IDENTIFICATION OF A NOVEL POST-TRANSLATIONALLY MODIFIED ANTIGEN IN THE NOD MOUSE MODEL OF AUTOIMMUNE DIABETES TIMOTHY A. WILES

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1 IDENTIFICATION OF A NOVEL POST-TRANSLATIONALLY MODIFIED ANTIGEN IN THE NOD MOUSE MODEL OF AUTOIMMUNE DIABETES by TIMOTHY A. WILES B.S., University of Evansville, 2010 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Immunology Program 2017

2 This thesis for the Doctor of Philosophy degree by Timothy A. Wiles has been approved for the Immunology Program by Jill Slansky, Chair Howard Davidson Jordan Jacobelli Nichole Reisdorph Lawrence Wysocki Kathryn Haskins, Mentor Date: May 19, 2017 ii

3 Wiles, Timothy A. (Ph.D., Immunology) Identification of a Novel Post-Translationally Modified Antigen in the NOD Mouse Model of Autoimmune Diabetes Thesis directed by Professor Kathryn Haskins ABSTRACT Autoimmunity arises from a failure of the immune system to establish or maintain self-tolerance. In the thymus, developing T cells are exposed to self-peptides derived from genetically encoded proteins, and self-reactive T cells are either deleted or develop a regulatory phenotype before venturing into the periphery. Post-translational modification (PTM) of self-peptides in the periphery may lead to the generation of neo-epitopes that are not displayed in the thymus. T cells specific for these modified peptides can bypass central tolerance mechanisms and escape into the periphery where they may potentially contribute to an autoimmune response. PTM of proteins is well documented in several autoimmune diseases (e.g., rheumatoid arthritis, multiple sclerosis, celiac disease), but has been little characterized in autoimmune diabetes. To facilitate the identification of autoantigens relevant to this disease, we have used a panel of islet-reactive, diabetogenic CD4 T cell clones isolated from spontaneously diabetic NOD mice. In this work, we demonstrate that the antigen for one of these clones, BDC-6.9, is a member of a novel class of post-translationally modified peptides we term hybrid insulin peptides (HIPs). The BDC-6.9 antigen, 6.9HIP, is formed by fusion of a normal cleavage product of islet amyloid polypeptide to the C- terminus of an insulin C-peptide fragment. Characterization of the CD4 T cell response to this antigen in the NOD mouse suggests an important role for HIPs as antigens in autoimmune diabetes. These findings hold great potential for improving our understanding of iii

4 the etiology and pathogenesis of autoimmune diabetes, promoting the development of disease biomarkers, and informing the design of disease interventions. The form and content of this abstract are approved. I recommend its publication. Approved: Kathryn Haskins iv

5 ACKNOWLEDGMENTS I express gratitude to Katie Haskins, who has been an exceptional and attentive mentor. She has exhibited wisdom and foresight in directing my work. Katie has demonstrated a personal interest in my training as a scientist, and I have observed her very selflessly dedicate considerable time and energy to helping other scientists both fledgling and experienced further their careers. I thank Thomas Delong, whose contagious enthusiasm, eternal optimism, and creative brilliance were the crucible for the discovery of hybrid insulin peptides; Rocky Baker for all of his teaching, direction, and discussion; Brenda Bradley for managing the lab, maintaining the mouse colony, and helping with experimental design and execution; Gene Barbour for being the cell culture expert and always being there to lend a hand; Janet Wenzlau, who is always around when you need to hear something positive; and all other members of the lab, past and present. I also thank all of the student workers who have helped over the years. I am grateful to my committee for their guidance throughout this process. I thank Roger Powell and Kevin Quinn for assistance with chromatography and mass spectrometry experiments and for their willingness to even come in late on a Friday night to salvage a critical experiment. Maki Nakayama first identified the peptide B:9-23(13Q) as a possible mimotope for BDC-6.9 and therefore played an important role in discovery of the BDC-6.9 hybrid insulin peptide. I thank Raul Torres and Ross Kedl for directing the graduate program and always being so friendly, encouraging, and approachable. I also thank Ross Kedl for allowing free use of his CyAn flow cytometer. I thank Phillip Pratt for islet isolation. I dedicate this work to my grandfather, Jimmie Daniel, one of the greatest men I know. v

6 This work was supported by the National Institutes of Health [R01 DK (KH)], the Juvenile Diabetes Research Foundation [ (KH)], and the American Diabetes Association [1-14-BS-089 (KH), 1-15-ACE-14 (TD), and 1-15-JF-04 (RB)]. The NIH tetramer core graciously provided tetramer reagents at no charge. vi

7 TABLE OF CONTENTS CHAPTER I. INTRODUCTION Type 1 diabetes (T1D)... 1 The non-obese diabetic (NOD) mouse... 1 MHC class II and autoimmune diabetes... 2 CD4 T cells in autoimmune diabetes... 3 The BDC panel of diabetogenic CD4 T cell clones... 4 A role for Th1 cells in pathogenesis... 6 Antigens for autoreactive CD4 T cells in autoimmune diabetes... 7 Heat-shock protein 60 (Hsp60)... 7 Glutamic acid decarboxylase 65 (GAD65)... 8 A primary antigen in T1D?... 8 Insulin as an autoantigen... 9 Proinsulin processing... 9 Insulin-reactive CD4 T cell clones The insulin B:9-23 epitope Other insulin epitopes The importance of insulin as an autoantigen Antigen-specific tolerance induction as a treatment for diabetes Post-translationally modified antigens in autoimmune diabetes Antigens for the BDC T cell clones vii

8 The BDC-6.9 antigen Overall objective of thesis II. GENERAL METHODS Mice Beta membrane (β-mem) preparation Culture of T cell clones Islet isolation Peptides T cell antigen assays Adoptive transfer of activated CD4 T cell clones III. BDC-6.9 AND IAPP PEPTIDES Background and rationale Specialized methods Mice RIP-TAg cells and RT-ftlys Citrullination of synthetic peptides Transglutaminase-mediated deamidation of synthetic peptides Oxidation of peptidyl methionine using H 2 O Mass spectrometric analysis of peptides Results Natural antigen sources for BDC IAPP peptides viii

9 Post-translationally modified IAPP peptides NOD IAPP Summary and conclusions IV. SEARCHING FOR THE BDC-6.9 ANTIGEN BY MASS SPECTROMETRY Background and rationale Specialized methods Size exclusion chromatography (SEC) High-performance liquid chromatography (HPLC) Mass spectrometry Analysis of mass spectrometry data Results Chromatography Mass spectrometric analysis of antigenic chromatographic fractions Comparing the distributions of peptides and the BDC-6.9 antigen Analysis of tryptically digested SEC fractions Summary and conclusions V. A BDC-6.9 MIMOTOPE Background and rationale Specialized methods Tolerance induction with Ag-SP Statistical analysis ix

10 Results B:9-23(13Q) Truncations Positional/alanine scanning Register trapping Inducing tolerance to B:9-23(13Q) Summary and conclusions VI. THE BDC-6.9 HYBRID INSULIN PEPTIDE (6.9HIP) Background and rationale Specialized methods Peptide cross-linking CFSE proliferation assay with transgenic T cells I-Ag7 peptide binding assay Results The BDC-2.5 hybrid insulin peptide (2.5HIP) B:9-23(13Q) and homology to natural sequence Antigenicity of an insulin-iapp hybrid peptide for BDC BALB/c 6.9HIP HIP truncation and mutational analysis I-Ag7 binding Summary and conclusions x

11 VII. DETECTING 6.9HIP IN BETA CELLS Background and rationale Specialized methods Antigen purification Mass spectrometry Results Identification of 6.9HIP in antigenic fractions by mass spectrometry Confirming identification of 6.9HIP by tandem mass spectrometry Summary and conclusions VIII. 6.9HIP-REACTIVE CD4 T CELLS IN DISEASE Background and rationale Specialized methods Tetramer analysis Intracellular staining Isolation of BDC-9.H Statistics Results The 6.9HIP tetramer (6.9HIP-tet) Prevalence of 6.9HIP-tet + cells in NOD mice Phenotype of 6.9HIP-tet + cells in NOD mice Isolation of a 6.9HIP-reactive CD4 T cell line Summary and conclusions xi

12 IX. SUMMARY AND DISCUSSION Summary The importance of HIPs as antigens in autoimmune diabetes The mechanism of HIP formation HIPs and diabetes-associated MHC class II molecules HIPs and human disease Identifying hybrid peptides in T1D and other autoimmune diseases Significance of the discovery of HIPs in T1D REFERENCES xii

13 LIST OF ABBREVIATIONS ABTS... 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) Ag-SP... antigen-coupled spleen cells APC... antigen presenting cell BDC... Barbara Davis Center beta-mem (β-mem)... beta-membrane CD... cluster of differentiation CFSE... carboxyfluorescein succinimidyl ester ChgA... chromogranin A CID... collision-induced dissociation CLIP... class II-associated invariant chain peptide CPE... carboxypeptidase E DMEM... Dulbecco s modified Eagle s medium DTT... dithiotreitol EDC... 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDTA... ethylene diamine tetraacetic acid EIC... extracted ion chromatogram ELISA... enzyme-linked immunosorbent assay ELISPOT... enzyme-linked immunospot ESI... electrospray ionization FBS... fetal bovine serum FoxP3... forkhead box P3 GABA... γ-aminobutyric acid GAD... glutamic acid decarboxylase GRP kda glucose-regulated protein HBSS... Hank s balanced salt solution HEL... hen egg lysozyme HEPES... 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid HIP... hybrid insulin peptide HLA... human leukocyte antigen HPLC... high-performance liquid chromatography HSP... heat shock protein I... ionomycin IA-2... insulinoma-associated protein 2 IAM... iodoacetamide IAPP... islet amyloid polypeptide IAPP1... IAPP propeptide 1 IAPP2... IAPP propeptide 2 IC... islet cells ICA69... islet cell autoantigen of 69kDa IL... interleukin IFN-γ... interferon- γ INS-SP... insulin-coupled spleen cells MHC... major histocompatibility complex MPP... Mass Profiler Professional software (Agilent) xiii

14 NOD... non-obese diabetic mouse PAD... peptidyl-arginine deiminase PAM... peptidylglycine alpha-amidating monooxygenase PBMC... peripheral blood mononuclear cell PBS... phosphate-buffered saline PC1... prohormone convertase 1 PC2... prohormone convertase 2 PD-L1... programmed death-ligand 1 PMA... phorbol 12-myristate 13-acetate PTM... post-translational modification Q-TOF... quadrapole time-of-flight RA... rheumatoid arthritis RIP... rat insulin promoter RT-ftlys... RIP-TAg freeze-thaw lysate SD... standard deviation SEC... size exclusion chromatography SOD... superoxide dismutase sulfo-nhs... N-hydroxysulfosuccinimide T1D... type 1 diabetes TAg... SV40 large T antigen TCEP... tris-(2-carboxyethyl)phosphine TCR... T cell receptor tet... tetramer TFA... trifluoroacetic acid TFE... trifluoroethanol Tg... transgenic TGase... transglutaminase Th1... type 1 T helper cell Th2... type 2 T helper cell TIC... total ion chromatogram xiv

15 CHAPTER I INTRODUCTION Type 1 diabetes (T1D) Type 1 diabetes (T1D) is a human autoimmune disease characterized by specific ablation of the insulin-producing beta cells of the pancreatic islets by infiltrating immune cells [1]. The consequent loss of insulin production results in chronic hyperglycemia. In this setting, cells suffer from starvation in the midst of plenty, as they are unable to uptake sufficient glucose for cellular metabolism even when this vital resource is plentiful in the blood. Prior to the advent of exogenous insulin administration as a treatment in the 1920s, T1D was fatal within weeks of onset [2]. The introduction of insulin analogs, insulin infusion pumps, continuous glucose monitors, and other advances have drastically improved T1D care. However, the required life-long treatment remains expensive and fails to maintain the level of control afforded by endogenous islet function. Recent progress in the development of closed-loop artificial pancreas systems bears promise [3], but commercial introduction of such a device could still be decades away and would only provide a treatment not a cure for T1D. Although islet transplantation has long been pursued as a cure, limited availability of human islets and the need for immunosuppression remain considerable hurdles [4,5]. If a cure for T1D is to be realized, the problem of persistent autoimmunity must be addressed. The non-obese diabetic (NOD) mouse The foremost animal model for studying the autoimmune process in diabetes is the non-obese diabetic (NOD) mouse [6 9]. NOD mice spontaneously develop a form of autoimmune diabetes very similar to T1D, and many of the autoantigens discovered in the NOD mouse have later been identified as antigens in the human disease [10]. Our laboratory 1

16 has used the NOD mouse for three decades to study the role of CD4 T cells in the pathogenesis and regulation of disease. We have also used the NOD mouse model to identify novel autoantigens in T1D with the aims of better understanding the etiology and T cell drivers of the disease, and with the view to ultimately developing an antigen-specific approach for reversing or even preventing T1D. MHC class II and autoimmune diabetes The strongest genetic association for T1D is with the major histocompatibility complex (MHC) class II locus [11 13], also known as the human leukocyte antigen (HLA) class II locus. In the NOD mouse, the MHC class II molecule I-Ag7 also plays a critical role in the development of disease. For example, spontaneous disease fails to develop in mice deficient in MHC class II expression [14]. The NOD.H2-b mouse, in which the NOD MHC genes (including the gene encoding the MHC class II molecule I-Ag7) have been replaced by the MHC genes of the C57Bl/10 strain, does not develop diabetes [15]. It is important to note, however, that a particular MHC haplotype is not sufficient to confer disease. B6.g7 mice, for example, which express I-Ag7 on the C57Bl/6 background, do not develop spontaneous diabetes [16]. Conventionally, I-Ag7 is conceptualized as having a peptide binding groove capable of accommodating a nine amino acid peptide core in a linear configuration [17 19]. The positions of these nine residues in relation to the I-Ag7 binding groove are referred to as p1- p9. Side chains of residues at p1, p4, p6, and p9 interact with pockets that are present in the groove at these positions. These interactions anchor the peptide to I-Ag7. Side chains of residues at other positions are typically oriented towards the T cell receptor (TCR) and provide TCR contacts [18 21]. A given peptide can potentially bind I-Ag7 in multiple 2

17 registers, with different registers placing the amino acid residues of the peptide in different positions. Numerous studies have established that the p9 pocket of I-Ag7 exhibits a strong preference for residues with negatively charged side chains (i.e., aspartic acid or glutamic acid) [17,20 24]. This preference is conferred by the substitution of a serine residue at position 57 of the I-Ag7 β chain for the conserved aspartic acid residue present at this position in most other MHC class II molecules. This substitution leaves an unpaired arginine in the p9 pocket, which interacts favorably with aspartic or glutamic acid side chains in p9 of bound peptides [20,25]. Interestingly, the disease-associated human HLA class II molecule DQ8 [26 28] expresses a similar variation that likewise confers preference for negatively charged residues in p9 [20,21,29,30]. It has been suggested that post-translational modification of self-peptides could generate neo-epitopes that preferentially bind to these unique MHC class II molecules, thus accounting for their association with disease susceptibility [31]. CD4 T cells in autoimmune diabetes Because MHC class II presents antigens to CD4 T cells, the association of certain MHC class II alleles with disease susceptibility suggests a role for CD4 T cells in pathogenesis. Indeed, CD4 T cells play a critical role in autoimmune diabetes. As a significant advance in the field, our laboratory was the first to isolate CD4 T cell clones from the NOD mouse [32,33] and demonstrate that adoptive transfer of a CD4 T cell clone could induce disease in very young NOD mice, long before spontaneous disease develops [34]. 3

18 The Barbara Davis Center (BDC) panel of diabetogenic CD4 T cell clones The Barbara Davis Center (BDC) T cell clones were so named because of the institution at which they were generated [32,33]. To generate the clones, spleen and lymph nodes were isolated from individual newly diabetic NOD mice, homogenized, and placed in culture with normal mouse serum and, as antigen, fresh dissociated NOD islet cells. Cells were harvested after five days and cultured with irradiated NOD spleen cells as antigen presenting cells (APCs), NOD islet cells as antigen, and supernatant from stimulated EL-4 thymoma cells [35] as a source of IL-2 in medium supplemented with fetal bovine serum. Cells were incubated for two weeks at 37 C and 10% CO 2. Every two weeks, T cells were counted and then restimulated in a fresh culture containing IL-2, islet cells, and APCs. When assay of T cell lines indicated an antigen-specific response, the T cell lines were cloned by limiting dilution. The first number in the name of a BDC clone indicates the diabetic NOD mouse from which the mouse was derived. All of the BDC clones are CD4 + [33],respond to islet cells from NOD mice [33], and are of a Th1 phenotype, producing IFN-γ upon stimulation [36]. Each of the BDC clones, after activation and expansion in culture, is capable of rapidly inducing disease when injected intraperitoneally into NOD pups 8-14 days old [34,37 39]. Most of the BDC clones are also capable of inducing disease when transferred into NOD.scid recipients [36,40]. As NOD.scid mice lack endogenous lymphocytes [41], this observation indicates that islet-specific CD4 T cell clones are capable of causing disease in the absence of CD8 T cells or B cells. T cell clones provide multiple advantages over primary T cells when identifying autoantigens. T cells specific for a particular antigen are few in number among polyclonal cell populations (e.g., in peripheral blood) and therefore can be difficult to detect. 4

19 Similarly, responses observed by T cells from a given patient can change over time as the individual ages, encounters various pathogens, etc. Furthermore, individual mice or patients can differ greatly in terms of T cell repertoire, leading inherently to inconsistency from one sample to the next, a problem that is again circumvented by the use of T cell clones. T cell clones are all of the same specificity and can provide a much more robust response to antigen in vitro. T cell clones are also more practical as reagents than primary cells. Isolation of primary T cells from NOD mice is often a terminal procedure, and isolation from human patients is limited by many factors, such as availability of the patient. Identifying novel autoantigens is a lengthy and involved process that is likely to be unfeasible if approached using primary T cells as a reagent. T cell clones, on the other hand, provide the reliability, consistency, robustness, and flexibility needed for extensive troubleshooting and investigation. Antigens identified using T cell clones can then be studied using primary cells to confirm that they are indeed relevant to disease. To further study CD4 T cells in pathogenesis, T cell receptor transgenic (TCR-Tg) mice have been generated. These mice carry transgenes encoding the α and β chains of the TCR expressed on a particular T cell clone. Thus the majority of T cells that develop in these mice express the transgenic TCR. The BDC-2.5 TCR-Tg mouse [42] carries the transgenes for the TCR of the BDC-2.5 clone and has been crossed with various backgrounds [42,43]. A NOD BDC-6.9 TCR-Tg mouse has also been developed [44]. The BDC-2.5 TCR-Tg mouse in particular has been used widely to study islet-specific CD4 T cells in the NOD mouse model of diabetes and continues to be a very useful asset in the field [45,46]. 5

20 A role for Th1 cells in pathogenesis The observation that all of the clones in the BDC panel of CD4 T cells are of a Th1 phenotype strongly suggests a role for Th1 cells in pathogenesis [36]. One of the primary cytokines produced by Th1 cells is IFN-γ. One study designed to address the role of Th1 cells made use of the BDC-2.5 TCR-Tg mouse. Because allelic exclusion of the TCR α chain is incomplete, T cells expressing the BDC-2.5 TCR β chain but a different α chain can develop in the BDC-2.5 TCR-Tg mouse. The investigators therefore generated a genetic variant strain of the TCR-Tg mouse, BDC-2.5 TCR-Tg Cα null, in which expression of endogenous TCR α chain genes was eliminated [47]. All of the T cells in this mouse therefore expressed the clonotypic BDC-2.5 TCR. Splenocytes from these mice were cultured under different conditions to induce either a Th1 or a Th2 phenotype. Upon transfer into young NOD recipients, only the Th1 cells induced diabetes [47], indicating that Th1 but not Th2 cells are pathogenic in NOD mice. Several studies have addressed the role of the Th1 cytokine IFN-γ in the development of diabetes. Splenocytes from diabetic NOD mice are capable of inducing diabetes when transferred into irradiated NOD recipients, but treatment with an anti-ifn-γ monoclonal antibody prevents this transfer of disease [48]. Another study demonstrated that administration of either anti-ifn-γ monoclonal antibody or soluble IFN-γ receptor can prevent spontaneous disease, although neither treatment protected from insulitis [49]. However, spontaneous diabetes is delayed but not prevented in NOD mice that do not express IFN-γ [50] or the IFN-γ receptor [51]. In another study, it was shown that NOD.scid mice lacking expression of the IFN-γ receptor were protected from transfer of disease by CD4 T cells from BDC-2.5 TCR-Tg Cα null mice, as the beta cells of the recipient mice were 6

21 resistant to IFN-γ-mediated killing [52]. In contrast, these mice were susceptible to disease induction by adoptive transfer of TCR transgenic CD8 T cells, which destroy beta cells primarily through a perforin/granzyme B mechanism [52]. Transfer of disease in IFN-γ receptor deficient recipients was delayed, however, compared to transfer in IFN-γ receptor sufficient mice. Thus, IFN-γ receptor expression on beta cells is necessary for CD4-mediated but not CD8-mediated killing of beta cells. However, due to conflicting results, whether or not IFN-γ is required for development of spontaneous disease remains in question. It is likely that IFN-γ production by Th1 cells plays an important role in pathogenesis, but that, under certain conditions, other mechanisms can compensate in the absence of IFN-γ signaling. Antigens for autoreactive CD4 T cells in autoimmune diabetes Multiple islet antigens have been discovered for CD4 T cells in both human T1D patients and NOD mice [10,53,54], only some of which will be discussed here. Heat-shock protein 60 (Hsp60) The first CD4 T cell autoantigen discovered in the NOD mouse was heat-shock protein 60 (Hsp60) [10,55]. T cells specific for this antigen were first identified based on their cross-reactivity for Hsp65 of Mycobacterium tuberculosis. It was later determined that both the mouse and human Hsp60 peptide p277, which differ by only one amino acid from one another, are antigenic [56,57]. Administration of p277 can prevent spontaneous disease in NOD mice [56,58]. T1D patients show a heightened T cell proliferative response to Hsp60 and Hsp60 peptides (including p277), implicating this protein as an autoantigen in human disease [59]. 7

