Characterization of Novel T-cell Epitopes on 65 kDa and 67 kDa Glutamic Acid Decarboxylase Relevant in Autoimmune Responses in NOD Mice

Journal of Autoimmunity (1998) 11, 83–95

Characterization of Novel T-cell Epitopes on 65 kDa and 67 kDa Glutamic Acid Decarboxylase Relevant in Autoimmune Responses in NOD Mice Marc A. Zechel1, John F. Elliott2, Mark A. Atkinson3 and Bhagirath Singh1 1

Department of Microbiology & Immunology, The University of Western Ontario, and The John P. Robarts Research Institute, London, Ontario, Canada 2 Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada 3 Department of Pathology and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, Florida, USA Received 4 April 1997 Accepted 21 November 1997 Key words: glutamic acid decarboxylase, epitope, peptide, immunity, autoantigen

It has recently been shown that the T-cell mediated immune responses to glutamic acid decarboxylase (GAD) play an important role in insulindependent diabetes mellitus (IDDM) in NOD mice. However, specific epitopes responsible for eliciting these responses remain unresolved. In this study, the T-cell epitopes involved in GAD-specific immune responses in NOD mice were characterized. By priming NOD mice with GAD65, three new GAD65 epitopes (GAD65 78–97, GAD65 202–221, GAD65 217–236) distinct from those previously reported were found. Furthermore, our investigations into the fine determinant specificity of GAD67 revealed two additional GAD67-specific peptide epitopes (GAD67 28–47, GAD67 42–61). Two of the GAD65 epitopes (GAD65 202–221 and GAD65 217–236) are shared between GAD65 and GAD67. Spontaneous immune responses to these peptides were found in pre-diabetic and diabetic mice and differential patterns of responses to these peptides were observed depending on the age of the mice, disease status, or if the mice were protected from diabetes by adjuvant immunotherapy. Characterization of these new epitopes will help in the elucidation of autoimmune responses to GAD in IDDM. © 1998 Academic Press Limited

Introduction

we aimed to determine autoreactive T-cell peptide epitopes by mapping the GAD molecule using overlapping synthetic peptides [3]. In this study, we report the characterization of GAD peptide epitopes responsible for T-cell mediated immune responses in prediabetic, diabetic, GAD-immunized, or CFA-protected NOD mice. We have identified three new GAD65 epitopes, different from those previously reported [3], and two GAD67-specific peptide epitopes which gave rise to immune responses in NOD mice. Identification of these epitopes will allow for the regulation of autoimmune responses to GAD in IDDM.

Insulin-dependent diabetes mellitus (IDDM) is a T-cell mediated disease which results from the destruction of the insulin-producing pancreatic â-cells, ultimately leading to the host’s inability to maintain homeostatic blood glucose. A breakdown in self-tolerance to â-cell antigens precedes progression to diabetes [1] and it has recently been found that NOD mice are more prone to autoimmunity than other mouse strains [2]. Many candidate autoantigens have been implicated in IDDM, however, it is believed that glutamic acid decarboxylase (GAD) is particularly relevant since autoantibodies and cell-mediated responses to GAD are strongly correlated with the disease process [1, 3–5]. Moreover, we have shown that immunization with GAD67 [4] prevents autoimmune diabetes in NOD mice, as does neonatal tolerization to GAD65 [5]. To this end, it is important to identify and confirm the specific regions of GAD responsible for the autoimmune response in IDDM [3, 6, 7]. Therefore,

Materials and Methods Mice NOD/Lt mice were originally obtained from the Jackson Laboratory (Bar Harbor, ME) and maintained in our animal facilities. Female NOD mice were used for this study and the incidence of diabetes in our colony for female mice is approx. 80% by 20 weeks of age.

Correspondence to: Dr Bhagirath Singh, Department of Microbiology & Immunology, The University of Western Ontario, London, Ontario, N6A 5C1, Canada. Fax: 519-661-3499. 83 0896-8411/98/010083+13 $25.00/0/au970178

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Figure 1. Alignment of protein amino-acid sequences of huGAD65, mGAD65, and mGAD67. Amino-acid sequences of GAD were downloaded from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) and are shown aligned for homology. Lines between amino acids indicate identity, asterisks indicate similarity. Regions of grey shadowing are areas containing peptide sequences shown to be immunoreactive in this study (GAD65p6, 14 and 15; GAD67p2 and 3) and their start and end sites are further demarcated by alternating solid or broken arrows.

