. c-Rel is Required for the Induction of pTregs in the Eye but Not in the Gut Mucosa. Immunol Invest 45: 776–786.

189

326. Ruan, Q., V. Kameswaran, Y. Tone, L. Li, H.-C. Liou, M. I. Greene, M. Tone, and Y. H. Chen. 2009. Development of Foxp3(+) regulatory t cells is driven by the c-Rel enhanceosome. Immunity 31: 932–940.

327. Visekruna, A., M. Huber, A. Hellhund, E. Bothur, K. Reinhard, N. Bollig, N. Schmidt, T. Joeris, M. Lohoff, and U. Steinhoff. 2010. c-Rel is crucial for the induction of Foxp3(+) regulatory CD4(+) T cells but not T(H)17 cells. Eur J Immunol 40: 671–676.

328. Hori, S. 2010. c-Rel: a pioneer in directing regulatory T-cell lineage commitment? Eur J Immunol 40: 664–667.

329. Anquetil, F., G. Mondanelli, N. Gonzalez, T. Rodriguez Calvo, J. Zapardiel Gonzalo, L. Krogvold, K. Dahl-Jørgensen, B. Van den Eynde, C. Orabona, U. Grohmann, and M. G. von Herrath. 2018. Loss of IDO1 Expression From Human Pancreatic β-Cells Precedes Their Destruction During the Development of Type 1 Diabetes. Diabetes 67: 1858–1866.

330. Moffett, J. R., and M. A. Namboodiri. 2003. Tryptophan and the immune response. Immunol Cell Biol 81: 247–265.

331. Lee, G. K., H. J. Park, M. Macleod, P. Chandler, D. H. Munn, and A. L. Mellor. 2002. Tryptophan deprivation sensitizes activated T cells to apoptosis prior to cell division. Immunology 107: 452–460.

332. Pallotta, M. T., C. Orabona, C. Volpi, C. Vacca, M. L. Belladonna, R. Bianchi, G. Servillo, C. Brunacci, M. Calvitti, S. Bicciato, E. M. C. Mazza, L. Boon, F. Grassi, M. C. Fioretti, F. Fallarino, P. Puccetti, and U. Grohmann. 2011. Indoleamine 2,3-dioxygenase is a signaling protein in long-term tolerance by dendritic cells. Nat Immunol 12: 870– 878.

333. Fallarino, F., U. Grohmann, and P. Puccetti. 2012. Indoleamine 2,3-dioxygenase: from catalyst to signaling function. Eur J Immunol 42: 1932–1937.

334. Nathan, D. M., and DCCT/EDIC Research Group. 2014. The diabetes control and complications trial/epidemiology of diabetes interventions and complications study at 30 years: overview. Diabetes Care 37: 9–16.

335. Vehik, K., D. Cuthbertson, H. Ruhlig, D. A. Schatz, M. Peakman, J. P. Krischer, and DPT-1 and TrialNet Study Groups. 2011. Long-term outcome of individuals treated with oral insulin: diabetes prevention trial-type 1 (DPT-1) oral insulin trial. Diabetes Care 34: 1585–1590.

336. Wolf, J. L., R. S. Kauffman, R. Finberg, R. Dambrauskas, B. N. Fields, and J. S. Trier. 1983. Determinants of reovirus interaction with the intestinal M cells and absorptive cells of murine intestine. Gastroenterology 85: 291–300.

190

337. Huarte, E., A. Rynda-Apple, C. Riccardi, J. A. Skyberg, S. Golden, M. F. Rollins, A. G. Ramstead, L. O. Jackiw, M. Maddaloni, and D. W. Pascual. 2011. Tolerogen-induced interferon-producing killer dendritic cells (IKDCs) protect against EAE. J Autoimmun 37: 328–341.

338. Nutsch, K., J. N. Chai, T. L. Ai, E. Russler-Germain, T. Feehley, C. R. Nagler, and C.-S. Hsieh. 2016. Rapid and efficient generation of regulatory T cells to commensal antigens in the periphery. Cell Rep 17: 206–220.

339. Schmidt, A., N. Oberle, and P. H. Krammer. 2012. Molecular mechanisms of treg- mediated T cell suppression. Front Immunol 3: 51.

340. Tarbell, K. V., L. Petit, X. Zuo, P. Toy, X. Luo, A. Mqadmi, H. Yang, M. Suthanthiran, S. Mojsov, and R. M. Steinman. 2007. Dendritic cell-expanded, islet- specific CD4+ CD25+ CD62L+ regulatory T cells restore normoglycemia in diabetic NOD mice. J Exp Med 204: 191–201.