22 Glutamic acid decarboxylase 65 (GAD65) The enzyme glutamic acid decarboxylase 65 (GAD65), which is involved in the conversion of glutamic acid to the neurotransmitter γ-aminobutyric acid (GABA), is also an antigen for CD4 T cells in both the NOD mouse and in humans [60 66]. The CD4 T cell response to GAD65 can be seen in 4-week-old NOD mice, making it one of the earlier responses to develop [61]. Inducing tolerance to GAD65 can prevent diabetes in NOD mice, suggesting an important role for GAD65-reactive T cells in pathogenesis [61]. GAD65 is expressed in islets and in the brain [10]. Ablation of GAD65 expression in islets prevents disease [67], but global ablation of GAD65 expression has no effect on disease incidence [68]. Thus, it is still uncertain if GAD65 expression is required for disease development. A primary antigen in T1D? A question of great interest is which autoantigens are critical for disease initiation and/or progression. As expression of Hsp60 [57] and GAD65 [10] is not islet-restricted, it is difficult to imagine how development of an autoimmune response to either of these unmodified antigens could be the initiating step in disease. Likewise, expression of most of the antigens identified in autoimmune diabetes thus far is not restricted to the islets, with many of the antigens being expressed in other neuroendocrine tissues or even displaying wide tissue expression [53]. As additional examples, insulinoma-associated protein 2 (IA-2) and islet cell autoantigen of 69kDa (ICA69), both autoantigens for CD4 T cells in human T1D and in the NOD mouse [53], are expressed in the brain as well as in islets [69,70]. Furthermore, genetic ablation of either IA-2 or ICA69 does not prevent spontaneous diabetes in NOD mice [71,72]. In the case of GAD65, it has been shown that some GAD65-reactive T cells are specific for a peptide with high sequence similarity to a Coxsackie B viral protein 8

23 and that these T cells also respond to peptides from the viral protein [73]. These findings suggest that molecular mimicry may account for the loss of tolerance to the self-peptide but still do not explain the islet-specificity of the autoimmune response. Furthermore, subsequent work has indicated that these T cell responses are not restricted to individuals with T1D [74], arguing against a key role for this epitope. Insulin as an autoantigen The dominant candidate for a primary autoantigen in diabetes is insulin [75]. Unlike most other antigens in autoimmune diabetes, insulin is expressed only in the beta cells of the pancreatic islets (and in small amounts in the thymus for the induction of central tolerance) [76]. Insulin produced in the beta cell is stored in secretory granules prior to secretion, where it exists in a dense crystalline core of insulin hexamers coordinated by two zinc ions and one calcium ion [77]. Insulin accounts for roughly half of the total secretory granule protein content [78,79]. It has been estimated that a single beta cell contains roughly two billion molecules of insulin, with each granule containing approximately 200,000 molecules [78,80]. Whereas humans express only one form of insulin, mice and rats express two forms, insulin 1 and insulin 2, encoded by different genes [81]. The insulin 2 gene, located on chromosome 7, is the ortholog of the human insulin gene [81]. The insulin 1 gene, located on chromosome 19 [82], is thought to have arisen from a duplication event involving the insulin 2 gene and is lacking the second intron [81,83,84]. Both forms of insulin are expressed in the islets, though evidence regarding the relative expression of the two forms is conflicting [85]. Proinsulin processing After removal of the signal peptide from the pre-proinsulin molecule to generate proinsulin, several steps occur to generate mature hormonally-active insulin [53,86]. 9

24 A schematic of the proinsulin molecule is provided (Fig. 1). Proinsulin is divided into three chains. In order from the N-terminus to the C-terminus, they are B-chain, C-peptide, and A- chain. Prohormone convertase 1 (PC1; also known as PC3) cleaves insulin at the B-chain/Cpeptide junction [87], and PC2 cleaves at the C-peptide/A-chain junction [88]. It has been suggested that each enzyme has the ability to cleave insulin at both sites to some extent, as some mature insulin is still generated in the absence of functional PC1 or PC2 [89 91]. Each of these endoproteases cleaves the prohormone following dibasic residues. The dibasic residues are removed from the C-terminus of B-chain and C-peptide by carboxypeptidase E (CPE; formerly known as carboxypeptidase H) [92]. The B-chain and A-chain remain linked as the mature insulin hormone by two disulfide bridges: one between a cysteine residue at position 7 in each chain and another between a cysteine at position 19 in the B-chain and one in position 20 in the A-chain [93]. A disulfide bridge also forms between cysteine residues at positions 6 and 11 of the insulin A-chain [93]. The proinsulin 1 and 2 sequences are very similar. Proinsulin 1 is 84 residues long and proinsulin 2 is 86 residues long. The A-chains are identical between the two molecules and the B-chains differ by only two residues [94 96]. Insulin 2 C-peptide contains two additional residues compared to insulin 1 C-peptide and differs at three other residues [94 96]. Human proinsulin is also very similar, differing from mouse proinsulin 2 by two residues in the B-chain, nine in C-peptide, and one in the A-chain [96,97]. Insulin-reactive CD4 T cell clones As discussed earlier, the development of diabetogenic CD4 T cell clones was an important advance in the study of autoimmune diabetes. Several years after the isolation of the BDC clones, Wegmann et al generated T cell lines from NOD mice by expanding 10

25 H 2 N - B-chain A-chain - COOH C-peptide Figure 1: The proinsulin molecule. A schematic of human proinsulin, with each square representing an amino acid residue. The representation is also accurate for the mouse insulin 2 molecule. Proinsulin consists of B-chain (light gray) and A-chain (dark gray) connected by C-peptide (white). The N-terminus and C-terminus are indicated. Endoprotease cleavage sites are indicated by dashed lines. Prohormone convertase 1 (PC1) cleaves between B-chain and C-peptide and prohormone convertase 2 (PC2) cleaves between C-peptide and A-chain. Dibasic residues (squares marked with an X) are removed by carboxypeptidase E (CPE). The mature insulin hormone consists of B-chain linked by two disulfide bridges (indicated by solid lines) to A-chain, which contains an intramolecular disulfide bridge. 11

26 islet-infiltrating T cells in culture using irradiated islet cells as an antigen source and irradiated NOD splenocytes as APCs [98]. Two lines were generated from NOD mice 7 weeks of age (little islet infiltration) and two were generated from mice 12 weeks of age (extensive islet infiltration). Each of the four lines was generated from a separate mouse. A proliferative response was seen by all four of the lines when cultured with either NOD islet cells or porcine insulin in the presence of irradiated NOD splenocytes as APCs [98]. When T cell clones were generated from the four lines, 24 out of 54 clones responded to insulin, with a much higher percentage of the clones isolated from 12-week-old mice being insulinreactive [98]. Ten of the insulin-reactive clones were analyzed further by antibody staining and flow cytometry. All of the clones were CD4 + α/β T cells. At least three different TCR β- chain variable regions (Vβ) were represented in the panel, indicating that the clones did not arise strictly from expansion of a single clone [98]. These findings were the first demonstration of islet-reactive CD4 T cell clones being specific for insulin and suggested that insulin-reactive cells are frequent in the pancreatic infiltrate of 12-week-old but not 7- week old NOD mice. The insulin B:9-23 epitope In order to determine the precise insulin epitope being recognized, the same group screened six of the insulin-reactive CD4 T cell clones isolated from 12-week-old NOD mice against a panel of synthetic peptides 15 amino acids in length spanning insulin A-chain and B-chain. All six of the clones responded exclusively to the B-chain peptide B:9-23 (SHLVEALYLVCGERG) [99]. The clones responded equally to the B:9-23 sequence from insulin 1 and insulin 2, which differ only by the most N-terminal amino acid (proline in insulin 1 and serine in insulin 2). Several heterogeneous islet-reactive T cell lines isolated in 12

27 the same fashion as the CD4 T cell clones were screened against the same peptide panel. A response to B:9-23 but no other peptides was detected with each of the lines, indicating that B:9-23 is a dominant insulin epitope for CD4 T cells in prediabetic NOD mice [99]. Five of the six clones secreted IFN-γ but not IL-4 in response to B:9-23, indicating that, like the BDC clones, they were of a Th1 phenotype. Each of the six clones was capable of inducing overt diabetes within four weeks when injected intraperitoneally into 6-14-day-old NOD mice, although incidence of diabetes following transfer varied greatly from clone to clone [99]. The ability of these clones to accelerate diabetes suggests a pathogenic role for B:9-23- reactive CD4 T cells. The human B:9-23 sequence, which is identical to the insulin 2 B:9-23 sequence in mice, is also an antigen for CD4 T cells in human T1D. The first demonstration of this was in 2001 by Alleva et al [100]. In this study, short-term T cell lines were established from the peripheral blood of new-onset T1D patients (defined as being within three months of diagnosis) and non-diabetic controls (age- or HLA-matched). The cell lines were screened for a proliferative response to B:9-23 peptide in the presence of irradiated autologous peripheral blood mononuclear cells (PBMCs) as APCs. T cells from 10 out of 12 new-onset patients, but none of the 13 control subjects, responded to B:9-23 [100]. It was also determined that B:9-23 binds the disease-associated MHC class II allele DQ8 with high affinity and that an anti-hla-dq antibody could block the response of a line to the peptide, thus indicating that the response was MHC class II-restricted [100]. ELISPOT assays of freshly isolated PBMCs were used to determine that T cells from seven out of eight T1D patients produced IFN-γ but not IL-2, IL-4, IL-5, or IL-13 in response to B:9-23 peptide [100]. PBMCs from 23 non-diabetic controls were also tested, none of which produced 13

28 cytokines in response to B:9-23. As all of the T1D patients had received insulin treatment, it remained possible that the response to B:9-23 was an artifact of an immune response developed to exogenous insulin. However, IFN-γ-secreting B:9-23-reactive cells were detected in PBMCs from three out of five non-diabetic individuals who had never received exogenous insulin but who were at high risk for developing diabetes (defined as being a firstdegree relative of someone with T1D and expressing a disease associated HLA-haplotype), arguing against this explanation [100]. Since the discovery of B:9-23 as a CD4 T cell antigen in NOD and human diabetes, the response to this epitope has been the subject of extensive study. Because insulin is expressed in both beta cells and the thymus, and since B:9-23 is an unmodified insulin peptide, it would seem that self-tolerance mechanisms would prevent the development of an autoimmune response to this epitope. It has been demonstrated that some B:9-23-reactive T cells recognize the peptide only when presented by I-Ag7 in a register (register 3) that is unfavorable for binding of the peptide to I-Ag7 [19]. Register three places an arginine residue, which carries a positively-charged side chain, in p9 of the binding register. As discussed previously, the p9 pocket of I-Ag7 is defined by an unpaired, positively-charged arginine residue and displays a preference for amino acids with negatively-charged side chains. As the arginine side chain of the B:9-23 peptide clashes with the positive charge in the p9 pocket, the peptide binds only weakly in register 3. It was suggested that inefficient thymic presentation of B:9-23 in register 3 allows CD4 T cells specific for the peptide presented in this register to elude the central tolerance checkpoint [19]. Two different fluorescently-labeled MHC class II tetramer reagents were developed in which I-Ag7 was loaded with B:9-23 variants designed for improved binding in register 3 [101]. 14

29 These reagents were able to stain insulin-reactive CD4 T cell hybridomas and were used to detect B:9-23-reactive cells in the prediabetic NOD pancreas by flow cytometry [101]. We also made use of one of these tetramers, which we refer to as insp8g-tet, in some of our studies, as will be shown in Chapter VIII of this thesis. It was later shown using a similar HLA-DQ8 tetramer reagent that CD4 T cells specific for B:9-23 bound in register 3 are also present in the blood of T1D patients but not healthy controls [102]. CD4 T cell clones specific for B:9-23 bound in register 3 were isolated from several of the T1D patients. These clones also responded to four different peptides, derived from microbial proteins, that share homology with B:9-23 [102]. Based on this observation, the authors suggested that exposure to certain microbes could elicit a T cell response to B:9-23. Other insulin epitopes Although B:9-23 has been the most studied insulin epitope in diabetes, other insulin peptides have also been identified as CD4 T cell epitopes. The insulin 2 B chain sequence VKQHLCGSHLVEALYL has been shown to be antigenic for CD4 T cells infiltrating the islets of 14-week-old female NOD mice [103]. CD4 T cells from either the spleen or pancreatic lymph nodes of 3-4-week-old NOD mice responded to the insulin 2 peptide FFYTPMSRRE, which spans the B-chain/C-peptide junction [104]. Another study demonstrated that PBMCs from human subjects at risk for developing T1D, but not from control subjects, responded to a homologous human proinsulin peptide (FFYTPKTRREAED) [105]. The insulin A-chain has also been shown to contain a human CD4 T cell epitope. A CD4 T cell clone isolated from a type 1 diabetic subject was shown to recognize the A-chain peptide KRGIVEQCCTSICS [106]. The dibasic residues, lysine and arginine, which originate from C-peptide, were requisite for activation of the clone. 15

30 The insulin 1 C-peptide sequence SPGDLQTLALEVARQK has been shown to be antigenic for CD4 T cells infiltrating the islets of 14-week-old female NOD mice [103]. This region of C-peptide has also been implicated as an epitope in human T1D by a study in which CD4 T cell clones were isolated from the residual islets of a deceased organ donor who had been diagnosed with T1D three years prior [107]. Almost all of the clones were capable of producing IFN-γ. The clones were screened for reactivity to synthetic peptide panels representing multiple known islet autoantigens, including HSP60, GAD65, insulin B- chain, insulin C-peptide, and insulin A-chain. Out of 53 clones, 14 responded to a peptide from the C-peptide region QVELGGGPGAGSLQPLALEGSL [107]. Surprisingly, none of the clones responded to any of the other peptides tested, including a peptide spanning the entirety of the B:9-23 sequence. To confirm this region of C-peptide as an epitope, PBMCs were isolated from eight individuals diagnosed with T1D within the previous three months and six healthy control subjects. Individuals in both groups were HLA-DQ8 +. PBMCs from three of the T1D patients but none of the control subjects responded to the peptide PGAGSLQPLALEGSL [107]. The importance of insulin as an autoantigen Several studies have attempted to determine if insulin is a critical autoantigen in diabetes. It has been shown that diabetes incidence is greatly reduced in mice lacking the insulin 1 gene [108], whereas mice lacking the insulin 2 gene show highly accelerated diabetes [109]. One study has indicated that only insulin 2 expression is detectable in the thymus at the transcript level [110]. Thus, deletion of the insulin 2 gene may prevent induction of central tolerance to insulin in the thymus, explaining the observed acceleration in disease. 16

31 The findings by Wegmann et al and Alleva et al indicate that B:9-23 is a CD4 T cell epitope in autoimmune diabetes in humans and in the NOD mouse model. As discussed earlier, Th1 cells seem to be important contributors to the pathogenesis of diabetes. The observations that B:9-23-reactive cells in both mice and humans produce IFN-γ and that IFNγ-producing B:9-23-reactive cells can accelerate disease in young NOD mice indicate that CD4 T cells specific for the B:9-23 epitope play an important role in pathogenesis. The most compelling evidence for B:9-23 as a critical epitope in diabetes came from Nakayama et al in Knock-out mice lacking both insulin 1 and insulin 2 were generated, and a mutated insulin transgene was inserted into these mice [111]. The genetic mutation resulted in a single amino acid substitution within the B:9-23 sequence that did not alter the hormonal activity of insulin but destroyed the critical CD4 T cell epitope. These mice failed to develop autoimmune diabetes, suggesting that expression of B:9-23 is required for development of autoimmune diabetes in the NOD mouse [111]. Antigen-specific tolerance induction as a treatment for diabetes An ultimate goal of identifying autoantigens in the NOD mouse is to target the immune response to these antigens as a treatment for disease. Antigen-specific tolerance induction is a preferred treatment for autoimmunity because it can specifically target the autoreactive component of the immune response while leaving the remainder intact to combat infection and other threats. Furthermore, antigen-specific strategies can potentially avoid some of the harsh side effects of more global treatments. Several approaches to inducing tolerance to islet antigens have been investigated for efficacy in T1D [112]. One approach that has been successful in treating several mouse 17

32 models of immune-mediated diseases is antigen-coupled spleen cell (Ag-SP) therapy [113,114]. Ag-SP therapy has been very successful as an antigen-specific approach for treating allergy [115], transplant rejection [116], and autoimmunity [117] in mouse models. In this approach, protein or peptide antigen is chemically cross-linked to splenocytes using the chemical agent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and the resulting Ag-SP are injected intravenously. A mechanism for tolerance induction by this treatment has been proposed [114,118]. The chemical cross-linking process induces apoptosis in the coupled cells. After injection, the cells undergo scavenger receptor-mediated uptake by host APCs. These APCs in turn produce IL-10 and increase expression of the regulatory molecule PD-L1. Presentation of the coupled antigen in this tolerogenic context favors induction of anergy in antigen-specific T cells and promotes the generation of regulatory T cells. Though limited, some work has been done demonstrating the efficacy of this approach in treating autoimmune diabetes in the NOD mouse [119,120]. Administration of insulin-coupled spleen cells (INS-SP) has been shown to reverse spontaneous diabetes in NOD mice, but only with a 50% rate of success [119]. Prevention of spontaneous disease could be accomplished with either INS-SP or B:9-23-SP when administered to mice 4-6 weeks of age but only INS-SP could prevent disease when administered at weeks of age [120]. The authors suggested that in older mice epitope spreading had diversified the epitopes being targeted by autoimmunity, necessitating the induction of tolerance to a broader spectrum of epitopes to accomplish disease prevention [120]. By the time that overt disease has developed, it is likely that epitope spreading has advanced even further in some mice, thus it may be necessary to induce tolerance to multiple autoantigens in order to reverse disease in all mice. 18

33 Post-translationally modified antigens in autoimmune diabetes As the CD4 T cell autoantigens discussed thus far are unmodified self-peptides, it is difficult to imagine how autoreactivity to these peptides could be the main driver of disease. In the thymus, developing T cells are exposed to self-peptides derived from genetically encoded proteins, and self-reactive T cells are either deleted or develop a regulatory phenotype before venturing into the periphery [ ]. It has been suggested that the posttranslational modification (PTM) of self-peptides in the periphery generates neo-epitopes that could act as critical antigens in autoimmune disease. As these modified peptides would not likely be expressed in the thymus, T cells specific for these modified peptides could bypass central tolerance mechanisms, escape into the periphery, and potentially contribute to an autoimmune response [ ]. PTM of proteins is well documented in several autoimmune diseases, including rheumatoid arthritis, multiple sclerosis, and celiac disease. The exploration of modified proteins as autoantigens in diabetes, however, is still in its infancy, though some examples of modified antigens have been identified [128]. For example, CD4 T cell clones isolated from a human donor with T1D were found to recognize a modified insulin A-chain peptide [129]. A disulfide bond between adjacent residues in the peptide was required for recognition by the clones. A PTM that has been studied extensively in the context of the autoimmune disease rheumatoid arthritis is citrullination. Citrullination is the deimination of arginine to form the modified amino acid citrulline and is catalyzed by the enzyme peptidyl arginine deiminase (PAD). Autoantibodies directed toward citrullinated self proteins are well documented as a hallmark of rheumatoid arthritis (RA) [ ], and T cell responses to citrullinated peptides have been documented in a mouse model of RA [133] and in human 19

34 RA patients [134]. More recently, citrullinated peptides have been identified as autoantigens in diabetes. In one study, it was determined that PAD expression is upregulated in NOD islets and that inflammatory stress results in citrullination of glucose-regulated protein 78 (GRP78) within the beta cell [135]. T cells specific for the modified protein were detected in prediabetic NOD mice [135]. Another group demonstrated the presence of CD4 T cells specific for a citrullinated GAD epitope in the peripheral blood of T1D patients [136]. Another modification that has recently been implicated in the generation of neoepitopes in T1D is deamidation of glutamine residues by the enzyme tissue transglutaminase (TGase). TGase is known to generate modified antigens in celiac disease [137]. Deamidation by TGase converts glutamine residues to glutamic acid residues, which has been shown to improve binding of some peptides to the disease-associated allele HLA-DQ8 by generating a negatively charged side chain on the peptide that interacts favorably with the p9 pocket of the HLA molecule [31]. CD4 T cells recognizing a deamidated GAD peptide have been detected at higher frequencies in the peripheral blood of T1D patients compared to controls, suggesting that deamidated peptides may be T cell epitopes in diabetes [136]. Recent work has also demonstrated that human islets can generate deamidated peptides and that human dendritic cells pulsed with native islet proteins present deamidated peptides, demonstrating that deamidated islet antigens are potentially generated and presented to CD4 T cells in vivo [138]. Antigens for the BDC T cell clones A primary goal of our lab has been the identification of novel autoantigens in T1D, using the NOD mouse as a model of disease. An ultimate objective is to use the discovery of antigens to develop our understanding of disease etiology and pathogenesis, promote the 20

35 identification of disease biomarkers, and inform the development of antigen-specific therapies for T1D. To facilitate these aims, we use the BDC panel of T cell clones to identify antigens in autoimmune diabetes. The first antigen identified by the lab was the protein chromogranin A (ChgA), which was found to be the source of an antigenic peptide for the T cell clone BDC-2.5. Dr. Thomas Delong in our lab developed the biochemical purification of beta cell tumors by sequential chromatography and tested the fractions for antigenic activity with T cell clones. From the antigenic fractions, he used mass spectrometry to identify ChgA as the principal candidate antigen. Later work in collaboration with John Kappler s group determined that WE14 (WSRMDQLAKELTAE), a naturally-occurring cleavage product of chromogranin A (ChgA), is a weakly antigenic ligand for the T cell clone BDC-2.5 [139]. However, several lines of data indicate that the peptide only fills half of the I-Ag7 binding groove. Testing the peptide EDKRWSRMDQLAKELTAE, which contains the naturally-preceding ChgA sequence, is not antigenic for BDC-2.5 [139]. Given this unusual binding and the weak antigenicity of the peptide, Dr. Delong hypothesized that the natural antigen for BDC-2.5 is a post-translationally modified version of WE14. It was found that treatment of WE14 (which contains a glutamine residue) with TGase rendered the peptide highly antigenic for BDC-2.5 [140]. The increased antigenicity was not the result of deamidation, as a peptide containing glutamic acid (the deamidation product) instead of glutamine was no more antigenic than the unmodified peptide. In addition to deamidating glutamine residues, TGase can also covalently link them to lysine residues via an isopeptide bond. As WE14 contains both a glutamine and a lysine residue, TGase should theoretically be capable of generating peptidepeptide aggregates by cross-linking multiple WE14 molecules. It was demonstrated that such 21

36 cross-linking did occur when the peptide was treated with TGase [140]. It was hypothesized that these peptide aggregates would be more efficiently taken up by APCs than individual WE14 molecules, thus explaining the increase in antigenicity. Testing of PBMCs indicated that T cells specific for WE14 are present in the blood of human T1D patients but not control subjects [141]. Furthermore, TGase-treated WE14 elicited a more robust response from the PBMCs of some patients [141], supporting the hypothesis that a post-translationally modified form of WE14 is an antigen in T1D. The most compelling evidence for ChgA as an important autoantigen in diabetes was provided when the lab recently demonstrated that NOD mice deficient in ChgA do not develop spontaneous diabetes [142]. Thus, like insulin, ChgA is a critical autoantigen in the pathogenesis of diabetes in the NOD mouse. As we suspected that the natural form of the antigen for BDC-2.5 was a cross-linked version of WE14, we continued investigating the antigen for this clone. Our lab has also in recent years identified the antigen for the T cell clone BDC as islet amyloid polypeptide (IAPP) [143], which had been previously implicated as a CD8 T cell autoantigen in human T1D [144]. The IAPP peptide KS20 is highly antigenic for BDC [143], and KS20-reactive CD4 T cells can be detected in the pancreas of both prediabetic and diabetic NOD mice [145]. IAPP is a hormone produced by beta cells and cosecreted with insulin [93]. Like insulin, IAPP (sometimes referred to as amylin) begins as a prohormone that must undergo several modifications prior to becoming hormonally active. In the proiapp molecule, the amylin region is flanked on either terminus by a propeptide region (Fig. 2). In this thesis, we will refer to the propeptide on the N-terminus as IAPP1 and to the propeptide on the C-terminus as IAPP2. The peptide KS20 falls entirely within the amylin region of the molecule. Each of the propeptide regions is separated from amylin by 22

37 amylin HOOC - IAPP2 - NH 2 IAPP1 Figure 2: Islet amyloid polypeptide (IAPP). A schematic of mouse proiapp. Each square represents an amino acid residue. ProIAPP consists of amylin (white) flanked by IAPP propeptide 1 (IAPP1; light gray) on the N-terminus and IAPP propeptide 2 (IAPP2; dark gray) on the C-terminus. Endoprotease cleavage sites are indicated by dashed lines. PC2 cleaves IAPP1 from amylin and IAPP2 is cleaved by PC1. Following removal of the dibasic residues (squares marked with an X) from the C-terminus of the amylin region by CPE, peptidylglycine alpha-amidating monooxygenase (PAM) removes the exposed C-terminal glycine and amidates the C-terminus of the trimmed peptide. An arrow marks the peptide bond between the tyrosine and glycine residues, which is attacked by PAM. Along with an amidated C-terminus, the mature amylin hormone also contains a disulfide bridge (indicated by the solid bracket) between cysteine residues at positions 2 and 7. 23

38 dibasic residues. PC2 and PC1 cleave proiapp at the dibasic residues to liberate IAPP1 and IAPP2, respectively [146]. CPE then removes the dibasic residues from the C-terminus of amylin [146]. The enzyme peptidylglycine alpha-amidating monooxygenase (PAM) then removes a C-terminal glycine from amylin, leaving behind an amidated C-terminus. To be fully biologically active, a disulfide bridge must also form between cysteine residues at position 2 and 7 of the amylin peptide [147]. The BDC-6.9 antigen Most of the T cell clones in the BDC panel respond to islets from both NOD and BALB/c mice [33]. However, the clones BDC-6.9 and BDC-9.3 respond only to NOD islets. BDC-6.9 and BDC-9.3 express the same TCR and have been used interchangeably in the lab. To identify the locus harboring the gene encoding the BDC-6.9 antigen, NOD and BALB/c mice were crossed and the F 1 progeny were backcrossed with BALB/c mice. Islets of the offspring were tested with BDC-6.9 for antigenicity. Genetic analysis revealed that antigenicity for BDC-6.9 was co-inherited with two microsatellite regions on chromosome 6 [148]. Because the Iapp gene was identified within this locus, IAPP became the lead candidate for the BDC-6.9 antigen. Islets from NOD mice deficient in IAPP were found to be non-antigenic for BDC-6.9, supporting the hypothesis that IAPP is the antigen for the clone. The coding sequences of Iapp in NOD and BALB/c mice differ by two nonsynonymous single-nucleotide differences [149,150], each resulting in a single amino acid difference between the NOD and BALB/c IAPP molecules. One amino acid substitution is in the IAPP1 region, while the other is in the IAPP2 region. It was hypothesized that the BDC- 6.9 epitope was a peptide from one of these two regions of IAPP and that the NOD, but not the BALB/c, variant of the peptide would be antigenic. However, when peptides spanning the 24

39 NOD proiapp molecule, including IAPP1 and IAPP2, were tested with BDC-6.9, none of the peptides stimulated the clone. It was therefore hypothesized that the antigen for BDC-6.9 was a modified version of IAPP. Overall objective of thesis CD4 T cells play an important role in autoimmune diabetes in both the NOD mouse and humans. Some of the CD4 T cell antigens implicated in the development of autoimmune diabetes have been discussed in this introduction. A possible role for post-translationally modified peptides as autoantigens has also been discussed. The BDC panel of clones has been an invaluable resource that has enabled the lab to identify both ChgA and IAPP for the first time as autoantigens in the NOD mouse. Because the BDC-2.5 clone responds only weakly to the ChgA peptide WE14, it seemed likely that the natural antigen for this clone is post-translationally modified. Compelling data suggested that IAPP was the BDC-6.9 antigen. However, no antigenic IAPP peptides could be identified. Thus, it was hypothesized that the BDC-6.9 antigen is a post-translationally modified IAPP peptide. The objective of this thesis was to identify the antigen for BDC-6.9 and investigate the role of CD4 T cells specific for the BDC-6.9 antigen in pathogenesis. This project was done in parallel with efforts to identify the BDC-2.5 antigen. These efforts were ultimately very rewarding, bringing an exciting conclusion to a decades-long investigation and revealing a novel class of autoantigens in T1D. 25

40 CHAPTER II GENERAL METHODS Methods standardized in the laboratory and used throughout my thesis work are presented in this chapter. Specialized methods are described in the chapters in which they are used. Mice BALB/c, non-obese diabetic (NOD), NOD.RIP-TAg, and NOD.IAPP-/- mice were bred and housed at National Jewish Health (Denver, CO) or University of Colorado Denver in specific pathogen-free conditions. Generation of NOD.RIP-TAg [151] and NOD.IAPP-/- [143] mice was described previously. The NOD mouse, which spontaneously develops autoimmune diabetes, is the premiere animal model for studying T1D. The transgenic NOD.RIP-TAg mouse spontaneously develops pancreatic beta cell tumors due to expression of the SV40 large T antigen under the control of the rat insulin promoter. NOD.RIP-TAg tumors were used extensively as an abundant source of beta cell antigen throughout this thesis. All experiments were conducted under protocols approved by the Institutional Animal Care and Use Committee. Beta membrane (β-mem) preparation Beta cell tumors were harvested from NOD.RIP-TAg mice and frozen at -80 C until use. Thawed tumors were homogenized through 40 µm strainers and cells were lysed by passing successively through 22, 27, and 30 gauge needles. Following removal of large cellular debris by low-speed centrifugation, secretory granules were pelleted by centrifugation at 18,400 x g and resuspended at a total protein concentration of 200 µg/ml. This preparation of control antigen is referred to as beta membrane (β-mem). 26

41 Culture of T cell clones T cell clones were cultured in high glucose Dulbecco s modified Eagle s medium (DMEM) supplemented with sodium bicarbonate (final concentration of 44 mm), L-arginine (0.55 mm), L-asparagine (0.27 mm), L-glutamine (1.5 mm), sodium pyruvate (1 mm), gentamicin sulfate (50 mg/l), 2-mercaptoethanol (50 µm), and HEPES (10 mm, ph 7.4). Fetal bovine serum (FBS) was added to a final concentration of 10% v/v. In each flask, one million T cells were cultured with 2.5 x 10 7 irradiated NOD splenocytes as antigen presenting cells (APCs), β-mem as antigen at a final concentration of 150 ng/ml, and supernatant from stimulated EL-4 thymoma cells as a source of IL-2 [35]. Cells were cultured on a two-week cycle at 37 C and 10% CO 2. Islet isolation To isolate pancreatic islet cells for assay with T cell clones, mice were euthanized and the pancreas was inflated with collagenase solution via the common bile duct. Following inflation, the pancreas was removed and incubated at 37 C to allow for digestion. Islets were then isolated by density centrifugation and were subsequently handpicked under a microscope. Islets were dissociated with trypsin to generate a single cell suspension and then islet cells were counted. Peptides Synthetic peptides were obtained commercially at >95% purity from CHI scientific. T cell antigen assays T cells (2 x 10 4 /well) were incubated with antigen and NOD thioglycolate-elicited peritoneal macrophages (2.5 x 10 4 /well) as APCs in a 96-well plate one to two days at 37 C. Following incubation, supernatant was collected and IFN-γ concentration was measured by 27

42 sandwich ELISA using a biotinylated anti-ifn-γ secondary antibody and streptavidincoupled peroxidase with 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as a substrate for detection. IFN-γ concentrations are reported either in units of absorbance or in nanograms of IFN-γ per ml of supernatant as calculated based on several concentrations of IFN-γ standard included in separate wells of the assay plate. Although the relative antigenicity of different peptides for a given T cell clone is consistent from one assay to the next, the absolute amount of IFN-γ produced by the clone can vary under the same conditions in different assays. Thus, data from one representative experiment are usually shown rather than the average and standard deviation for multiple assays. Adoptive transfer of activated CD4 T cell clones BDC T cell clones were cultured on a two-week restimulation cycle. For adoptive transfer, five days following restimulation cells were transferred to larger flasks containing additional fresh medium supplemented with IL-2 at twice the concentration used for restimulation (exact IL-2 concentrations were different for each clone). Cells were cultured for four days at 37 C and 10% CO 2 then harvested from flasks using enzyme-free dissociation buffer. Mice were injected i.p. with 1x10 7 cells in Hank s balanced salt solution (HBSS) then monitored daily for diabetes onset by urine glucose testing. Diabetes was confirmed by blood glucose testing. Mice were considered diabetic after one blood glucose reading >15 mm (270 mg/dl). 28

43 CHAPTER III BDC-6.9 AND IAPP PEPTIDES 1 Background and rationale In earlier work to characterize the antigen response of the BDC-6.9 T cell clone [33], it was determined that this clone responds to islets from NOD mice but not to islets from BALB/c mice. Subsequent genetic linkage analysis led to the identification of the beta cellspecific hormone IAPP as a candidate antigen for BDC-6.9 [148]. Of particular interest were the two propeptide regions of IAPP, each of which differs between the NOD and BALB/c mouse by one amino acid residue [ ]. It was postulated that the BDC-6.9 epitope was a peptide from one of these two regions of IAPP and that the NOD, but not the BALB/c, variant of the peptide would be antigenic. However, when BDC-6.9 was tested with a panel of IAPP peptides spanning the full proiapp sequence, it was found that none of the peptides were antigenic, leading to the hypothesis that the epitope recognized by BDC-6.9 is a posttranslationally modified IAPP peptide. In this chapter, we document our initial experiments to identify a modified IAPP peptide antigen for BDC-6.9. Specialized methods Mice NOD.RIP-SOD mice were bred and housed at National Jewish Health (Denver, CO) in specific pathogen-free conditions. NOD.RIP-SOD mice overexpress Cu/Zn superoxide dismutase, a radical-scavenging enzyme, specifically in the beta cells of the pancreas. Generation of NOD.RIP-SOD mice was described previously [152]. All experiments were conducted under protocols approved by the Institutional Animal Care and Use Committee. 1 Portions of this chapter have been published [163,165] and are included here with the permission of Science and Journal of Autoimmunity. 29

44 RIP-TAg cells and RT-ftlys RIP-TAg cells were prepared by harvesting tumors from NOD.RIP-TAg mice, generating a single cell suspension, counting the cells, and freezing aliquots in freezing medium [90% fetal bovine serum (FBS), 10% dimethyl sulfoxide (DMSO)]. Aliquots were thawed and used in antigen assays with T cell clones. The amount of antigen tested in assays was expressed in terms of cell numbers. RIP-TAg freeze-thaw lysate (RT-ftlys) was prepared by aliquoting and freezing dissociated NOD.RIP-TAg tumor cells in standard culture medium. Freeze-thaw in standard medium led to lysis of cells, thus the name RIP-TAg freeze-thaw lysate. For quantitative purposes, amounts were expressed as cell equivalents (based on the concentration of cells prior to freezing) or as total protein concentration (as determined by bicinchoninic acid assay). Citrullination of synthetic peptides Peptides (final concentration = 2 mg/ml) were incubated with rabbit type II peptidyl arginine deiminase (PAD) at a substrate:enzyme ratio of 1mg peptide:2 units enzyme in 0.1 M Tris-HCl ph 7.5, 10 mm CaCl2, and 5 mm dithiothreitol (DTT). Reactions were incubated at 37 C for 2 hours then stopped by addition of ethylene diamine tetraacetic acid (EDTA) to a final concentration of 20 mm. Reaction mixtures were tested with T cells at a final peptide concentration of 86.5 µg/ml. Transglutaminase-mediated deamidation of synthetic peptides Peptides (final concentration = 500 µg/ml) were incubated with 0.1 U/ml transglutaminase (TGase) in 50 mm Tris-HCl ph 6, 10 mm CaCl2, and 5 mm dithiothreitol (DTT). Reactions were incubated at 37 C for 6 hours. Reaction mixtures were tested with T cells at a final peptide concentration of 50 µg/ml. 30

45 Oxidation of peptidyl methionine using H 2 O 2 Peptide was incubated with hydrogen peroxide (H 2 O 2 ) at the indicated concentration for 2 hours at 37 C, then excess hydrogen peroxide was vacuum evaporated and samples were tested with T cells. Mass spectrometric analysis of peptides Samples were diluted in aqueous solution containing 3% v/v acetonitrile and 0.1% formic acid and analyzed on either a 6340 LCMS ion-trap mass spectrometer or a 6520 LCMS Q-TOF mass spectrometer (Agilent) in MS or tandem MS (MS/MS) mode. Results Natural antigen sources for BDC-6.9 Before testing modified IAPP peptides for antigenicity with BDC-6.9, it was important to establish a source of natural antigen that could be used as a reliable positive control to confirm that the T cell clone was responding properly in each assay. To measure the antigenicity of samples (e.g., peptides, islets) for the BDC clones, the T cell clones, various concentrations of the sample, and thioglycolate-elicited peritoneal macrophages from NOD mice as antigen presenting cells (APCs) are cultured in wells of a 96-well plate. If a sample is antigenic for a T cell clone, the T cells will produce IFN-γ, as all of the BDC clones are of a Th1 phenotype. Following incubation at 37 C for one to two days, IFN-γ in the supernatant is detected by sandwich ELISA using a biotinylated anti-ifn-γ secondary antibody and streptavidin-coupled peroxidase with ABTS as a substrate for detection. IFN-γ concentration is reported either in units of absorbance or in ng/ml as calculated based on a standard curve. 31

46 All clones in the BDC panel respond to islets isolated from NOD mice. As mentioned previously, the BDC T cell clones also respond to islets from BALB/c mice, with the exception of the clones BDC-6.9 and BDC-9.3. Because BDC-6.9 and BDC-9.3 have the same TCR, the two are used interchangeably in assays. Islets from mice deficient in IAPP (NOD.IAPP-/-) are not antigenic for either BDC-6.9 or BDC-9.3. Responses by these two clones and the insulin B:9-23-reactive clone BDC-4.38 to islet cells from various mouse strains are provided (Fig. 1). Because islet isolation is labor intensive and costly, it is impractical to use NOD islet cells as a positive control on a routine basis. The NOD.RIP-TAg mouse expresses the SV40 large T antigen under the control of the rat insulin promoter [151]; the T antigen is expressed specifically in beta cells in this mouse, leading to the spontaneous development of beta cell tumors. The beta cell line NIT-1 [151], which was established from the beta cell tumors of a NOD.RIP-TAg mouse, was originally considered as a source of antigen for the BDC T cell clones. However, early work by our lab demonstrated that NIT-1 cells lose antigenicity over time in culture [153]. As an alternative, a crude preparation of beta cell secretory granule proteins isolated from NOD.RIP-TAg tumors was developed. Tumors are harvested from NOD.RIP-TAg mice and the tissue is ground through a strainer to generate a single cell suspension. The tumor cells are then mechanically lysed by successive passage through needles with progressively smaller inner diameters and large cellular debris is removed by low-speed centrifugation. The supernatant is then centrifuged at high speed to pellet smaller cellular components. The pellet is enriched for secretory granules, which contain the antigens for the BDC clones [153]. This preparation is designated as beta membrane (β-mem). 32

47 IFN-γ (A415) BDC-4.38 BDC-6.9 BDC-9.3 NOD BALB/c NOD.IAPP-/- Figure 1: Islets from NOD.IAPP-/- mice are not antigenic for BDC-6.9/BDC-9.3. T cell clones were cultured with APCs and 1 x 10 4 dissociated islet cells from NOD, BALB/c, or NOD.IAPP-/- mice. IFN-γ production was measured by ELISA as a readout of T cell activation. The average +/- S.D. of triplicate wells is reported. Results are representative of at least two independent experiments. 33

48 Since β-mem, which is used as a positive control antigen for other clones in the BDC panel, is only weakly antigenic for BDC-6.9, a cruder preparation was tested for antigenicity. Tumors were forced through a cell strainer to generate a single cell suspension and then the cells were frozen in cell culture medium for later use. Because freezing and thawing these cells in media causes most of the cells to lyse, this antigen prep was called RIP-TAg freezethaw lysate (RT-ftlys). The control clone BDC-2.5 responded equally well to β-mem or RTftlys (Fig. 2a). BDC-6.9 did not respond to β-mem, but did respond to RT-ftlys (Fig. 2b), thus RT-ftlys became one of the standard positive control antigens for BDC-6.9. As an alternative form of antigen, the RIP-TAg tumor cells could be frozen in freezing medium, which maintained the integrity of the cells. Intact RIP-TAg cells were antigenic for BDC-2.5, BDC (specific for the IAPP peptide KS20 [143]), and BDC-6.9 (Fig. 3). IAPP peptides After establishing that RIP-TAg cells and RT-ftlys could serve as good positive control antigens, we sought to confirm earlier observations that BDC-6.9 does not recognize unmodified IAPP peptides. The Iapp DNA sequence and the resulting IAPP protein sequence in NOD and BALB/c mice are provided (Table I). Note the single amino acid difference between the two strains in each of the propeptide regions of IAPP. We tested several commercially synthesized, high purity peptides spanning the entire pre-proiapp molecule (Table II) and found that none of these peptides was antigenic for BDC-6.9 (Fig. 4c), confirming earlier results. The IAPP signal peptide MA23 (MMCISKLPAVLLILSVALNHLRA), though not shown in Table II, was also tested but was not antigenic for BDC-6.9 (Fig. 4c). The KS20-reactive clone BDC was included in the assay as a control (Fig. 4b). 34

49 A IFN-γ (A415) BDC protein (µg/ml) β-mem RT-ftlys B IFN-γ (A415) BDC protein (µg/ml) β-mem RT-ftlys Figure 2: RT-ftlys, but not β-mem, is antigenic for BDC-6.9. (A) BDC-2.5 and (B) BDC- 6.9 were cultured with APCs and either β-mem or RT-ftlys. IFN-γ production was measured by ELISA as a readout of T cell activation. Each antigen source was tested in at least three experiments. 35

50 IFN-γ (A415) # of RIP-TAg cells BDC-2.5 BDC BDC-6.9 Figure 3: Primary cells isolated from RIP-TAg tumors are antigenic for BDC-6.9. The T cell clones BDC-2.5, BDC-5.2.9, and BDC-6.9 were cultured with APCs and RIP-TAg cells. Results are representative of two independent experiments. 36