341. Mokdad-Gargouri, R., S. Abdelmoula-Soussi, N. Hadiji-Abbès, I. Y.-H. Amor, I. Borchani-Chabchoub, and A. Gargouri. 2012. Yeasts as a tool for heterologous gene expression. Methods Mol Biol 824: 359–370.

342. Kent, S. C., S. I. Mannering, A. W. Michels, and J. A. B. Babon. 2017. Deciphering the Pathogenesis of Human Type 1 Diabetes (T1D) by Interrogating T Cells from the “Scene of the Crime”. Curr Diab Rep 17: 95.

343. Mannering, S. I., F. S. Wong, I. Durinovic-Belló, B. Brooks-Worrell, T. I. Tree, C. M. Cilio, N. C. Schloot, R. Mallone, and Immunology of Diabetes Society T-Cell Workshop Committee. 2010. Current approaches to measuring human islet-antigen specific T cell function in type 1 diabetes. Clin Exp Immunol 162: 197–209.

344. Bresson, D., L. Togher, E. Rodrigo, Y. Chen, J. A. Bluestone, K. C. Herold, and M. von Herrath. 2006. Anti-CD3 and nasal proinsulin combination therapy enhances remission from recent-onset autoimmune diabetes by inducing Tregs. J Clin Invest 116: 1371–1381.

345. Ludvigsson, J. 2009. Therapy with GAD in diabetes. Diabetes Metab Res Rev 25: 307–315.

346. Sgouroudis, E., A. Albanese, and C. A. Piccirillo. 2008. Impact of protective IL-2 allelic variants on CD4+ Foxp3+ regulatory T cell function in situ and resistance to autoimmune diabetes in NOD mice. J Immunol 181: 6283–6292.

347. Noble, J. A., and H. A. Erlich. 2012. Genetics of type 1 diabetes. Cold Spring Harb Perspect Med 2: a007732.

191

348. Caminiti, L., G. B. Pajno, G. Crisafulli, F. Chiera, M. Collura, G. Panasci, P. Ruggeri, F. Guglielmo, and G. Passalacqua. 2015. Oral Immunotherapy for Egg Allergy: A Double-Blind Placebo-Controlled Study, with Postdesensitization Follow-Up. J Allergy Clin Immunol Pract 3: 532–539.

349. Longo, G., E. Barbi, I. Berti, R. Meneghetti, A. Pittalis, L. Ronfani, and A. Ventura. 2008. Specific oral tolerance induction in children with very severe cow’s milk-induced reactions. J Allergy Clin Immunol 121: 343–347.

350. Clark, A. T., S. Islam, Y. King, J. Deighton, K. Anagnostou, and P. W. Ewan. 2009. Successful oral tolerance induction in severe peanut allergy. Allergy 64: 1218–1220.

351. Gottlieb, P. A., T. Delong, R. L. Baker, L. Fitzgerald-Miller, R. Wagner, G. Cook, M. R. Rewers, A. Michels, and K. Haskins. 2014. Chromogranin A is a T cell antigen in human type 1 diabetes. J Autoimmun 50: 38–41.

352. Bermúdez-Humarán, L. G. 2009. Lactococcus lactis as a live vector for mucosal delivery of therapeutic proteins. Hum Vaccin 5: 264–267.

353. Casalta, E., and M.-C. Montel. 2008. Safety assessment of dairy microorganisms: the Lactococcus genus. Int J Food Microbiol 126: 271–273.

354. Ferris, S. T., J. A. Carrero, J. F. Mohan, B. Calderon, K. M. Murphy, and E. R. Unanue. 2014. A minor subset of Batf3-dependent antigen-presenting cells in islets of Langerhans is essential for the development of autoimmune diabetes. Immunity 41: 657–669.

355. Madhavan, T. P. V., J. D. Riches, M. J. Scanlon, G. C. Ulett, and H. Sakellaris. 2016. Binding of CFA/I Pili of Enterotoxigenic Escherichia coli to Asialo-GM1 Is Mediated by the Minor Pilin CfaE. Infect Immun 84: 1642–1649.

192

BIOGRAPHICAL SKETCH

Andrew Nelson received his Ph.D. from the University of Florida in the fall of

2018. Prior to doctoral study, he earned a Bachelor of Science degree in microbiology from South Dakota State University in Brookings, SD, USA. His degree is specialized in infectious diseases and he minored in biology and chemistry.

193