51 Table I: IAPP coding sequence and amino acid sequence in NOD and BALB/c mice. BALB CDS CTG NOD CDS ATGATGTGCATCTCCAAACTGCCAGCTGTCCTCCTCATCCTCTCTGTGGCACTAAACCACTTGAGAGCT NOD pep M M C I S K L P A V L L I L S V A L N H L R A BALB pep L BALB CDS AGC NOD CDS ACACCTGTCAGAAGTGGTACCAACCCTCAGATGGACAAACGGAAGTGCAACACGGCCACGTGTGCCACA NOD pep T P V R S G T N P Q M D K R K C N T A T C A T BALB pep S BALB CDS NOD CDS CAACGCCTGGCAAACTTTTTGGTTCGTTCCAGCAACAACCTTGGTCCAGTCCTCCCACCAACCAACGTG NOD pep Q R L A N F L V R S S N N L G P V L P P T N V BALB pep BALB CDS GGG NOD CDS GGATCGAATACATATGGCAAGAGGAATGCGGCAAGGGATCCAAATAGGGAATCCTTGGATTTCTTACTCGTT NOD pep G S N T Y G K R N A A R D P N R E S L D F L L V BALB pep G The nucleotide coding sequence and peptide amino acid sequence are shown for NOD and BALB/c IAPP. Sequences are identical except where noted for BALB/c. Propeptide regions are highlighted. Sequences obtained from Ensembl and BALB = BALB/c. CDS = coding sequence. pep = peptide sequence. 37

52 Table II: IAPP peptide panel tested with BDC-6.9 and BDC-9.3. TPVRSGTNPQMDKRKCNTATCATQRLANFLVRSSNNLGPVLPPTNVGSNTYGKRNAARDPNRESLDFLLV TPVRSGTNPQMDKRKCNTATCATQRLANFLVRSSNNLGPVLPPTNVGSNTYGKRNAARDPNRESLDFLLV TPVRSGTNPQMDKRKCNTATCATQRLANFLVRSSNNLGPVLPPTNVGSNTYGKRNAARDPNRESLDFLLV RSGTNPQMDKR KCNTATCATQRLANFLVRSSNNLGPVLPPTNVGSNTYGKRNAARDPNRESLDFLLV MMCISKLPAVLL KCNTATCATQRLANFLVRSSNNLGPVLPPTNVGSNTYGKRNAARDPNRESLDFLLV MMCISKLPAVLLILSVALNHLR RLANFLVRSSNNLGPVLPPTNVGSNTYGKRNAARDPNRESLDFLLV MMCISKLPAVLLILSVALNHLRATPVRSGTNP NNLGPVLPPTNVGSNTYGKRNAARDPNRESLDFLLV MMCISKLPAVLLILSVALNHLRATPVRSGTNPQMDKRKCNTATCATQRLANFLVNAARDPNRESLDFLLV IAPP1 proiapp KS20 amylin* RT20 NG18 IAPP2 The NOD proiapp sequence is shown at top (highlighted in gray). Key peptides tested with BDC-6.9 and BDC-9.3 are shown below the full-length sequence. Each peptide was tested in at least two independent experiments. Multiple concentrations of each peptide, starting at a highest concentration of 100 µg/ml, were tested in each experiment. Peptides listed represent the NOD sequence, which differs slightly from control strains. Bolded residues are specific to the NOD mouse. BDC-6.9 and BDC-9.3 did not respond to any of the peptides at the concentrations tested. The IAPP signal peptide MA23, though not shown, was also tested and found to not be antigenic for BDC-6.9 or BDC-9.3. IAPP1 = IAPP propeptide 1. IAPP2 = IAPP propeptide 2. *The amylin peptide was synthesized with two modifications found naturally in the mature IAPP hormone: a disulfide bridge between C2 and C7 and an amidated C-terminus. 38

53 A 60 RT-ftlys IFN-γ (ng/ml) BDC BDC-6.9 4x10 5 cell equivalents 2x10 5 cell equivalents B IFN-γ (ng/ml) BDC peptide (µg/ml) MA23 NOD IAPP1 KS20 RT20 NG18 NOD IAPP2 C IFN-γ (ng/ml) 70 BDC peptide (µg/ml) MA23 NOD IAPP1 KS20 RT20 NG18 NOD IAPP2 Figure 4: BDC-6.9 does not respond to IAPP peptides. (A) BDC and BDC-6.9 were cultured with RT-ftlys (4x10 5 or 2x10 5 cell equivalents) as a positive control of T cell responsiveness. (B) BDC and (C) BDC-6.9 were cultured with various concentrations of peptides spanning the NOD proiapp molecule. The IAPP signal peptide, MA23, was also tested. Each peptide was tested in at least two experiments. 39

54 Post-translationally modified IAPP peptides After confirming previous findings that BDC-6.9 does not respond to unmodified IAPP peptides, we began testing modified IAPP peptides for antigenicity. Following cleavage of the propeptide regions, IAPP undergoes three post-translational modifications prior to secretion that are requisite for hormonal activity: formation of a disulfide bond between cysteine residues two and seven of the peptide, removal of the C-terminal glycine, and amidation of the newly exposed carboxy terminus of a tyrosine residue. This modified form of IAPP, referred to as amylin, had not yet been tested with BDC-6.9, so we decided to test it for antigenicity. We also tested the full NOD proiapp molecule in case the epitope for BDC-6.9 spanned the junction between either propeptide region and the amylin sequence. Like the amylin peptide KS20, both amylin and NOD proiapp were antigenic for BDC (Fig. 5), but neither of these peptides stimulated BDC-6.9. As previously mentioned, a modified NOD IAPP2 peptide seemed the most likely candidate for the BDC-6.9 antigen. Because the NOD IAPP2 sequence contains an arginine, we hypothesized that the BDC-6.9 antigen was citrullinated. Citrullination, also known as deimination, converts the amino acid arginine to citrulline and is catalyzed by the enzyme peptidyl arginine deiminase (PAD) [154]. Citrullinated proteins have been identified as autoantigens in NOD mice [135,136]. We treated NOD IAPP1 and NOD IAPP2, which both contain an arginine residue, with PAD enzyme and confirmed that modification of NOD IAPP2 was successful by mass spectrometric analysis (Fig. 6). Citrullination leads to a mass loss of one Dalton, which is easily detectable by mass spectrometry. In the total ion chromatogram (TIC) for the treated peptide, peaks corresponding to doubly-charged unmodified and citrullinated peptide, which differ by approximately 0.5 m/z, can be seen, 40

55 IFN-γ (A415) no antigen RIP-TAg cells (9x10 4 ) NOD proiapp (35 µm) amylin (26 µm) BDC BDC-6.9 Figure 5: NOD proiapp and amylin are not antigenic for BDC-6.9. BDC (an IAPP-reactive clone) and BDC-6.9 were tested with the NOD version of the full-length proiapp molecule and the mature full-length amylin peptide containing a disulfide bridge and an amidated C-terminus. RIP-TAg cells were used as a positive control. Though not shown for the sake of clarity, BDC-6.9 also failed to respond to NOD proiapp and amylin at multiple other concentrations. Each peptide was tested in at least three experiments. 41

56 spectral intensity - PAD + PAD m/z = z = +2 m/z = z = +2 m/z = z = +2 retention time Figure 6: Mass spectrometric analysis confirms citrullination of PAD-treated peptides. NOD IAPP1 peptide was treated with PAD enzyme. A total ion chromatogram (TIC) shows appearance of a doubly charged species with a mass/charge ratio of 652.2, corresponding to citrullinated NOD IAPP1. The other two labeled peaks correspond to unmodified peptide (m/z = 651.7; z = +2) and peptide containing a spontaneously oxidized methionine residue (m/z = 659.7; z = +2). Y-axes are scaled equally. Results are from one experiment. 42

57 indicating that some of the peptide was modified. Since different molecules ionize with different efficiency, it is not possible to use the area under the peaks to make quantitative comparisons between the percentages of modified and unmodified peptide without the use of labeled peptides as internal references. Because we were using highly purified peptides as a substrate in the citrullination reaction and a pure T cell clone population as a responder in the antigen assay, we expected that enough citrullinated peptide was present in the modified sample to elicit a measurable T cell response if the epitope recognized by BDC-6.9 was indeed the citrullinated form of IAPP2. However, when tested, neither modified peptide stimulated BDC-6.9 (Fig. 7). At low ph, the enzyme transglutaminase (TGase) catalyzes the deamidation of glutamine residues to form glutamic acid [137]. Deamidated peptides have been identified as autoantigens in both celiac disease and T1D, with TGase being implicated as the modifying enzyme [31, ]. We tested whether treatment of IAPP peptides with TGase at low ph could render IAPP peptides antigenic for BDC-6.9, but NOD IAPP1 and amylin were not antigenic following TGase treatment (Fig. 8). NOD IAPP2 does not contain a glutamine residue and is therefore not a substrate for TGase but was included as a control. Oxidative stress seems to play a role in beta cell death during the pathogenesis of autoimmune diabetes [152, ], and there is evidence that oxidative PTM of peptides can lead to the generation of neoantigens [ ]. To investigate whether the BDC-6.9 antigen was an oxidized IAPP peptide, we obtained NOD.RIP-SOD mice, which overexpress the radical-scavenging enzyme Cu/Zn superoxide dismutase specifically in beta cells [152]. We predicted that if the BDC-6.9 antigen contained an oxidative PTM, then less of the 43

58 IFN-γ (ng/ml) no antigen RIP-TAg cells (1x10 6 ) BDC-6.9 NOD IAPP1 NOD IAPP2 - PAD + PAD Figure 7: Citrullinated IAPP peptides are not antigenic for BDC-6.9. The T cell clone BDC-6.9 was tested with unmodified NOD IAPP1 and NOD IAPP2 (both argininecontaining peptides) or with the same peptides treated with PAD enzyme to citrullinate arginine residues. - PAD control reactions were incubated in the same reaction buffer as PAD-treated samples. RIP-TAg cells were used as a positive control. Results are shown for a final peptide concentration of 86.5 µg/ml, but multiple concentrations of the unmodified and modified peptides were tested with negative results. Results are from one experiment. 44

59 IFN-γ (A415) no antigen NOD IC (1x10 4 ) NOD IAPP1 BDC-6.9 amylin NOD IAPP2 - TGase + TGase Figure 8: Deamidated IAPP peptides are not antigenic for BDC-6.9. The T cell clone BDC-6.9 was tested with unmodified NOD IAPP1, amylin, and NOD IAPP2 or with the same peptides treated with transglutaminase (TGase) enzyme at low ph to deamidate glutamine residues. NOD IAPP2, which does not contain a glutamine residue, was used as a control. - TGase control reactions were incubated in the same reaction buffer as TGasetreated samples. Dissociated NOD islet cells (IC) were used as a positive control. Results are shown for a final peptide concentration of 50 µg/ml, but multiple concentrations of the unmodified and modified peptides were tested with negative results. Results are from one experiment. 45

60 antigen would form in islets from NOD.RIP-SOD mice. However, contrary to our hypothesis, these islets were still highly antigenic for all of the BDC clones tested (Fig. 9). NOD IAPP1 After repeated testing of IAPP peptides with BDC-6.9, we found that one commercial batch of NOD IAPP1 peptide weakly stimulated BDC-6.9 (Fig. 10). The same batch of peptide did not stimulate BDC-5.2.9, suggesting that the BDC-6.9 response was not due to a contaminant that generally activated T cells. However, the peptide did not stimulate BDC-9.3. Because BDC-9.3 expresses the same TCR as BDC-6.9, the results were suspect. To further investigate, we obtained a new batch of NOD IAPP1. The new batch was not antigenic for BDC-6.9 (Fig. 11). We also tested two extended versions of NOD IAPP1 containing naturally flanking sequence, TR14 and LN23. These peptides were also not antigenic (Fig. 11). An aspartic acid residue (D) can greatly influence the configuration in which a peptide binds to the NOD MHC class II molecule I-Ag7 and in turn which residues are presented to the TCR [17,19 24]. We questioned if the real epitope was lacking the aspartic acid residue and if NOD IAPP1, which contains the residue, was being presented sub-optimally to the TCR. We therefore tested the peptide TM11, in which the C-terminal aspartic acid had been omitted. This peptide likewise did not stimulate BDC-6.9 (Fig. 11). We next examined the antigenic and non-antigenic batches of NOD IAPP1 by mass spectrometry to search for any differences. Although both batches appeared very pure, we determined that peptide containing an oxidized methionine residue was present in both batches (Fig. 12). Spectrum Mill software estimated that a higher percentage of the peptide in the older batch was oxidized, which provided a possible explanation for the antigenicity of this batch. To test if the modified species was antigenic, we incubated NOD IAPP1, as well 46

61 A BDC-2.5 2x10 4 IC IFN-γ (A415) NOD BALB/c 3x10 3 IC C57Bl/6 NOD.IAPP-/- NOD.RIP-SOD B IFN-γ (A415) NOD BALB/c BDC C57Bl/6 NOD.IAPP-/- NOD.RIP-SOD C IFN-γ (A415) NOD BALB/c BDC-6.9 C57Bl/6 NOD.IAPP-/- NOD.RIP-SOD Figure 9: Overexpression of superoxide dismutase does not prevent formation of the BDC-6.9 antigen. (A) BDC-2.5, (B) BDC-5.2.9, and (C) BDC-6.9 were tested with islet cells (IC) isolated from NOD, BALB/c, C57Bl/6, NOD.IAPP-/-, or NOD.RIP-SOD mice (which overexpress the radical scavenging enzyme Cu/Zn superoxide dismutase specifically in beta cells). Results are from one experiment. 47

62 A IFN-γ (ng/ml) BDC BDC-6.9 BDC-9.3 no antigen RIP-TAg cells (2.5x10 5 ) RIP-TAg cells (6.25x10 4 ) B 60 BDC KS20 IFN-γ (ng/ml) NOD IAPP1 C IFN-γ (ng/ml) D IFN-γ (ng/ml) BDC-6.9 BDC peptide (µg/ml) KS20 NOD IAPP1 KS20 NOD IAPP1 Figure 10: A batch of NOD IAPP1 is weakly antigenic for BDC-6.9. (A) T cell clones were tested with RIP-TAg cells as a positive control. (B) BDC-5.2.9, (C) BDC-6.9, and (D) BDC-9.3 were tested with KS20 and NOD IAPP1. Results are representative of two experiments. BDC-9.3 was only tested in one experiment. 48

63 A IFN-γ (ng/ml) BDC BDC-6.9 RIP-TAg cells (1x10 6 ) RIP-TAg cells (5x10 5 ) B IFN-γ (ng/ml) BDC peptide (µg/ml) NOD IAPP1 NOD IAPP1 (new batch) NOD IAPP1 TR14 NOD IAPP1 LN23 NOD IAPP1 TM11 C IFN-γ (ng/ml) BDC peptide (µg/ml) NOD IAPP1 NOD IAPP1 (new batch) NOD IAPP1 TR14 NOD IAPP1 LN23 NOD IAPP1 TM11 Figure 11: A new batch of NOD IAPP1 is not antigenic for BDC-6.9. (A) BDC and BDC-6.9 were tested with RIP-TAg cells as a positive control antigen. (B) BDC and (C) BDC-6.9 were tested with NOD IAPP1, which previously showed weak antigenicity for BDC-6.9, and a new batch of NOD IAPP1. A truncated version, TM11, and two extended versions including natural N- and/or C-terminal flanking residues, TR14 and LN23, were also tested. TM11 = TPVRSGTNPQM; TR14 = TPVRSGTNPQMDKR; LN23 = LRATPVRSGTNPQMDKRKCNTAT. Results are from one experiment. 49

64 Figure 12: Mass spectrometric analysis reveals that both batches of NOD IAPP1 are highly pure but contain some peptide with an oxidized methionine residue. (A) Total ion chromatograms (TIC) showing the purity of two batches of NOD IAPP1. Y-axes are scaled equally. (B) Extracted ion chromatograms (EIC) demonstrating the presence of a doublycharged species with a mass/charge ratio of , corresponding to peptide with oxidized methionine, in both batches. Y-axes are scaled equally. (C) Spectrum Mill analysis of MS/MS data from each sample confirmed the modified species to be oxidized peptide. Oxidized methionine is indicated by a lower-case letter m. RT = retention time. m/z = mass/charge ratio. Da = Dalton. Spectral intensity provides an estimate of abundance. Results are from one experiment. 50

65 as NOD IAPP2 (which does not contain methionine), with various concentrations of hydrogen peroxide as an oxidizing agent [158]. Mass spectrometric analysis confirmed that treatment of NOD IAPP1 generated peptide with oxidized methionine (Fig. 13), but no antigenicity was observed (Fig. 14). Summary and conclusions Earlier genetic linkage analysis performed by our lab suggested that one of the two propeptide regions of IAPP was the antigen for BDC-6.9 [148]. Failure of NOD.IAPP-/- islets to stimulate the clone further supported this hypothesis. However, none of the IAPP peptides tested was antigenic for the clone. We thus tested if introducing modifications citrullination, deamidation, or oxidation could render IAPP peptides antigenic. Each of the modified peptides tested also failed to stimulate BDC-6.9. The list of possible PTMs we could potentially test, multiplied by the number of IAPP peptides that could be modified, would make it impractical to exhaustively test modified peptides. Furthermore, examining each modification would often necessitate the acquisition of expensive reagents, painstaking optimization of reaction conditions, and labor-intensive validation of modification by mass spectrometry, Western blot, or other means. We therefore focused on an alternative but parallel approach: identifying the natural BDC-6.9 antigen in beta cells by mass spectrometry. 51

66 Figure 13: Treatment of NOD IAPP1 with hydrogen peroxide results in methionine oxidation. NOD IAPP1 peptide was incubated with hydrogen peroxide (H 2 O 2 ) at a final concentration of 0, 1, 4, or 400 mm. EICs demonstrating mass spectrometric identification of oxidized peptide (m/z = ; z = +2) following treatment with various concentrations of hydrogen peroxide are shown. Y-axes are scaled equally. Sequences were confirmed by MS/MS analysis (data not shown). Results are representative of two independent experiments. 52

67 A 80 RIP-TAg cells IFN-γ (ng/ml) no antigen 1x10 6 5x x x10 5 B 80 NOD IAPP1 IFN-γ (ng/ml) no H 2 O 2 4 mm 40 mm 400 mm C 80 NOD IAPP2 IFN-γ (ng/ml) no H 2 O 2 4 mm 40 mm 400 mm Figure 14: Treatment of NOD IAPP1 with hydrogen peroxide to oxidize the methionine residue does not increase antigenicity for BDC-6.9. Peptides were incubated with the indicated concentration of hydrogen peroxide. Following removal of unreacted hydrogen peroxide by vacuum evaporation, samples were tested with BDC-6.9 for antigenicity. The T cell clone was tested with (A) various numbers of RIP-TAg cells as a positive control or with (B) NOD IAPP1 or (C) NOD IAPP2 treated with hydrogen peroxide. Results are shown for a final peptide concentration of 124 µg/ml, but multiple concentrations of the unmodified and modified peptides were tested with similar results. Results are from one experiment. 53

68 CHAPTER IV SEARCHING FOR THE BDC-6.9 ANTIGEN BY MASS SPECTROMETRY Background and rationale As described in Chapter III, the testing of a variety of modified IAPP peptides did not reveal an antigen for BDC-6.9. As a parallel approach, we sought to identify the natural antigen by mass spectrometry in beta cells. Beta cell proteins can be fractionated by chromatography, and the T cell clone can be used to identify fractions containing the antigen. Peptides present in antigenic fractions can then be identified by mass spectrometry. Our lab had previously used such methodology to establish WE14 as a weak antigen for BDC-2.5 [139] and the IAPP peptide KS20 as a highly antigenic peptide for BDC [143]. Despite the great power of mass spectrometry as a technique for identifying peptides in complex samples, it also has inherent shortcomings. Even the purified antigenic fractions will still be a complex mixture of peptides; therefore, this approach will yield at best a small list of peptides that are candidate antigens. Further information is needed to narrow down the list. Additionally, mass spectrometry yields very large datasets that can only be efficiently analyzed using computer software. The software analyzes the spectral data obtained for peptides in the sample and searches a known protein database (or a custom database designed by the user) for peptides that could be described by each spectrum. The investigator can designate certain post-translational modifications to be considered in the search, but if a particular peptide in the sample is not present in the database or if the peptide contains an unexpected modification then it will not be identified by the software. Therefore, the more that is known about the antigen from the beginning of the experiment, the more likely it can be identified by mass spectrometry. Our experimental choices were generally informed by 54

69 the preexisting evidence supporting IAPP as the antigen for BDC-6.9. In this chapter, I recount some early attempts to identify the BDC-6.9 antigen using mass spectrometric analysis of beta cells and present some of the challenges we encountered. The chromatography and mass spectrometry experiments presented in this thesis were done in collaboration with Dr. Thomas Delong, Dr. Nichole Reisdorph (director of the mass spectrometry facility), and several members of the Reisdorph lab. Specialized methods Size exclusion chromatography (SEC) Beta cell tumors were harvested from NOD.RIP-TAg mice and homogenized through 40 µm strainers. Cells were lysed by passing successively through 22, 27, and 30 gauge needles. Following removal of large cellular debris by low-speed centrifugation, secretory granules were pelleted by centrifugation at 18,400 x g and then solubilized in 2% octyl-βglucoside. Insoluble debris was removed by high-speed centrifugation. The soluble fraction was then resolved by SEC on a Superdex /60 column (Amersham Biosciences) at room temperature at a flow rate of 1 ml/minute with either phosphate-buffered saline (PBS) or 100 mm Tris (ph9, 100 mm NaCl) as a running buffer. Fractions (100 fractions, 1.25 ml each) were collected and tested directly in an antigen assay with T cell clones. High-performance liquid chromatography (HPLC) Antigenic SEC fractions were separated on a reverse-phase high-performance liquid chromatography (RP-HPLC) Extend C18 column (Agilent) at a flow rate of 0.15 ml/minute. The following gradient was used: 97% buffer A (30 mm ammonium acetate in water, ph 7.65) and 3% buffer B (30 mm ammonium acetate in 97% acetonitrile) from 0-20 min, 3-15% B from minutes, 15% B from minutes, 15-35% B from minutes, 55

70 35% B from minutes, % B from minutes, 100% B from minutes, 100-3% B from minutes, and 3% B from minutes. Thirty-six fractions were collected over a 120 minute run. Solvents were removed by vacuum evaporation prior to analysis by T cell antigen assay or mass spectrometry. Mass spectrometry Proteins in SEC or HPLC fractions were reduced with either dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) and were in some experiments denatured with trifluoroethanol (TFE) and treated with iodoacetamide (IAM) to alkylate reduced cysteines. Digestion with trypsin was performed overnight at 37 C at a 1:30 enzyme:substrate ratio, followed by inactivation of trypsin by the addition of acid. Digested or undigested samples were resolved by online chromatography (C18 column) at a flow rate of 0.45 µl/minute. For the experiment represented in Table I, the following gradient was used: 97% buffer A (water, 0.1% formic acid) and 3% buffer B (90% acetonitrile/water, 0.1% formic acid) to 10% B by 0.5 minutes, 10-50% B from minutes, 50-80% B from minutes, 80% B from minutes, and 80-3% B from 9-10 minutes. For the experiments represented in Tables II-VI, the following gradient was used: 3-10% buffer B from minutes, 10-50% B from minutes, 50-80% B from minutes, 80% B from minutes, and 80-3% B from minutes. Samples were analyzed on an electrospray ionization (ESI) quadrupole timeof-flight (Q-TOF) mass spectrometer in positive ion mode (Agilent). Peptide fragmentation was done by collision-induced dissociation (CID) using nitrogen as the collision gas. Samples were run either with fragmentation (tandem MS; MS/MS) or without fragmentation (MS only). An acquisition range of m/z was used for MS only experiments and a 56

71 range of m/z was used for the MS scan in MS/MS experiments. Multiply-charged parent ions were selected automatically for fragmentation based on abundance. Collision energy was determined automatically based on the charge state and mass of the parent ion. Parent ions of specific interest were preferentially targeted by addition to a preferred ion list. Analysis of mass spectrometry data MS/MS spectra were analyzed using Spectrum Mill software (Agilent) to determine the sequence of fragmented peptides. Spectrum Mill searches were performed using the Swiss-Prot mouse protein database. Because the NOD proiapp sequence differs from the reference sequence in the database, a custom database including the NOD proiapp sequence was also used. Peptide matches were considered valid when the score was >10 and the percent scored peak intensity (%SPI), which indicates the percentage of the spectrum that could be explained by the interpretation, was >70%. When Spectrum Mill could not interpret a spectrum of interest, manual interpretation was attempted. Mass Profiler Professional software (Agilent) was used to identify peptides with a distribution matching the profile of the T cell clone response. Results Chromatography Because screening of modified synthetic peptides did not identify the BDC-6.9 antigen, we sought to purify the natural antigen from beta cells. Because islets are difficult to obtain in large quantities, we used beta cell tumors from NOD.RIP-TAg mice as starting material. Tumors were mechanically dissociated using a cell strainer to generate a single cell suspension, then cells were lysed by forcing them successively through needles of decreasing bore size. Our lab had previously demonstrated that the antigens for the BDC T cell clones 57

72 are associated with the insulin secretory granule [153]. To enrich for secretory granule proteins, we first removed cellular debris and large organelles by low speed centrifugation. The remaining cellular components were pelleted at high speed and then solubilized in the detergent octyl-β-glucoside. To further purify the antigen, soluble material was fractionated by size exclusion chromatography (SEC; also known as gel filtration chromatography). Chromatographic fractions were collected and tested with BDC-6.9 for antigenicity (Fig. 1). Larger molecules, which have a shorter retention time, elute from SEC columns in earlier fractions. Most of the protein in our samples eluted in early fractions, whereas the BDC-6.9 antigen eluted much later, and thus much of the irrelevant protein in the sample was removed. We further separated antigenic SEC fractions by reverse-phase high-performance liquid chromatography (HPLC). In this technique, a buffer gradient is used; hydrophilic peptides elute earlier in the gradient, followed by hydrophobic peptides. After collecting HPLC fractions, we transferred them to the wells of a 96-well plate and removed solvent by vacuum evaporation. We then tested the fractions for antigenicity with BDC-6.9 (Fig. 2). The BDC-6.9 antigen again eluted in fractions with low total protein content, suggesting that this chromatographic step provided further purification of the antigen. Mass spectrometric analysis of antigenic chromatographic fractions Having purified the BDC-6.9 antigen from beta cells by sequential chromatography, our goal was to identify peptides present in antigenic fractions that could be candidates for the BDC-6.9 antigen. The antigenic HPLC fractions were analyzed using a quadrupole timeof-flight (Q-TOF) mass spectrometer. Parent ions were selected automatically for fragmentation based on abundance. Spectrum Mill software (Agilent) was used to search the 58

73 A IFN-γ (A415) total protein BDC-6.9 response fraction Figure 1: Purification of the BDC-6.9 antigen from NOD.RIP-TAg tumor cells by size exclusion chromatography (SEC). Proteins isolated from lysed NOD.RIP-TAg tumor cells were solubilized in detergent and fractionated by SEC. Fractions were tested with BDC-6.9 for antigenicity. The absorbance by each fraction at 280nm is reported as an indication of total protein content. IFN-γ production was measured by ELISA. Representative of at least three independent experiments. 59

74 A IFN-γ (ng/ml) total protein BDC-6.9 response fraction Figure 2: Separation of an antigenic SEC fraction by high-performance liquid chromatography (HPLC). An antigenic SEC fraction was further resolved using HPLC. HPLC fractions were vacuum evaporated to remove organic solvents, resuspended in culture medium, and tested with BDC-6.9 for antigenicity. The absorbance by each fraction at 210nm is reported as an indication of total protein content. IFN-γ production was measured by ELISA. Representative of at least three independent experiments. 60

75 resulting MS/MS data for spectra matching proteins found in the Swiss-Prot mouse protein database or a custom database including the NOD proiapp sequence. A list of peptides identified in antigenic fractions and the parent protein from which each peptide originated is provided (Table I). We identified several insulin 1 and insulin 2 peptides, but only two IAPP peptides. As many as 50 molecules of insulin are present in the secretory granule for each molecule of IAPP [93], and thus this finding was not surprising. We also searched the data for post-translational modifications, including citrullination, glutamine deamidation, methionine oxidation, and C-terminal amidation. Only one modified peptide an insulin C-peptide fragment with an amidated C-terminus was identified in the samples. We had hypothesized that the antigen for BDC-6.9 was a post-translationally modified version of IAPP1 or IAPP2, but no IAPP1 or IAPP2 peptides modified or unmodified were identified by mass spectrometry. As we only considered a very limited list of possible modifications, the possibility remained that the antigen contained a modification we had not accounted for in our search. Comparing the distributions of peptides and the BDC-6.9 antigen Because the antigenic fractions contained multiple peptides, many of which could not be identified, we needed an approach to narrow the list of peptides to be considered as possible antigens. We chose to search for peptides with a distribution across HPLC fractions that matched the profile of the BDC-6.9 response. If a particular peptide is indeed the antigen for BDC-6.9, the abundance of the peptide in a fraction should correlate with the antigenicity of the fraction. Due to differences in ionization efficiency of different peptides, accurate comparisons cannot be made regarding the abundance of one peptide versus another, 61

76 Table I: Peptides identified by mass spectrometry in antigenic HPLC fractions. Protein Insulin 2 Insulin 1 IAPP (amylin) (B-chain) (C-peptide) (A-chain) (C-peptide) Peptide Sequence LVRSSNNLGPVLPPTNVGSN FVKQHLCG YLVCGERGF LVCGERGFF LVCGERGFF NLGPVLPPTNVGSN REVEDPQVEQLELGGSPGDLQTLALEVARQ REVEDPQVEQLELGGSPGDLQTLALEVARQ EVEDPQVEQLELGG EVEDPQVEQLELGGSPGDLQTL EVEDPQVEQLELGGSPGDLQTLALEVARQ VEDPQVEQLELGGSPGDLQTLALEVARQ GIVDQCCTSICSL IVDQCCTSICSL SRREVEDPQVAQLELGGGPGAGDLQTLALEVAQQ EVEDPQVAQLELGGGPGAGDLQTLA EVEDPQVAQLELGGGPGAGDLQTLALEVAQQ VEDPQVAQLELGGGPGAGDLQTLALEVAQQ ELGGGPGAGDLQTLALEVAQQ Modification amidated C-terminus HPLC fractions were analyzed by tandem MS at an MS scan rate of 5 spectra/second and an MS/MS scan rate of 3 spectra/second. A maximum of 6 precursors were selected for fragmentation per cycle. Precursors were actively excluded for 0.5 minutes after 2 spectra were collected for the same precursor. The protein from which each peptide originated is given. Post-translational modifications are indicated when applicable. Sequences spanning two regions of a protein (e.g., insulin B-chain and C-peptide) are categorized according to the dominant contributor. Sequences were identified based on a Spectrum Mill search of MS/MS data against the Swiss-Prot mouse protein database and a custom database including the NOD proiapp sequence. Matches were validated using automatic thresholds. C-terminal amidation, methionine oxidation, glutamine deamidation, and arginine citrullination were considered as possible modifications in the search. Only proteins identified by two or more peptides are shown. Results are from one experiment. 62

77 but spectral intensity can be used as a surrogate of abundance when comparing the same peptide present in different samples. To analyze the distribution of peptides across HPLC fractions, we used Mass Profiler Professional (MPP) software (Agilent). We identified several peptides as having the correct distribution across fractions, but because the spectral intensities of the parent ions corresponding to these peptides were very low, we were unable to determine the peptide sequences by MS/MS analysis. We therefore decided to analyze SEC fractions by mass spectrometry. We predicted that although these samples would be more complex than HPLC fractions, the total amount of antigen in these fractions would be greater as some antigen is lost with each purification step. To identify peptides with the appropriate distribution, we analyzed all antigenic SEC fractions and some flanking non-antigenic fractions in triplicate MS only runs. Analysis in MS only mode does not involve fragmentation of peptides and thus does not provide information on peptide sequence. However, MS only analysis is more quantitative, allowing more accurate assessment of peptide distribution. Using the MPP software, we discovered seven peptides with the appropriate distribution across fractions (Table II). We then analyzed one of the antigenic fractions in MS/MS mode, specifically targeting these parent ions for fragmentation. Because of the large size of many of these peptides, the fragmentation spectra were difficult to interpret. As the Spectrum Mill software was unable to identify most of the peptides, we attempted to manually interpret the spectra. Although low spectrum quality made it difficult to confidently identify the full sequence of these peptides, four of the seven could be described by insulin C-peptide sequence (Table II). Three of these seemed to contain some sort of modification that prevented the peptide from being completely described by native insulin sequence alone. 63

78 Table II: Peptides matching the distribution of the BDC-6.9 antigen across SEC fractions. Mass/Charge Ratio Charge Retention Time Spectral Intensity Protein (m/z) (z) (minutes) (x10 4 ) insulin 1 C-peptide* n.d insulin 2 C-peptide* insulin 2 C-peptide/A-chain n.d insulin 1 C-peptide* n.d. SEC fractions were analyzed in triplicate by mass spectrometry without fragmentation (MS only). Data were examined using MPP software. Peptides with distributions matching the profile of the BDC-6.9 response across fractions are listed. Masses are in Daltons. Approximate spectral intensities are based on data from MS only runs. An antigenic fraction was then run in fragmentation (MS/MS mode), targeting the peptides listed. Manual interpretation of the spectra allowed prediction of the protein from which the peptide originated. *Peptide seemed to contain a post-translational modification. n.d. = not determined. Results are from one experiment. 64

79 Many peptides not in our candidate list were identified by MS/MS analysis and are shown in Tables III, IV, and V. Most of the peptides were from either insulin 1 (Table III) or insulin 2 (Table IV). The most striking quality of the data was the preponderance of insulin C-peptide fragments differing by truncation of one amino acid from the C-terminus. One fragment, residues 1-26 (C:1-26), has been described in the literature [146,161,162]. The enzyme responsible for generating this fragment is unknown. In addition to insulin peptides, we also discovered several other peptides (Table V), including a series of chromogranin A peptides displaying a similar C-terminal truncation pattern. We also detected the chromogranin A peptide DQLAKELTAE, which is a fragment of the peptide WE14, a weak antigen for the BDC-2.5 clone [139]. The IAPP peptide KCNTATCATQRLAN, part of the epitope for the BDC clone [143], was also discovered. The peptide DPNRESLDFL, which originates from IAPP2, was also present. Since we hypothesized that a modified version of IAPP2 was the antigen for BDC-6.9, this finding was intriguing. Analysis of tryptically digested SEC fractions Our results suggested that the BDC-6.9 antigen might be too large in its native form to sequence by MS/MS analysis. This obstacle is commonly circumvented in mass spectrometry experiments by digesting samples with a protease prior to analysis. Trypsin, a protease commonly used for this purpose, cleaves peptides on the carboxyl side of lysine or arginine residues. We tryptically digested the same SEC fractions characterized in Tables II- V, performed triplicate analysis of each fraction in MS only mode, identified peptides with the appropriate distribution using MPP software, and then targeted these candidates in 65

80 Table III: Insulin 1 peptides identified in an antigenic SEC fraction. Chain B C A Peptide Sequence FVKQHLCGP FVKQHLCGPH FVKQHLCGPHLVEALYLVCGERGFFYTPKS LYLVCGERGF LYLVCGERGFF LYLVCGERGFFYTPKS YLVCGERGF YLVCGERGFF FYTPKS SRREVEDPQVEQLELGGSPGDLQ SRREVEDPQVEQLELGGSPGDLQTLALEVARQ EVEDPQVEQLELGGSPGDL EVEDPQVEQLELGGSPGDLQ EVEDPQVEQLELGGSPGDLQT EVEDPQVEQLELGGSPGDLQTL EVEDPQVEQLELGGSPGDLQTLA EVEDPQVEQLELGGSPGDLQTLAL EVEDPQVEQLELGGSPGDLQTLALE EVEDPQVEQLELGGSPGDLQTLALEVARQ VEDPQVEQLELGGSPGDLQ VEDPQVEQLELGGSPGDLQT VEDPQVEQLELGGSPGDLQTL VEDPQVEQLELGGSPGDLQTLA VEDPQVEQLELGGSPGDLQTLAL DPQVEQLELGGSPGDLQT VEQLELGGSPGDLQ VEQLELGGSPGDLQTLALEVARQ ELGGSPGDLQTLALEVARQ GGSPGDLQTLALEVARQ QTLALEVARQ TLALEVARQ LALEVARQ ALEVARQ GIVDQCCTSICSL GIVDQCCTSICSLYQLENYCN SLYQLENYCN SEC fractions were analyzed by tandem MS at an MS scan rate of 3 spectra/second and an MS/MS scan rate of 2 spectra/second. A maximum of 10 precursors were selected per cycle. Sequences were identified based on a Spectrum Mill search of MS/MS data against the Swiss-Prot mouse protein database. C-terminal amidation, methionine oxidation, glutamine deamidation, and citrullination were considered as possible modifications in the search, but no modified insulin 1 peptides were identified. Results from one experiment. B = B-chain. C = C-peptide. A = A-chain. 66

81 Table IV: Insulin 2 peptides identified in an antigenic SEC fraction. Chain B C A Peptide Sequence FVKQHLCGSHLVEALYLVCGERGFFYTPMS FVKQHLCGSHLVEALYLVCGERGFFYTPMSRREVEDPQ SRREVEDPQVAQLELGGGPGAGDLQ REVEDPQVAQLELGGGPGAGDLQ REVEDPQVAQLELGGGPGAGDLQT EVEDPQVAQL EVEDPQVAQLEL EVEDPQVAQLELGGGPGAGD EVEDPQVAQLELGGGPGAGDL EVEDPQVAQLELGGGPGAGDLQ EVEDPQVAQLELGGGPGAGDLQT EVEDPQVAQLELGGGPGAGDLQTL EVEDPQVAQLELGGGPGAGDLQTLA EVEDPQVAQLELGGGPGAGDLQTLAL EVEDPQVAQLELGGGPGAGDLQTLALEVAQ EVEDPQVAQLELGGGPGAGDLQTLALEVAQQ VEDPQVAQLELGGGPGAGDL VEDPQVAQLELGGGPGAGDLQ VEDPQVAQLELGGGPGAGDLQT VEDPQVAQLELGGGPGAGDLQTLA EDPQVAQLELGGGPGAGDLQ PQVAQLELGGGPGAGDLQT VAQLELGGGPGAGDLQ VAQLELGGGPGAGDLQT VAQLELGGGPGAGDLQTL QLELGGGPGAGDLQ ELGGGPGAGDLQTLALEVAQQ GGGPGAGDLQTLALEVAQQ QTLALEVAQQ TLALEVAQ TLALEVAQQ TLALEVAQQKR EVAQQKRGIVDQCCTSICSLYQLENYCN SEC fractions were analyzed by tandem MS at an MS scan rate of 3 spectra/second and an MS/MS scan rate of 2 spectra/second. A maximum of 10 precursors were selected for fragmentation per cycle. The region of insulin from which each peptide originates is given. Sequences spanning two regions of insulin are categorized according to the dominant contributor. Sequences were identified based on a Spectrum Mill search of MS/MS data against the Swiss-Prot mouse protein database. C-terminal amidation, methionine oxidation, glutamine deamidation, and citrullination were considered as possible modifications in the search, but no modified insulin 2 peptides were identified. Results are from one experiment. B = B-chain. C = C-peptide. A = A-chain. 67

82 Table V: Other peptides identified by mass spectrometry in an antigenic SEC fraction. Protein Peptide Sequence Modification Chromogranin A AEDQELESLSAIEAELEKVAHQL Chromogranin A AEDQELESLSAIEAELEKVAHQLQ Chromogranin A AEDQELESLSAIEAELEKVAHQLQA Chromogranin A AEDQELESLSAIEAELEKVAHQLQAL Chromogranin A AEDQELESLSAIEAELEKVAHQLQALRR amidated C-terminus Chromogranin A AEDQELESLSAIEAELEKVAHQLQALRRG Chromogranin A (WE14) DQLAKELTAE IAPP (amylin) KCNTATCATQRLAN IAPP (IAPP2) DPNRESLDFL Neuroendocrine convertase 2 QELEEELDEAVERSLQSILRKN ProSAAS AVPRGEAAGAVQELARALAHLLEAERQE Secretogranin 2 TNEIVEEQYTPQSLATLESVFQELGKLTGPS Secretogranin 2 TNEIVEEQYTPQSLATLESVFQELGKLTGPSNQ Secretogranin 2 SVFQELGKLTGPSNQ Secretogranin 3 FPKPEGSQDKSLHNRELSAERPLNEQIAEAEADKI Serine racemase TIALEVLNQ SEC fractions were analyzed by tandem MS at an MS scan rate of 3 spectra/second and an MS/MS scan rate of 2 spectra/second. A maximum of 10 precursors were selected for fragmentation per cycle. The protein from which each peptide originates is given. Posttranslational modifications are indicated when applicable. Sequences were identified based on a Spectrum Mill search of tandem mass spectrometry data against the Swiss-Prot mouse protein database and a custom database including the NOD proiapp sequence. C-terminal amidation, methionine oxidation, glutamine deamidation, and arginine citrullination were considered as possible modifications in the search. Results are from one experiment. 68

83 MS/MS analysis of the digested samples. The list of peptides selected based on distribution is provided (Table VI). The spectral intensities of the parent ions corresponding to these peptides were low, resulting in poor product ion spectra. We were only able to identify two of the eight peptides: one was an insulin 1 C-peptide fragment, and the other was a secretogranin peptide. Summary and conclusions Based on compelling evidence, we hypothesized that the antigen for BDC-6.9 was a modified form of IAPP1 or IAPP2. However, a limited screening of modified peptides did not identify a ligand that could activate the clone. As a parallel approach, we tried to identify the natural antigen in beta cells using mass spectrometry. We began with beta cell tumors from NOD.RIP-TAg mice as an antigen source, and then purified the antigen from these cells by SEC and HPLC, using the T cell clone to track the antigen. SEC and HPLC fractions were then analyzed by mass spectrometry, looking for peptides whose distribution matched the distribution of the antigen across the fractions as measured by the T cell response. The analysis returned several candidates, but most of these peptides could not be fully sequenced by tandem mass spectrometry. However, our analyses did return a few insulin C-peptide fragments as possible antigens, with some of these appearing to be post-translationally modified. Although the genetic linkage analysis implicated IAPP as the BDC-6.9 antigen, we began to consider the possibility that a modified insulin peptide might be the antigen for BDC

84 Table VI: Tryptically-digested peptides matching the distribution of the BDC-6.9 antigen across SEC fractions. Mass/Charge Ratio Charge Retention Time Spectral Intensity Protein (m/z) (z) (minutes) (x10 4 ) n.d n.d n.d insulin 1 C-peptide n.d n.d n.d secretogranin Tryptically-digested SEC fractions were analyzed in triplicate by mass spectrometry without fragmentation (MS only). Data were examined using MPP software. Peptides with distributions matching the profile of the BDC-6.9 response across fractions are listed. Masses are in Daltons. Approximate spectral intensities are based on data from MS only runs. An antigenic fraction was then run in fragmentation (MS/MS) mode, targeting the peptides listed. Manual interpretation of the spectra allowed prediction of the protein from which the peptide originated. n.d. = not determined. Results are from one experiment. 70

85 CHAPTER V A BDC-6.9 MIMOTOPE Background and rationale Our efforts to identify the BDC-6.9 antigen by screening synthetic IAPP peptides or by mass spectrometric analysis of beta cell proteins had generated conflicting results. Additional information about the antigen was needed to inform our next steps. The discovery of a peptide mimotope for BDC-6.9 could provide additional clues about the natural antigen, but no such mimotope had yet been identified for the clone. Mimotopes, so called because they mimic the sequence of epitopes, are peptide sequences that are antigenic for a T cell but are not present in vivo. Mimotopes can be used to make predictions about the natural antigen. This is generally done by comparing multiple mimotopes for homology to identify certain conserved motifs or by mutating the mimotope to identify critical residues. This information can then be used to identify natural peptides with these key features. Discovery of WE14 as a weak antigen for BDC-2.5 was greatly aided by the prior identification of several peptide mimotopes [139]. Several years ago, a collaborator, Dr. Maki Nakayama, was screening mutated insulin peptides with B:9-23-reactive T cell hybridomas and she used a hybridoma expressing the BDC-6.9 TCR as a control responder. As expected, this hybridoma did not respond to B:9-23, but it was noted that there was a response to a few mutated peptides. Because B:9-23 did not stimulate BDC-6.9, and because the other peptides were not naturally occurring peptides, we initially assumed that these responses were artifacts of the hybridoma system. However, after we failed to identify the antigen for BDC-6.9 by mass spectrometry or by screening IAPP 71

86 peptides, we decided to test if any of the B:9-23 variants antigenic for the BDC-6.9 hybridoma were likewise antigenic for BDC-6.9. Specialized methods Tolerance induction with Ag-SP Mice adoptively transferred with activated CD4 T cell clones were treated one day later with i.p. injection of antigen-coupled spleen cells (Ag-SP) in PBS. Ag-SP were prepared by incubating splenocytes (3.2x10 8 /ml) for one hour at 4 C with periodic gentle mixing in the presence of 1 mg/ml peptide and mg/ml 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC). Reactions were performed in PBS. Prior to injection, cells were washed with PBS to remove unreacted EDC and peptide, filtered to remove cell clumps, and then counted. Mice were injected with 5x10 7 coupled leukocytes. Injections were performed as soon as possible following completion of the coupling reaction. Statistical analysis Survival analysis was performed by applying the log-rank test using Prism 7.0a software. All possible pairwise comparisons were evaluated for significance within each figure (Fig. 9, 10, 11). Comparisons were considered significant when p < When comparing more than two groups (Fig. 9, 10), the Bonferroni-corrected threshold was calculated and used to evaluate significance in order to compensate for multiple comparisons. Corrected thresholds are reported in the figure legends. P-values are only shown for comparisons in which statistical significance was achieved. 72

87 Results B:9-23(13Q) Based on Dr. Nakayama s evidence that some mutated B:9-23 peptides stimulated a hybridoma expressing the BDC-6.9 TCR, we decided to test the mutated peptides with the BDC-6.9 T cell clone. We observed that the mutated insulin B:9-23 peptide B:9-23(13Q) (SHLVQALYLVCGERG) was antigenic for BDC-6.9 and BDC-9.3 but not for the B:9-23- reactive clone PD [98] (Fig. 1). Because conversion of peptidyl glutamic acid to form glutamine is not known to happen in vivo, it was apparent that this peptide was not the natural antigen for BDC-6.9 but rather a mimotope. Nonetheless, this finding was seminal for two key reasons. First, it identified a peptide that could be used as a positive control in antigen assays, eliminating the need for using a natural antigen source. Secondly, and more importantly, this provided a critical piece of evidence in our effort to elucidate the BDC-6.9 antigen as no other peptide had ever been identified that could consistently stimulate BDC We next began testing modified versions of B:9-23(13Q) to determine which residues were critical for recognition in the hope that we might use this information to identify a natural sequence with potential for antigenicity. A list of these peptides and their sequences is provided (Table I). The T cell clone BDC-9.3, which has the same TCR as BDC-6.9, was used in many of these assays because it grows better in culture than BDC-6.9 and responds more reliably to antigen. Truncations We first sought to identify a minimal epitope required for antigenicity. This would then allow us to restrict future mutations to those residues likely involved in TCR recognition or MHC binding. Our tests showed that the first three residues, SHL, could be truncated with 73

88 A PD B: B:9-23(13Q) peptide (µg/ml) IFN-γ (ng/ml) B 100 BDC IFN-γ (ng/ml) peptide (µg/ml) C BDC IFN-γ (ng/ml) peptide (µg/ml) Figure 1: The mutated insulin peptide B:9-23(13Q) is antigenic for BDC-6.9 and BDC The insulin B-chain epitope B:9-23 and the mutated peptide B:9-23(13Q) were tested with (A) the insulin-reactive clone PD12-4.4, (B) BDC-6.9, or (C) BDC-9.3. Results are representative of at least three independent experiments. 74

89 Table I: Sequences of B:9-23 variants tested with BDC-6.9/BDC-9.3. Peptide Name Peptide Sequence B: 9-23 SHLVEALYLVCGERG B: 9-23(13Q) SHLVQALYLVCGERG B:12-23(13Q) VQALYLVCGERG B:13-23(13Q) QALYLVCGERG B:14-23 ALYLVCGERG B: 9-22(13Q) SHLVQALYLVCGER B: 9-21(13Q) SHLVQALYLVCGE B: 9-20(13Q) SHLVQALYLVCG B: 9-23(13Q16A) SHLVQALALVCGERG B: 9-23(13Q19A) SHLVQALYLVAGERG B: 9-23(13A) SHLVAALYLVCGERG B: 9-23(13N) SHLVNALYLVCGERG B: 9-23(13K) SHLVKALYLVCGERG B: 9-23(13R) SHLVRALYLVCGERG B: 9-23(13Q)r1 SHLVQALYAVCEERG B: 9-23(13Q)r3 SHLVQVLYLVAGEEG Names and sequences of B:9-23 peptide variants tested with BDC-6.9/BDC-9.3 for antigenicity. Residues that differ from the natural B:9-23 sequence are indicated by bold lettering. I-Ag7 anchor residues introduced in register-trapped peptides are underlined. 75

90 little effect on antigenicity (Fig. 2). However, removal of only one amino acid from the C- terminus drastically reduced antigenicity (Fig. 3). We therefore considered the sequence VQALYLVCGERG [B:12-23(13Q)] to be the minimal epitope. Positional/alanine scanning Using B:9-23(13Q) as a base peptide, we did a limited alanine scan to determine residues important for antigenicity. Substitution of an alanine for the tyrosine at position 16 or the cysteine at position 19 had little effect on antigenicity (Fig. 4). However, substitution of alanine for the glutamine in position 13 completely abolished activity (Fig. 5a). We next questioned if asparagine (N), which is chemically very similar to glutamine, could suffice in position 13. This mutation also destroyed antigenicity (Fig. 5b), as did substitution with lysine (K) or arginine (R) (Fig. 5c). The BDC-6.9 TCR thus seemed highly sensitive to the glutamine in position 13. Register trapping We next were interested in determining which amino acids in the B:9-23(13Q) sequence were critical for TCR recognition and which were important for anchoring the peptide to I-Ag7. To do this, we used a technique called register trapping [19]. Based on knowledge of residues preferred in each binding pocket of I-Ag7, one can modify a peptide to bind strongly in different registers. In naming register-trapped B:9-23(13Q) peptides, we followed the conventional nomenclature for the B:9-23 registers [19]. Based on published data [21], we chose valine (V), leucine (L), alanine (A), and glutamic acid (E) to anchor the peptides to the p1, p4, p6, and p9 pockets of I-Ag7, respectively. Thus register-trapped peptides will contain the sequence VxxLxxAxxE. Residues that are not fixed presumably interact with the TCR. We tested the B:9-23(13Q) peptide trapped in either register 1 76

91 A IFN-γ (ng/ml) peptide (µg/ml) SHLVQALYLVCGERG VQALYLVCGERG B IFN-γ (ng/ml) peptide (µg/ml) SHLVQALYLVCGERG QALYLVCGERG C IFN-γ (ng/ml) peptide (µg/ml) SHLVQALYLVCGERG ALYLVCGERG Figure 2: The first three N-terminal amino acids of B:9-23(13Q) are dispensable for antigenicity. BDC-9.3 was tested with N-terminally truncated variants of B:9-23(13Q). Results are representative of at least two experiments. 77

92 A IFN-γ (ng/ml) peptide (µg/ml) SHLVQALYLVCGERG SHLVQALYLVCGER B IFN-γ (ng/ml) peptide (µg/ml) SHLVQALYLVCGERG SHLVQALYLVCGE SHLVQALYLVCG Figure 3: BDC-9.3 is sensitive to C-terminal truncation of B:9-23(13Q). BDC-9.3 was tested with C-terminally truncated variants of B:9-23(13Q). Results are representative of two experiments. 78

93 A IFN-γ (ng/ml) peptide (µg/ml) B:9-23(13Q) B:9-23(13Q16A) B IFN-γ (ng/ml) peptide (µg/ml) B:9-23(13Q) B:9-23(13Q19A) Figure 4: BDC-9.3 is permissive of alanine residues at positions 16 and 19 of B:9-23(13Q). BDC-9.3 was tested with variants of B:9-23(13Q) in which alanine had been substituted for (A) the tyrosine at position 16 or (B) the cysteine at position 19. Results are representative of at least two experiments. 79

94 A IFN-γ (ng/ml) peptide (µg/ml) B:9-23(13Q) B:9-23(13A) B IFN-γ (ng/ml) peptide (µg/ml) B:9-23(13Q) B:9-23(13N) C IFN-γ (ng/ml) peptide (µg/ml) B:9-23(13Q) B:9-23(13K) B:9-23(13R) Figure 5: A glutamine residue at position 13 is critical for antigenicity. BDC-9.3 was tested with variants of B:9-23(13Q) in which the glutamine in position 13 had been replaced with (A) alanine, (B) glutamine, asparagine, (C) lysine, or arginine. Results are representative of two experiments. 80

95 [B:9-23(13Q)r1] or register 3 [B:9-23(13Q)r3]. Schematics of these registers are shown (Fig. 6). Trapping B:9-23(13Q) in register 1 abrogated antigenicity, whereas trapping the peptide in register 3 maintained antigenicity (Fig. 7), suggesting that this is a register in which the peptide is recognized by the T cell clone. Inducing tolerance to B:9-23(13Q) As discussed in the introduction to this thesis, antigen-coupled spleen cell (Ag-SP) therapy is a promising approach for inducing antigen-specific tolerance in autoimmunity [114]. A knowledge of which antigens are major targets of autoreactivity in disease is important in the design of antigen-specific tolerance induction strategies. Although insulin has been shown to be an important antigen in autoimmune diabetes, administration of insulin-coupled spleen cells (INS-SP) was only successful in reversing spontaneous disease in roughly half of recent-onset diabetic NOD mice [119]. One possible explanation for this result is that insulin was not the dominant autoantigen in the mice that were refractory to treatment. We hypothesized that concomitantly targeting multiple antigens would result in a higher success rate. Having discovered that B:9-23(13Q) was antigenic for the diabetogenic T cell clones BDC-6.9 and BDC-9.3, we were interested in investigating if inducing tolerance to this peptide could augment treatment with INS-SP. Disease reversal experiments in spontaneously diabetic NOD mice are technically difficult because onset of disease cannot be predicted. As a more controlled approach to initially test peptides for their ability to induce tolerance, we used an adoptive clone transfer system. The BDC clones transfer disease generally within two weeks when transferred into NOD pups one to two weeks of age [38,39]. We initially tested the ability of KS20-SP to prevent induction of disease by BDC-5.2.9, a clone reactive to the IAPP peptide KS20 [143]. 81

96 Register 1 BDC-6.9 TCR S H L Q A Y V C E R G V L L G Register 3 I-Ag7 BDC-6.9 TCR S H L V Q L Y V G E G A L C R I-Ag7 Figure 6: I-Ag7 binding registers one (r1) and three (r3) for the peptide B:9-23(13Q). Schematics representing two possible binding registers for the BDC-6.9 mimotope B:9-23(13Q) showing the interaction of amino residues with the BDC-6.9 TCR and the pockets of the I-Ag7 binding register. 82

97 IFN-γ (ng/ml) peptide (µg/ml) B:9-23(13Q) B:9-23(13Q)r1 B:9-23(13Q)r3 Figure 7: B:9-23(13Q) is antigenic for BDC-9.3 when trapped in register 3 (r3). BDC-9.3 was tested with B:9-23(13Q) peptides containing substitutions that trapped the peptide in specific binding registers. Results are representative of two experiments. 83

98 Ten million activated BDC T cells were injected i.p. into 1-2-week-old NOD mice. The following day, 5x10 7 KS20-SP were administered i.p. Whereas almost all of the untreated mice developed disease, half of the mice receiving KS20-SP treatment were protected (Fig. 8). Treatment with splenocytes coupled to the irrelevant peptide WE14 or to the peptide CF14 (CNTATCATQRLANF), a truncated version of KS20 that is only weakly antigenic for BDC-5.2.9, did not offer protection from disease. Treatment with amylin-sp seemed to offer some protection compared to untreated mice, but this difference was not significant. We next tried preventing transfer of disease by BDC-6.9 and BDC-9.3 using B:9-23(13Q)-SP. Treatment prevented transfer of disease by BDC-6.9 with almost 100% efficiency, whereas treatment with B:9-23-SP was ineffective (Fig. 9). Treatment was unsuccessful in pups receiving BDC-9.3 (Fig. 10). As this clone responds more robustly than BDC-6.9 to B:9-23(13Q), the requirements for inducing tolerance may be different. After obtaining the results shown in Figure 9, a fourth experiment failed to demonstrate protection from disease induced by transfer of BDC-6.9, suggesting that this treatment may not be as robust as we originally thought. Due to this and other difficulties that arose with the experimental system, we focused our efforts on identifying the natural antigen for BDC-6.9. The use of Ag-SP treatment to prevent or reverse autoimmune diabetes remains an intriguing approach that merits further investigation in the context of newly-defined autoantigens. Summary and conclusions As a major advance in our efforts to identify the BDC-6.9 antigen, we determined that the mutated insulin peptide B:9-23(13Q) was antigenic for BDC-6.9. However, this is not a naturally occurring sequence. By testing truncated and mutated versions of this peptide, we found that the minimal epitope needed for recognition by the T cell was VQALYLVCGERG 84

99 BDC % diabetes free days post disease transfer untreated (n=20) WE14-SP (n=2) KS20-SP (n=14) amylin-sp (n=6) CF14-SP (n=10) p=0.001 p=0.003 Figure 8: Treatment with KS20-SP prevents disease induction by adoptive transfer of activated BDC T cells. Ten million activated BDC T cells were adoptively transferred into young (1-2-week-old) NOD mice. One day later, mice were treated with 5x10 7 Ag-SP. Mice were monitored daily for disease onset. P-values for comparisons between groups are indicated in the legend when statistically significant. A Bonferronicorrected threshold of p = was used for evaluating significance. Results for each group are pooled from at least two independent experiments, with the exception of the results for WE14-SP treatment, which are from one experiment. 85

100 BDC % diabetes free 50 untreated (n = 10) B:9-23-SP (n = 2) B:9-23(13Q)-SP (n = 9) p= p< days post clone transfer Figure 9: Treatment with B:9-23(13Q)-SP prevents disease induction by adoptive transfer of activated BDC-6.9 T cells. Ten million activated BDC-6.9 T cells were adoptively transferred into young (1-2-week-old) NOD mice. One day later, mice were treated with 5x10 7 Ag-SP. Mice were monitored daily for disease onset. P-values for comparisons between groups are indicated in the legend when statistically significant. A Bonferroni-corrected threshold of p = was used for evaluating significance. Results are pooled from three independent experiments. B:9-23-SP were only tested in one experiment. 86

101 BDC % diabetes free days post clone transfer untreated (n = 5) B:9-23(13Q)-SP (n = 5) Figure 10: Treatment with B:9-23(13Q)-SP does not prevent disease induction by adoptive transfer of activated BDC-9.3 T cells. Ten million activated BDC-9.3 T cells were adoptively transferred into young (1-2-week-old) NOD mice. One day later, mice were treated with 5x10 7 Ag-SP. Mice were monitored daily for disease onset. The difference between the two groups was not significant. Results are pooled from two independent experiments. 87

102 and that the glutamine residue was essential for antigenicity. Using the Ag-SP technique, we demonstrated that B:9-23(13Q)-SP could prevent transfer of disease by BDC-6.9 into NOD pups. However, these results were not reproducible in every experiment, suggesting that further optimization was needed to improve the reliability of the technique. At this point, we began to devote our full attention to identifying the natural antigen for BDC

103 CHAPTER VI THE BDC-6.9 HYBRID INSULIN PEPTIDE (6.9HIP) 2 Background and rationale A major goal in the lab s research on autoantigens for the diabetogenic BDC T cell clones was to identify the antigen for the BDC-2.5 clone, the best known clone in the panel and the first used to develop a TCR transgenic NOD mouse [42]. It had been previously demonstrated that the peptide WE14 (WSRMDQLAKELTAE), a natural cleavage product of chromogranin A, is weakly antigenic for BDC-2.5 [139]. It was shown that this peptide was likely to fill only half of the I-Ag7 binding groove, with the first five amino acids, WSRMD, situated in p5 through p9. Prior to cleavage from the chromogranin A protein, the WE14 sequence is preceded by the amino acids EDKR. An N-terminal extension of WE14 containing these residues, which would presumably fill p1 through p4 of the I-Ag7 binding groove, was synthesized for testing but was not antigenic [139]. Based on these findings, it was hypothesized that the natural antigen for BDC-2.5 was a modified chromogranin A peptide that contained the WE14 sequence and yet filled more of the I-Ag7 peptide binding groove. Dr. Thomas Delong in our group began pursuing the possibility that peptide sequence originating from a different protein might complement the WE14 sequence in the I-Ag7 binding groove to form the complete BDC-2.5 antigen. Analysis of chromatographically fractionated beta cell extract by mass spectrometry had identified several insulin C-peptide fragments that followed the distribution of the BDC-2.5 antigen across the fractions [163], and preliminary data indicated that some of these peptides had reactive C-termini 2 Portions of this chapter have been published [163,165] and are included here with the permission of Science and Journal of Autoimmunity. 89

104 (Thomas Delong, unpublished data). One such peptide, des(27-31)c-peptide [161,162,164], which had been described previously in the literature and which we will refer to here as C:1-26, was of particular interest because it shared homology with several previously defined mimotopes for BDC-2.5 (data not shown). This peptide appeared consistently in mass spectrometric analysis of our antigenic chromatographic fractions. Thomas Delong hypothesized that the natural antigen for BDC-2.5 consisted of WE14 covalently bound at the N-terminus to the C-terminus of the insulin fragment C:1-26, forming a hybrid insulin peptide (HIP). He further posited that different HIPs might be antigenic for other T cell clones in the BDC panel. In this chapter, we recount how the pursuit of this hypothesis led to the discovery of the BDC-6.9 antigen. Specialized methods Peptide cross-linking The peptides Ac-SSTLAL and Ac-RRALQTLAL were obtained commercially. The two serine or arginine residues were included at the N-terminus of the peptide to improve solubility, and the N-terminus was acetylated to protect it from cross-linking. The C-terminus of the peptide (2 mm) was activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC; 2 mm) and N-hydroxysulfosuccinimide (sulfo-nhs; 5 mm) for 20 minutes at room temperature. Excess EDC was quenched by addition of beta-mercaptoethanol. An excess of either WE14 or NOD IAPP2 was then added and incubated at 37 C for 2 hours. Reactions were tested with T cell clones for antigenicity. CFSE proliferation assay with transgenic T cells Splenocytes from NOD BDC-6.9 TCR-Tg mice were stained with carboxyfluorescein succinimidyl ester (CFSE). Cells (1 x 10 6 /well) were transferred to a 96-well plate and 90

105 α-cd28 and recombinant IL-2 were added to final concentrations of 100 ng/ml and 10 U/ml, respectively. Cells were tested with the indicated peptides or with α-cd3 (200 ng/ml) as a positive control. Cells were cultured four days at 37 C, harvested, counterstained with antibodies, and analyzed for CFSE dilution by flow cytometry. I-Ag7 peptide binding assay I-Ag7:CLIP monomer (NIH tetramer core) was treated with thrombin to cleave linked CLIP peptide. The biotinylated HEL peptide biotin-gggmkrhgldnyrgysl (bio-hel; 66.9 µm or 22.2 µm) was mixed 1:1 with 2 µm I-Ag7 monomer (NIH tetramer core) and 10 µl aliquots were transferred to 96-well polypropylene plates. Competitor peptide dilutions (5 µl) were then added and samples were incubated overnight at room temperature. Binding competition was performed in citrate buffer (500 mm, ph 5.3). Following incubation, samples were diluted in Tris buffer and transferred to plates coated with α-i-ag7 antibody (OX6), and bound I-Ag7:bio-HEL complex was detected using streptavidin-coupled peroxidase. Results The BDC-2.5 hybrid insulin peptide (2.5HIP) To test if hybrid insulin peptides (HIPs) were antigenic for any of the BDC clones, Dr. Delong devised a cost-effective method for generating HIP libraries. We obtained a commercially prepared selection of short peptides representing the C-termini of insulin C- peptide fragments and began coupling these to the N-termini of other peptides using the chemical cross-linking agent EDC. To do this, the C-terminus of each short insulin peptide was activated by incubation with sulfo-nhs and EDC, generating a stable NHS ester intermediate. Activated peptides were then incubated with other peptides such as WE14 and 91

106 IAPP2 to form HIPs. In this reaction, the activated C-terminus of the insulin peptide forms an amide bond (or fuses) with the N-terminal amine group of the other peptide present in the reaction. By obtaining short peptide sequences and coupling them to various peptides in this manner, we were able, for a limited cost, to generate large panels of hybrid peptides for screening with the BDC clones. Although the work done by the lab to screen large HIP panels with multiple BDC T cell clones is outside the scope of this thesis, the earliest experiments in which a limited selection of cross-linked peptides were tested with BDC-2.5 provided the first evidence implicating HIPs as autoantigens. Therefore, some of these initial results are discussed here. One of the first short insulin sequences tested with the EDC crosslinking technique was TLAL, representing the C-terminus of C:1-26. The peptide was obtained with serine residues added to the N-terminus to improve water solubility, and the N-terminus was acetylated to prevent cross-linking of the activated peptide molecules to one another. The peptide acetyl-sstlal was linked to either WE14 or NOD IAPP2. When we tested the resulting hybrid peptides, the WE14 hybrid was much more antigenic than unmodified WE14 for BDC-2.5 (Fig. 1). This was our first evidence that hybrid peptides may be antigens for CD4 T cells in the NOD mouse. B:9-23(13Q) and homology to natural sequence Our earlier data indicated that the peptide sequence VQALYLVCGERG [B:9-23(13Q)] was the minimal sequence from the mimotope B:9-23(13Q) that maintained full antigenicity for BDC-6.9. While searching for peptide sequences that shared homology with this core mimotope sequence, we obtained our first data implicating a HIP as an antigen for BDC-2.5 (Fig. 1). We noted that forming a hybrid peptide between the C:1-26 LQTLAL 92

107 peptide (µm) IFN-γ (ng/ml) BDC-2.5 WE14 Ac-SSTLAL-WE14 Ac-SSTLAL-IAPP2 Figure 1: Cross-linking the insulin C-peptide sequence TLAL to the N-terminus of WE14 increases antigenicity for BDC-2.5. WE14 or NOD IAPP2 was cross-linked to the C-terminus of the peptide Ac-SSTLAL. Cross-linked peptides were tested with BDC-2.5 for antigenicity. Results are from one experiment. The peptide Ac-RRALQTLAL, which contains two naturally preceding C-peptide residues (leucine and glutamine) and two arginine residues to improve solubility, was used in a second experiment. The second experiment yielded similar results. Concentrations for cross-linked peptides were calculated assuming 100% yield for the cross-linking reaction. Ac = acetylated N-terminus. 93

108 fragment and the N-terminus of NOD IAPP2 would generate the sequence LQTLAL- NAARDP (hyphenation is used for convenience to denote the transition from insulin to IAPP sequence). We had observed that alanine substitution at positions 16 and 19 of B:9-23(13Q) had little effect on antigenicity. Alignment of LQTLAL-NAARDP and the sequence for B:9-23(13Q16A19A) revealed roughly 40% homology (Fig. 2). Antigenicity of an insulin-iapp hybrid peptide for BDC-6.9 Based on this homology, we hypothesized that a hybrid insulin peptide formed by linkage of C:1-26 to the N-terminus of NOD IAPP2 would be antigenic for BDC-6.9. We designated this hypothetical BDC-6.9 antigen as 6.9HIP. To test our hypothesis, we obtained commercially synthesized hybrid insulin peptides (HIPs). A list of peptides obtained for testing is provided (Table I). We confirmed that full-length 2.5HIP:2, which is composed of the full insulin 2 C-peptide fragment (C2:1-26) linked to the full WE14 peptide through a traditional peptide bond, is highly antigenic for BDC-2.5 (Fig. 3a). C2:1-26 by itself was not antigenic for BDC-2.5, nor was the full-length hybrid consisting of C2:1-26 fused to NOD IAPP2 (6.9HIP:2). On the other hand, 6.9HIP:2, but not C2:1-26 or 2.5HIP:2, was highly antigenic for both BDC-6.9 (Fig. 3b) and BDC-9.3 (Fig. 3c). We next tested if full-length insulin 1 or insulin 2 C-peptide (C1 or C2, respectively) was antigenic and found that neither was antigenic for BDC-6.9 (Fig. 4a) or BDC-9.3 (Fig. 4b). To test if covalent linkage of the insulin and IAPP sequences was necessary for antigenicity, we tested the clones with a combination of C:1-26 and IAPP2 that were not linked. This peptide mixture was not antigenic for the clones (Fig. 4a,b), indicating that the hybrid peptides were not a product simply resulting from combination in the APC. Homology between B:12-23(13Q) and 6.9HIP suggested that the BDC-6.9 epitope spanned 94

109 B:9-23(13Q16A19A) 6.9HIP V Q A L A L V A G E R G L Q T L A L N A A R D P Figure 2: 6.9HIP shares sequence homology with B:9-23(13Q16A19A). Although not directly tested, mutational analysis indicated that the peptide B:9-23(13Q16A19A) should be antigenic for BDC-6.9. The sequences for this peptide and for the BDC-6.9 hybrid insulin peptide (6.9HIP) are provided for comparison. Residues shared by both peptides are indicated. 95

110 Table I: Sequences of hybrid insulin peptides and their constituents. Peptide insulin 1 C-peptide (C1) insulin 2 C-peptide (C2) insulin 1 C:1-26 (C1:1-26) insulin 2 C:1-26 (C2:1-26) WE14 NOD IAPP2 BALB/c IAPP2 2.5HIP:2 6.9HIP:1 6.9HIP:2 6.9HIP:core 6.9HIP:R G (BALB/c) Sequence EVEDPQVEQLELGGSPGDLQTLALEVARQ EVEDPQVAQLELGGGPGAGDLQTLALEVAQQ EVEDPQVEQLELGGSPGDLQTLAL EVEDPQVAQLELGGGPGAGDLQTLAL EVEDPQVAQLELGGGPGAGDLQTLAL-WSRMDQLAKELTAE EVEDPQVAQLELGGGPGAGDLQTLAL-NAARDPNRESLDFLLV EVEDPQVAQLELGGGPGAGDLQTLAL-NAAGDPNRESLDFLLV EVEDPQVAQLELGGGPGAGDLQTLAL-WSRMDQLAKELTAE EVEDPQVEQLELGGSPGDLQTLAL-NAARDPNRESLDFLLV EVEDPQVAQLELGGGPGAGDLQTLAL-NAARDPNRESLDFLLV LQTLAL-NAARDP LQTLAL-NAAGDP The sequences of HIPs and HIP constituents tested for antigenicity are provided. BALB/cspecific residues are underlined. Hyphenation is used to denote the transition from insulin sequence to WE14 or IAPP2 sequence. 96

111 A B C BDC peptide (µm) IFN-γ (ng/ml) BDC peptide (µm) IFN-γ (ng/ml) BDC peptide (µm) IFN-γ (ng/ml) WE14 C2: HIP:2 6.9HIP:2 IAPP2 C2: HIP:2 6.9HIP:2 IAPP2 C2: HIP:2 6.9HIP:2 Figure 3: The full-length hybrid peptide consisting of insulin 2 C:1-26 on the N- terminal side and NOD IAPP2 on the C-terminal side is highly antigenic for BDC-6.9 and BDC-9.3. The insulin 2 version of full-length 2.5HIP and 6.9HIP, as well as their constituent peptides, were tested with (A) BDC-2.5, (B) BDC-6.9, and (C) BDC-9.3 for antigenicity. Results are representative of at least three independent experiments. 97

112 A IFN-γ (ng/ml) BDC peptide (µm) NOD proiapp C1 C2 C1: IAPP2 C2: IAPP2 6.9HIP:1 6.9HIP:2 B IFN-γ (ng/ml) BDC peptide (µm) NOD proiapp C1 C2 C1: IAPP2 C2: IAPP2 6.9HIP:1 6.9HIP:2 Figure 4: The insulin 1 and insulin 2 versions of full-length 6.9HIP are equally antigenic for BDC-6.9. (A) BDC-6.9 and (B) BDC-9.3 were tested with full-length NOD proiapp, insulin 1 and insulin 2 C-peptide (C1 and C2, respectively), and the insulin 1 and insulin 2 versions of full-length 6.9HIP. Additionally, IAPP2 was tested in combination with either C1:1-26 or C2:1-26 to confirm that covalent linkage of the C-peptide fragment and IAPP2 is required for antigenicity. Representative of at least two independent experiments. C1: IAPP2 was tested in only one experiment. 98

113 the hybrid peptide junction. Because insulin 1 and 2 C-peptide are identical in the region that formed this junction, we predicted that the clones would not discriminate between full-length 6.9HIP formed with C1:1-26 or C2:1-26. Testing of these two forms of the hybrid, 6.9HIP:1 and 6.9HIP:2, confirmed this hypothesis (Fig. 4). Furthermore, the minimal 12-mer sequence LQTLAL-NAARDP (6.9HIP:core) was only slightly less antigenic than 6.9HIP:2 for both clones (Fig 5). BALB/c 6.9HIP (6.9HIP:R G) BDC-6.9 and BDC-9.3 do not respond to islets from BALB/c mice. We therefore hypothesized that the BALB/c counterpart of 6.9HIP, in which glycine (G) would be substituted for an arginine (R), would not be antigenic for these clones. We tested BDC-6.9 and BDC-9.3 with the 6.9HIP:core sequence and the corresponding BALB/c core sequence 6.9HIP:R G (LQTLAL-NAAGDP). 6.9HIP:R G was only weakly antigenic for BDC-6.9 and BDC-9.3 (Fig. 5b,c). These results were confirmed using splenocytes from the NOD BDC-6.9 TCR-Tg mouse as responder T cells (Fig. 6). Failure of 6.9HIP:R G to stimulate BDC-6.9 and BDC-9.3 at low concentrations provides a plausible explanation as to why these clones do not respond to BALB/c islets. 6.9HIP truncation and mutational analysis To determine a minimal epitope for 6.9HIP-reactive T cell clones, we tested BDC-6.9 and BDC-9.3 with N- and C-terminally truncated versions of 6.9HIP:core. Removal of the N- terminal leucine (L) from 6.9HIP notably reduced antigenicity, whereas removal of the N- terminal leucine and glutamine (Q) entirely abolished antigenicity (Fig. 7a). Removal of the C-terminal proline (P) had little effect, but removal of both the proline and aspartic acid (D) 99

114 A BDC IFN-γ (ng/ml) peptide (µm) B:9-23 C2: IAPP2 6.9HIP:2 6.9 HIP:core 6.9HIP:R G B BDC peptide (µm) IFN-γ (ng/ml) B:9-23 C2: IAPP2 6.9HIP:2 6.9 HIP:core 6.9HIP:R G C BDC peptide (µm) IFN-γ (ng/ml) B:9-23 C2: IAPP2 6.9HIP:2 6.9 HIP:core 6.9HIP:R G Figure 5: A core 12-mer peptide of 6.9HIP containing the NOD (but not BALB/c) IAPP sequence is highly antigenic for BDC-6.9. T cell clones were tested with the 12-mer sequence LQTLAL-NAARDP (6.9HIP:core) to confirm that the BDC-6.9 epitope is centered on the hybrid peptide junction. The BALB/c version of the core, LQTLAL-NAAGDP (6.9HIP:R G), was also tested. Results are representative of at least three independent experiments. 100

115 50 µm LQTLAL-NAARDP 62.3 % CFSE LQTLAL-NAAGDP 61.8 % Figure 6: T cells from NOD BDC-6.9 TCR-Tg mice proliferate in response to 6.9HIP:core (but not 6.9HIP:R G) at very low concentrations. Splenocytes from BDC- 6.9 TCR-Tg mice were labeled with CFSE and then cultured with varying concentrations of 6.9HIP:core or 6.9HIP:R G. After four days, cells were analyzed by flow cytometry for CFSE dilution as an indicator of proliferation. The percentage of the original population that was induced to proliferate was calculated using the FlowJo (Tree Star) proliferation platform and is indicated in the figure. Results are representative of two independent experiments. 101

116 A BDC peptide (µm) IFN-γ (ng/ml) LQTLAL-NAARDP QTLAL-NAARDP TLAL-NAARDP B BDC peptide (µm) IFN-γ (ng/ml) LQTLAL-NAARDP LQTLAL-NAARD LQTLAL-NAAR C BDC peptide (µm) IFN-γ (ng/ml) LQTLAL-NAARDP LATLAL-NAARDP LQTLAL-NAARAP LQTLAL-NAARRP Figure 7: Amino acid residues in both the insulin and IAPP regions of 6.9HIP are critical for antigenicity. BDC-6.9 was tested with truncated or mutated 6.9HIP peptides to define a minimal epitope and residues critical for antigenicity. (A) Peptides truncated at the N-terminus. (B) Peptides truncated at the C-terminus. (C) Peptides with substitutions at critical residues. Results in (A-C) are from the same assay plate; accordingly, values for the LQTLAL-NAARDP peptide are identical in all three figures. Results are representative of three independent experiments. The peptide LQTLAL-NAARAP was only tested in two experiments. 102

117 eliminated activity (Fig. 7b). Therefore, the 11-mer peptide LQTLAL-NAARD appeared to be the minimal epitope for BDC-6.9. To confirm the importance of the glutamine near the N-terminus of the peptide, the mutant peptide LATLAL-NAARDP, in which the glutamine had been replaced with an alanine (A), was also tested. This substitution led to a complete loss of peptide antigenicity (Fig. 7c), which could be predicted based on our results from testing mutated B:9-23(13Q) peptides. The peptide LQTLAL-NAARAP, in which aspartic acid (negatively charged side chain) had been replaced with an alanine (uncharged side chain), was less antigenic than the natural sequence (Fig. 7c). The peptide LQTLAL-NAARRP, in which aspartic acid had been replaced with arginine (positively charged side chain), was only weakly antigenic at very high concentrations (Fig. 7c), confirming that this position in the peptide is critical for antigenicity and indicating that an amino acid with a negatively charged side chain may be favorable for activity. I-Ag7 binding We were next interested in determining which residues in the 6.9HIP:core peptide were important for binding and which were interacting with the BDC-6.9 TCR. Due to homology between B:9-23(13Q) and 6.9HIP, we could predict a binding register for 6.9HIP based on our proposed binding register for B:9-23(13Q) (Fig. 8). To investigate the validity of this model, we performed a competitive binding assay. In this assay, I-Ag7 monomer was co-incubated with a fixed concentration of biotinylated HEL peptide (bio-hel) and varying concentrations of a competitor peptide. Following incubation, monomer was captured on a plate coated with anti-i-ag7 antibody (OX-6). Bound bio-hel was detected with a streptavidin-peroxidase detection system. Competitor peptides with a higher binding affinity 103

118 B:12-23(13Q) 6.9HIP BDC-6.9 TCR V Q L Y V G E G A L C R I-Ag7 BDC-6.9 TCR L Q L A N A R P T L A D I-Ag7 Figure 8: Models for presentation of B:12-23(13Q) and 6.9HIP to the BDC-6.9 TCR. Schematics are provided showing the putative I-Ag7 binding register for B:12-23(13Q) and 6.9HIP. Numbers indicate the position within the register. Pockets in the I-Ag7 binding cleft are indicated. 104

119 for I-Ag7 would more effectively out-compete bio-hel for binding to I-Ag7, resulting in the formation of less peroxidase reaction product (lower absorbance). The peptide ps3 (SRLGLWVRME), a mimotope for BDC-2.5, was previously shown to bind I-Ag7 [139] and was therefore used as a positive binding control. Results from these experiments confirmed binding of 6.9HIP:core to I-Ag7 (Fig. 9). Furthermore, substitution of arginine for aspartic acid greatly reduced binding of 6.9HIP to I- Ag7, confirming a role for this residue in anchoring the peptide to I-Ag7. Alanine substitution at this position did not affect binding. 6.9HIP:R G showed a slight decrease in binding. The decreased antigenicity of this BALB/c version of 6.9HIP is likely a combined effect of both decreased binding to I-Ag7 and diminished recognition by the TCR. Replacing glutamine with alanine had no effect on binding, suggesting that the critical role of this residue is in recognition by the TCR. It is important to note, however, that the peptides LATLAL-NAARDP and LQTLAL-NAARAP were only tested once for binding. Therefore, caution must be used in interpreting the results for these peptides. The results of the binding assays seemed to provide preliminary support for our proposed model, but further work is needed as confirmation. Results for most of the peptides are representative of at least four experiments. However, we periodically performed assays that yielded conflicting results or in which we failed to detect binding by any peptides, suggesting unreliability of a reagent or some other confounding factor for which we could not account. Attempts to optimize the assay did not prevent these occasional outcomes. Furthermore, we were puzzled by the consistent observation that competition with 6.9HIP:D R increased absorbance of the sample compared to the control sample that contained bio-hel but lacked competitor peptide (Fig. 9). This unexpected but consistent 105

120 A415nm competitor peptide (µm) ps3 LQTLAL-NAARDP LATLAL-NAARDP LQTLAL-NAAGDP LQTLAL-NAARAP LQTLAL-NAARRP no bio-hel no competitor Figure 9: Substitution of arginine for aspartic acid abrogates binding of 6.9HIP:core to I-Ag7. Peptides were tested for ability to compete with a biotinylated hen egg lysozyme peptide (bio-hel) for binding to I-Ag7 monomer. Lower absorbance values correspond to more efficient displacement of bio-hel by the competitor peptide and therefore indicate higher binding affinity. The peptide ps3 was used as a positive binding control. The average value for duplicate wells is reported. Error bars indicate the range of the duplicate data points. Results are representative of at least four independent experiments. LATLAL-NAARDP and LQTLAL-NAARAP were only tested in one experiment. 106

121 result would suggest that 6.9HIP:D R was actually enhancing binding of bio-hel. Similar results were seen for other peptides at low concentrations. Because of these perplexing observations, we decided to discontinue use of the competitive binding assay. We explored the use of other techniques, such as a direct binding assay, surface plasmon resonance, and isothermal titration calorimetry, but factors such as cost, time and effort required, and technical limitations made it impractical to pursue these methods. Summary and conclusions Previous work to identify the BDC-2.5 antigen determined that the chromogranin A peptide WE14 was a weak antigen for BDC-2.5. Several lines of evidence led us to the discovery that a hybrid peptide, or HIP, containing both insulin C-peptide and WE14 sequence, was much more antigenic for the clone. It was recognized that a similar HIP, formed by fusion of the same C-peptide fragment to IAPP2, would share homology with the BDC-6.9 mimotope B:9-23(13Q). We therefore tested this peptide and found it to be highly antigenic for BDC-6.9. A core peptide of this HIP, centered on the hybrid peptide junction, was likewise highly antigenic for BDC-6.9. This core region contained the NOD-specific IAPP2 arginine residue. Replacement of the arginine residue with glycine (BALB/c sequence) greatly reduced antigenicity, likely explaining the lack of antigenicity of BALB/c islets for BDC-6.9. These results made a strong case for 6.9HIP as the antigen for BDC

122 CHAPTER VII DETECTING 6.9HIP IN BETA CELLS 3 Background and rationale The results presented in the previous chapter provided strong evidence for 6.9HIP as the antigenic epitope for BDC-6.9. However, the existence of hybrid insulin peptides had never been demonstrated. To determine if 6.9HIP was indeed the natural antigen, it was necessary to investigate whether or not HIPs are present in pancreatic beta cells. To address this, we returned to our proteomics approach. We now were looking for one specific peptide, allowing us to optimize our workflow to be best suited for identification of the antigen. Specialized methods Antigen purification Beta cell tumors were harvested from NOD.RIP-TAg mice and homogenized through 40 µm strainers. Cells were lysed by passing successively through 22, 27, and 30 gauge needles. Following removal of large cellular debris by low-speed centrifugation, secretory granules were pelleted by centrifugation at 18,400 x g and then solubilized in 2% octyl-βglucoside. Insoluble debris was removed by centrifugation. The soluble fraction was then resolved by SEC on a Superdex /60 column (Amersham Biosciences) at room temperature at a flow rate of 1 ml/minute with PBS as a running buffer. Fractions (100 fractions, 1.25 ml each) were collected and tested directly in an antigen assay with T cell clones. Peak antigenic fractions were pooled and reduced with dithiotreitol (DTT; final concentration of 8 mm) for 1 hr. at 65 C. Reduced samples were fractionated on a reversephase HPLC Extend C18 column (Agilent) at a flow rate of 0.1 ml/minute using the 3 Portions of this chapter have been published [163,165] and are included here with the permission of Science and Journal of Autoimmunity. 108

123 following gradient: 97% buffer A (water, 0.1% formic acid) and 3% buffer B (90% acetonitrile/water, 0.1% formic acid) from 0-10 min, 3-15% B from minutes, 15-30% B from minutes, 30-50% B from minutes, % B from minutes, and 100% B from minutes. Thirty-six fractions of equal volume were collected over the course of the run. Solvents were removed by vacuum evaporation prior to analysis of fractions by T cell antigen assay or mass spectrometry. Mass spectrometry Proteins in chromatographic fractions were digested with the protease AspN in Tris buffer (50 mm, ph 8.4, 1 mm ZnSO 4 ) prior to analysis by mass spectrometry. Resulting peptides were resolved by online chromatography (C18 column, 1200 Series HPLC system) at a flow rate of 0.45 µl/minute using the following gradient: 97% buffer A (water, 0.1% formic acid) and 3% buffer B (90% acetonitrile/water, 0.1% formic acid) to 10% B by 0.5 minutes, 10-50% B from minutes, 50-80% B from minutes, 80% B from minutes, and 80-3% B from 9-10 minutes. Samples were analyzed with a 6550 ifunnel Q- TOF LC/MS mass spectrometer (Agilent) in positive ion mode using nitrogen as the collision gas for fragmentation of peptides by CID. Multiply-charged parent ions were selected automatically for fragmentation based on abundance. Collision energy was determined automatically based on the charge state and mass of the parent ion. Parent ions of specific interest were preferentially targeted by addition to a preferred ion list. MS/MS spectra were analyzed using Spectrum Mill software (Agilent) to search the Swiss-Prot mouse protein database and a custom database containing the NOD proiapp and 6.9HIP sequences. Peptide matches were considered valid when the score was >10 and the percent scored peak intensity (%SPI), which indicates the percentage of the spectrum that 109

124 could be explained by the interpretation, was >70%. Using data from separate MS only runs, spectral intensity was calculated with Mass Profiler Professional software (Agilent). Results Identification of 6.9HIP in antigenic fractions by mass spectrometry We previously used BDC-6.9 as the primary T cell clone for screening chromatographic fractions for antigenicity. However, this clone often failed to detect antigen in the fractions or would respond non-specifically to multiple fractions (Fig. 1). In contrast, BDC-9.3, which expresses the same TCR as BDC-6.9, responded much more robustly and reliably. We therefore chose to use BDC-9.3 to screen for antigen in all future chromatography experiments. As before, we used NOD.RIP-TAg tumors as a source of antigen. After mechanically lysing the tumor cells and enriching for secretory granule proteins, we fractionated the sample by SEC and identified antigenic fractions using BDC-9.3 as the responder T cell clone (Fig. 2a). We then pooled antigenic fractions and performed a second fractionation by HPLC. In some experiments, the antigen eluted in multiple peaks (Fig. 2b,c), whereas in other experiments it eluted as one peak (Fig. 2d), indicating that the antigen may sometimes exist in different forms. For example, if the antigen is indeed a hybrid insulin peptide, the existence of hybrids formed by linkage to the two different forms of insulin, insulin 1 and insulin 2, could account for the presence of two of the antigen peaks. We learned previously that the natural antigen may be too large for efficient MS/MS analysis, thus necessitating prior proteolytic digestion of the sample. We chose to use the protease AspN, which cleaves peptides on the N-terminal side of aspartic acid (D) residues. Cleavage of 6.9HIP would thus yield the fragment DLQTLAL-NAAR. This peptide would 110

125 A SEC fraction IFN-γ (ng/ml) BDC-6.9 BDC-9.3 A280 Figure 1: BDC-9.3 responds more reliably than BDC-6.9 to chromatographic fractions of antigen purified from NOD.RIP-TAg tumors. Proteins isolated from NOD.RIP-TAg tumors were fractionated by SEC and tested for antigenicity with BDC-6.9 and BDC-9.3. Absorbance at 280nm is reported as an indication of total protein content. Results are representative of at least two independent experiments. 111

126 A B C D A280 A spectral intensity (x10 6 ) spectral intensity (x10 6 ) SEC HPLC mass spectrometry mass spectrometry chromatographic fraction IFN-γ (ng/ml) IFN-γ (ng/ml) IFN-γ (ng/ml) IFN-γ (ng/ml) total protein T cell response total protein T cell response DLQTLAL-NAAR T cell response DLQTLAL-NAAR T cell response Figure 2: The abundance of the insulin-iapp hybrid in chromatographic fractions of beta cell proteins correlates with the magnitude of the BDC-9.3 response to the fractions. (A) Secretory granule proteins, isolated from pancreatic beta cell tumors of NOD.RIP-TAg mice, were fractionated by SEC. Fractions were then tested for antigenicity with BDC-9.3. (B) Antigenic SEC fractions were pooled and further fractionated by reversephase HPLC; HPLC fractions were then tested for antigenicity with BDC-9.3. (C) Following digestion with the protease AspN, HPLC fractions were analyzed by mass spectrometry in MS only mode, and the spectral intensity of the parent ion m/z = , z = +2, corresponding to the peptide DLQTLAL-NAAR, was analyzed using MPP software. Reported spectral intensities represent the mean of two duplicate MS runs of the same biological sample. The IFN-γ response to each fraction, as shown in (B), is also displayed here for ease of comparison. Results for (A-C) are representative of two independent experiments. (D) Results from the second experiment showing both the response by BDC-9.3 to HPLC fractions and the spectral intensity of the DLQTLAL-NAAR parent ion in each fraction. 112

127 be small enough for analysis but at the same time would contain enough sequence from both insulin and IAPP to enable confident characterization as a hybrid. Because our HPLC data suggested that the antigen may exist in multiple forms, digestion with AspN provided another benefit. The AspN core of 6.9HIP roughly describes the minimal epitope for BDC-6.9, which we defined earlier as the sequence LQTLAL-NAARD. Thus, various forms of the antigen would probably contain this same core sequence, with any differences being in other regions of the molecule. Digestion with AspN would excise a defined fragment from various unknown forms of the antigen, therefore we would know precisely what species to look for by mass spectrometry. Following digestion with AspN, we analyzed antigenic and flanking non-antigenic HPLC fractions by mass spectrometry in MS only mode and searched the data for a peptide with a mass corresponding to the sequence DLQTLAL-NAAR. Such a peptide did exist. Analysis of the data indicated that the distribution of this peptide matched the profile of the BDC-9.3 response to the various fractions (Fig. 2c,d), suggesting that the peptide from which this sequence was fragmented could be the natural antigen. Confirming identification of 6.9HIP by tandem mass spectrometry To confirm the identity of the peptide, we performed MS/MS analysis and searched the data using Spectrum Mill software against a custom database containing the hybrid peptide sequence. Tandem mass spectrometry data confirmed that the sequence of the peptide was DLQTLAL-NAAR (Fig. 3). Searching the data against the Swiss-Prot mouse protein database failed to identify a match for the spectrum of this peptide, indicating that the spectrum could only describe the hybrid peptide sequence. To validate this interpretation of 113

128 A spectral intensity (x10 3 ) b 2 y 2 b 3 y 4 2 y1 y3 b 5 b 6 b m/z y 6 y 5 DLQTLAL-NAAR y 7 y 8 y 9 B predicted observed ppm predicted observed ppm b y b y b y b y b y b y b y b y b y b y Figure 3: Tandem mass spectrometry confirms the presence of 6.9HIP in antigenic HPLC fractions. AspN-digested antigenic HPLC fractions were analyzed by mass spectrometry in MS/MS mode. The parent ion m/z = , z = +2, corresponding to the peptide DLQTLAL-NAAR, was specifically targeted for fragmentation. (A) MS/MS spectrum for the targeted parent ion. Both b- and y-ions are labeled. (B) Predicted m/z values for b- and y-ions of the peptide DLQTLAL-NAAR, as well as the observed values (when detected) in the spectrum of the peptide found in HPLC fractions. The differences between the predicted and observed values are reported in parts-per-million (ppm). Results are representative of three independent experiments. 114

129 the spectrum, we analyzed synthetic DLQTLAL-NAAR peptide by tandem mass spectrometry. The MS/MS spectrum of the synthetic peptide was nearly identical to the spectrum of the peptide identified in the antigenic HPLC fractions (Fig. 4), confirming that 6.9HIP was present in the antigenic fractions. Summary and conclusions Although we had shown that 6.9HIP was highly antigenic for BDC-6.9 and BDC-9.3, it was critical to determine if this peptide is generated within the beta cell. To do this, proteins were isolated from NOD.RIP-TAg tumors and sequentially fractionated by SEC and HPLC. Following digestion of the samples with AspN, mass spectrometric analysis was performed. We confidently identified 6.9HIP in antigenic HPLC fractions and we demonstrated that the abundance of this peptide in fractions corresponded to antigenicity. These findings provide compelling evidence that 6.9HIP is the natural antigen for the CD4 T cell clones BDC-6.9 and BDC

130 1 natural normalized spectral intensity 0-1 m/z synthetic Figure 4: MS/MS analysis of synthetic peptide confirms identification of 6.9HIP in antigenic HPLC fractions. Commercially obtained synthetic peptide with the sequence DLQTLAL-NAAR was analyzed by tandem mass spectrometry. A representative product ion spectrum is plotted on the negative y-axis. A representative product ion spectrum for the parent ion m/z = , z = +2 identified in antigenic HPLC fractions is plotted on the positive y-axis. Each spectrum is normalized to the most abundant ion within the spectrum. Results are from one experiment. 116

131 CHAPTER VIII 6.9HIP-REACTIVE CD4 T CELLS IN DISEASE 4 Background and rationale Having identified 6.9HIP as an antigen for BDC-6.9 and BDC-9.3, and after confirming that this hybrid peptide forms naturally in beta cells, we were interested in evaluating the role of 6.9HIP-reactive T cells in autoimmune diabetes in the NOD mouse model. To do this, we obtained a custom MHC class II tetramer. This reagent enabled us to detect and analyze CD4 T cells specific for 6.9HIP ex vivo by flow cytometry. Furthermore, using another tetramer specific for B:9-23-reactive CD4 T cells, we compared the frequency and phenotype of 6.9HIP-reactive and B:9-23-reactive cells. Specialized methods Tetramer analysis The APC-conjugated MHC class II tetramers HEL-tet, insp8g-tet, and 6.9HIP-tet were obtained from the NIH tetramer core. These tetramers were loaded with the peptides AMKRHGLDNYRGYSL, HLVERLYLVCGGEG, and LQTLALNAARDP, respectively. Pancreas and spleen were harvested from prediabetic (14-19-week-old) female NOD mice or diabetic female NOD or NOD IAPP-/- mice. Spleen samples were homogenized to yield single cell suspensions and pancreata were minced with surgical scissors and digested in 5 mg/ml collagenase in HBSS supplemented with FBS (15%) for 13 minutes in a 37 C water bath with periodic shaking. Tetramer staining was done with a 1:100 dilution of APC-labeled tetramers in culture medium for 1 hr at 37 C. Staining with antibodies was done for 20 minutes at room temperature. For tetramer analysis of T cell clones and lines, cells were 4 Portions of this chapter have been published [165] and are included here with the permission of Science and Journal of Autoimmunity. 117

132 expanded in culture with IL-2 for four days prior to staining to upregulate TCR expression and generate sufficient cell numbers. Intracellular staining After harvesting and homogenizing tissues, cells were aliquoted in a 96-well plate and stimulated at 37 C with phorbol 12-myristate 13-acetate (PMA; 0. 1µg/ml) and ionomycin (1 µg/ml) in the presence of the protein transport inhibitor brefeldin A. After 90 minutes of incubation, the supernatant was removed and cells were resuspended in medium containing tetramer. One hour later, antibodies to surface antigens were added and samples were incubated an additional 20 minutes at room temperature. After washing, cells were fixed overnight at 4 C. The following day, cells were washed and stained with α-ifn-γ and α-foxp3 antibodies for 30 minutes at room temperature in a commercial permeabilization buffer. Cells were analyzed on a CyAn (Beckman Coulter) flow cytometer. Data analysis was performed using FlowJo software (Tree Star, Ashland, OR). Isolation of BDC-9.H1 The polyclonal BDC-9.0 line was isolated previously, as described [33]. To determine if a population of 6.9HIP-reactive cells existed within this line, the line was stained with 6.9HIP-tet and α-tcr Vβ antibodies and analyzed by flow cytometry. A 6.9HIP-tet + Vβ6 + population was identified. Vβ6 + cells were sorted on a Sony icyt SYNERGY cell sorter and placed in culture. After expanding sorted cells for several weeks in culture, a second sort was performed on a MoFlo XDP cell sorter (Beckman Coulter) selecting for 6.9HIP-tet + Vβ6 + cells. After further expansion in culture, a third sort was performed selecting for 6.9HIP-tet + cells using the MoFlo XDP sorter. This sorted population was cultured and used for all BDC- 9.H1 experiments. For adoptive transfer experiments, cells were screened prior to transfer 118

133 with 6.9HIP-tet and α-tcr Vβ6 antibody. The clonality reported is the percentage of cells that were Vβ6 +. Statistics Data were analyzed using GraphPad Prism Version 7.0a software. Statistical significance was determined using the Friedman test and Dunn s multiple comparisons test (Figures 2, 4, and 5) or the Mann-Whitney test (Figure 3). Statistical significance was defined as p<0.05. Results The 6.9HIP tetramer (6.9HIP-tet) A custom designed I-Ag7 tetramer, 6.9HIP-tet, loaded with the 6.9HIP:core peptide sequence (LQTLALNAARDP), was obtained from the NIH tetramer core for the purpose of conducting ex vivo analysis on cells from NOD mice. To confirm the specificity of the tetramer, it was first used to stain individual T cell clones. 6.9HIP-tet specifically stained the 6.9HIP-reactive clones BDC-6.9 and BDC-9.3 but not an insulin B:9-23-reactive clone, PD [99] (Fig. 1). The insulin B:9-23 tetramer insp8g-tet [101] stained PD but not BDC-6.9 or BDC-9.3. Prevalence of 6.9HIP-tet + cells in NOD mice The tetramers were next used to assess the frequency of cells specific for 6.9HIP in the pancreas and spleen of diabetic and prediabetic NOD mice. HEL-tet, which was loaded with a hen egg lysozyme (HEL) peptide, was used as a negative control. Few, if any, T cells from the NOD mouse should stain with this tetramer, as HEL is a foreign antigen. In the pancreas of diabetic NOD mice, cells staining with 6.9HIP-tet were far more abundant than insp8g-tet + cells (Fig. 2a), whereas the frequencies were comparable in the spleen (Fig. 2b). 119

134 PD BDC-6.9 BDC-9.3 insp8g-tet 6.9HIP-tet tetramer Figure 1: 6.9HIP-tet specifically stains BDC-6.9 and BDC-9.3. The control clone PD (insulin B:9-23-reactive) and the 6.9HIP-reactive clones BDC-6.9 and BDC-9.3 were stained with either the control insulin tetramer insp8g-tet or 6.9HIP-tet. Gated on CD4 + cells. Representative of at least two independent experiments. 120

135 A DIABETIC PANCREAS HEL-tet insp8g-tet 6.9HIP-tet 0% 0% 0.32% 1.15% CD45 tetramer % of CD4 cells p < p = 0.02 HEL-tet + insp8g-tet + 6.9HIP-tet + B DIABETIC SPLEEN HEL-tet insp8g-tet 6.9HIP-tet CD E-3% 0.009% 0.029% 0.034% tetramer p = % of CD4 cells0.06 p = HEL-tet + insp8g-tet + 6.9HIP-tet + Figure 2: CD4 T cells specific for 6.9HIP are prevalent in the pancreas of diabetic NOD mice. Cells from the (A) pancreas (n=15) and (B) spleen (n=15) of diabetic NOD mice were stained with antibodies and tetramer (HEL-tet, insp8g-tet, or 6.9HIP-tet) and analyzed by flow cytometry. Representative flow plots and summary statistics are shown for each tissue. Data are pooled from eight independent experiments. Gated on live CD4 + CD45 + CD8/CD11b/CD11c/CD19 - cells. 121

136 Islets deficient in IAPP should not express 6.9HIP, thus fewer 6.9HIP-reactive cells should be present in the tissues of these mice. Analysis of both the pancreas (Fig. 3a) and spleen (Fig. 3b) of diabetic NOD.IAPP-/- mice confirmed this prediction. The frequency of B:9-23- specific CD4 T cells, however, was the same in both NOD and NOD.IAPP-/- mice, suggesting that IAPP deficiency does not noticeably influence CD4 T cell populations specific for other antigens. To determine if cells specific for 6.9HIP are detectable prior to disease onset, non-diabetic week old NOD mice were analyzed. Cells staining with 6.9HIP-tet were detected in both the pancreas (Fig. 4a) and spleen (Fig. 4b), indicating that 6.9HIP-reactive CD4 T cells are present before overt disease is established and are thereby potential contributors to pathogenesis. Phenotype of 6.9HIP-tet + cells in NOD mice To characterize how CD4 T cells specific for 6.9HIP-tet might contribute to disease, we next examined the phenotype of these cells in NOD mice prior to the onset of overt diabetes. The pancreas and spleen were harvested from prediabetic female mice ( weeks old). After processing the tissues, cells were stained with tetramer and stimulated with PMA/I in the presence of a protein transport inhibitor to stimulate cytokine production. Following stimulation, samples were fixed and stained with antibodies to IFN-γ and FoxP3. Representative staining is shown (Fig. 5a). In the pancreas, the percentage of cells that were FoxP3 + was lower in the 6.9HIP-tet + population than in the insp8g-tet + population (Fig. 5b). In the spleen, the 6.9HIP-tet + population showed an increased percentage of IFN-γ + cells relative to both the insp8g-tet + and the bulk CD4 population (Fig. 5c). A decreased percentage of 6.9HIP-tet + cells in the spleen were FoxP3 + compared to the bulk 122

137 A DIABETIC PANCREAS % of CD4 cells n.s. p = insp8g-tet + 6.9HIP-tet + NOD (n = 15) IAPP-/- (n = 5) B DIABETIC SPLEEN % of CD4 cells n.s. p = insp8g-tet + 6.9HIP-tet + NOD (n = 15) IAPP-/- (n = 5) Figure 3: The frequency of 6.9HIP-tet + cells is significantly reduced in NOD.IAPP-/- mice. The (A) pancreas (n = 5) and (B) spleen (n = 5) of diabetic NOD.IAPP-/- mice were analyzed by tetramer staining for the presence of CD4 T cells specific for 6.9HIP. NOD.IAPP-/- data are pooled from three independent experiments. NOD data are the same as from Figure 2. Gated on live CD4 + CD45 + CD8/CD11b/CD11c/CD19 - cells. n.s. = not significant. 123

138 A PREDIABETIC PANCREAS 0.8 p = % of CD4 cells HEL-tet + insp8g-tet + 6.9HIP-tet + B PREDIABETIC SPLEEN % of CD4 cells p = HEL-tet + insp8g-tet + 6.9HIP-tet + Figure 4: CD4 T cells specific for 6.9HIP are present in prediabetic NOD mice. Cells from the (A) pancreas (n = 7) and (B) spleen (n = 7) of non-diabetic week old NOD mice were stained with antibodies and tetramer and analyzed by flow cytometry. Data are pooled from two independent experiments. Gated on live CD4 + CD45 + CD8/CD11b/CD11c/CD19 - cells. 124

139 A PREDIABETIC PANCREAS CD4 + insp8g-tet + 6.9HIP-tet % 0.13% 3.17% 1.59% 9.33% 0% IFN-γ 6.70% 30.2% 0% FoxP3 B C PREDIABETIC PANCREAS % IFN-γ CD4 + IFN-γ insp8g-tet HIP-tet + PREDIABETIC SPLEEN % IFN-γ CD4 + IFN-γ p = insp8g-tet + p = HIP-tet + % FoxP3 + % FoxP CD4 + insp8g-tet + CD4 + insp8g-tet + FoxP3 p = HIP-tet + FoxP3 p = HIP-tet + Figure 5: The 6.9HIP-tet + CD4 T cell population is skewed towards an inflammatory phenotype in the spleen. The phenotype of tet + CD4 T cells from week-old prediabetic female mice was assessed following stimulation with PMA/I. For each tissue, the percentage (average +/- S.D.) of bulk CD4, insp8g-tet +, or 6.9HIP-tet + cells that were either IFN-γ + or FoxP3 + is reported. (A) Representative flow plot for pancreas samples. (B) Pancreas (n = 12). (C) Spleen (n = 12). Data are pooled from multiple experiments. Gated on CD4 + CD45 + CD8/CD11b/CD11c/CD19 - cells. 125

140 CD4 population (Fig. 5c). These data suggest a skewing of the 6.9HIP-reactive population away from a regulatory (FoxP3 + ) phenotype and towards a pathogenic Th1 (IFN-γ + ) phenotype. Isolation of a 6.9HIP-reactive CD4 T cell line The established clones BDC-6.9 and BDC-9.3 are capable of inducing disease in young NOD mice. To further evaluate the contribution of 6.9HIP-reactive cells to disease, 6.9HIP-tet was used to isolate a 6.9HIP-reactive CD4 T cell line. To circumvent the time required to derive a line from primary cells, the pre-existing polyclonal BDC-9.0 line was screened with 6.9HIP-tet. This line, previously isolated from the spleen and lymph nodes of a diabetic NOD mouse [33], contained a population of 6.9HIP-tet + cells, which were sorted and grown in culture (BDC-9.H1). Although BDC-9.H1 stained with 6.9HIP-tet (Fig. 6a), it expressed a unique TCR: BDC-9.H1 expressed Vβ6, whereas BDC-6.9 expresses Vβ4 (Fig. 6b). These data indicated that the T cell response to the insulin-iapp hybrid in NOD mice is heterogeneous with regard to TCR repertoire. Like BDC-6.9 and BDC-9.3, BDC-9.H1 did not respond to islet cells from IAPP-deficient NOD mice (Fig. 7a). When tested with synthetic peptides, BDC-9.H1 responded to 6.9HIP but not to the BALB/c version, 6.9HIP:R G (Fig. 7b). The combination of C:1-26 and IAPP2 was not antigenic for BDC- 9.H1 (Fig. 7b), indicating that the line is specific for the hybrid peptide. In contrast to BDC- 6.9, which responds similarly to 6.9HIP and 6.9HIP:core, BDC-9.H1 was more responsive to the full-length 6.9HIP molecule than to the core peptide (Fig. 7b), indicating that BDC-6.9 and BDC-9.H1 have different fine epitope specificity. To investigate whether BDC-9.H1 had pathogenic potential, cells were expanded and adoptively transferred into young NOD mice. Clonality of each batch of transferred cells was confirmed by flow cytometry. Mice receiving 126

141 A BDC-9.H1 B BDC-6.9 BDC-9.H1 0% 99.2% Vβ6 99.2% 0.034% insp8g-tet 6.9HIP-tet Vβ4 Figure 6: A 6.9HIP-reactive line, BDC-9.H1, isolated using 6.9HIP-tet, expresses a distinct TCR. The 6.9HIP tetramer was used to sort a population from the pre-existing cell line BDC-9.0, which was isolated from a diabetic NOD mouse. (A) The sorted line, BDC- 9.H1, was stained with either 6.9HIP-tet or the control insulin tetramer insp8g-tet. (B) BDC- 6.9 and BDC-9.H1 were stained with α-tcr Vβ antibodies to demonstrate distinct Vβ usage. Gated on CD4 + cells. Representative of at least two independent experiments. 127