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Table 1. Sequences of GAD peptides synthesized Peptide nomenclature

Corresponding protein sequence

Peptide amino acid sequence N- to C-terminus

Human GAD65 peptides GAD65p6

GAD65(78–97)

KPCSCSKVDVNYAFLHATDL

GAD65p14 GAD65p14a GAD65p14b GAD65p14c

GAD65(202–221) GAD65(202–216) GAD65(210–221) GAD65(207–221)

TNMFTYEIAPVFVLLEYVTL TNMFTYEIAPVFVLL APVFVLLEYVTL YEIAPVFELLEYVTL

GAD65p15 GAD65p15a GAD65p15b GAD65p15c

GAD65(217–236) GAD65(217–229) GAD65(218–229) GAD65(222–236)

EYVTLKKMREIIGWPGGSGD EYVTLKKMREIIG YVTLKKMREIIG KKMREIIGWPGGSGD

Mouse GAD67 peptides GAD67p1 GAD67p2 GAD67p3 GAD67p4 GAD67p5 GAD67p6 GAD67p7

GAD67(13–32) GAD67(28–47) GAD67(42–61) GAD67(57–76) GAD67(72–91) GAD67(87–106) GAD67(102–121)

NAGADPNTTNLRTTYDTWCG DTWCGVAHGCTRKLGLKICG LKICGFLQRTNSLEEKSRL EKSRLVSAFRERQSSKNLLS KNLLSCENSDQGARFRRTET RRTETDFSNLFAQDLLPAKN LPAKNGEEQTAQFLLEVVDI

Antigens Recombinant mouse GAD67 (mGAD) and human GAD65 (huGAD) were cloned and expressed as we have previously described [4]. Overlapping GAD65 peptides of 18–23 amino acids spanning the entire GAD molecule were prepared as described in [3]. Peptides which were found to give rise to stimulatory responses after initial screening were subsequently synthesized in our laboratory by solid-phase peptide synthesis [8]. To test for the presence of additional GAD determinants, peptide sequences were also synthesized which spanned the 100 amino acids nearest the N-terminal of mGAD67. This region represents the area of least homology between mGAD67 and huGAD65 (Figure 1). Mouse GAD67 and huGAD65 peptide sequences synthesized are summarized in Table 1. Peptides were reconstituted in media before use and 0.22 ìm-filter sterilized. Complete (CFA) or incomplete (IFA) Freund’s adjuvant used for immunizations was purchased from Sigma Chemical Co (St Louis, MO).

Immunization and T-cell proliferation assays Immunization and T-cell proliferation assays were performed as previously described [4]. Briefly, for primed responses, female NOD mice were immunized in the hind footpads with GAD protein (50 ìg) or GAD peptides (50 ìg) in CFA. After 10 days, pooled draining lymph nodes or spleens from at least three mice per group were removed, single cell suspensions prepared, and, in the case of spleens, red blood cells were lysed. Cell suspensions were incubated at 37°C for 72 h in 96-well flat-bottom microtitre plates in

triplicate (Becton Dickinson Co, Rutherford, NJ) at 2×105 cells/well in a total volume of 200 ìl culture medium (RPMI 1640 (Life Technologies, Grand Island NY) supplemented with 10% FCS (Bockneck, Canada), 10 mM HEPES, 2 mM L-glutamine, 5× 10 −5 M 2-ME, and 1 U/ml penicillin–streptomycin) using various concentrations of challenge protein or peptide antigen for 3 (peptide) or 4 days (protein). For unprimed responses, spleens from female NOD mice were removed, pooled (three per group), single cell suspensions prepared, and red blood cells lysed. Cell suspensions were incubated as above for 5 days with protein or peptide antigen. In either instance, cell proliferation was assessed by the addition of 1 ìCi/well [3H]-Tdr (New England Nuclear, DuPont, Boston, MA) during the last 18 h of culture. The results are presented as mean cpm±SEM.

Cytokine assays Quantities of IL-4 and IFN-ã in culture supernatants harvested at 24, 48, and 72 h from primed mice were determined by sandwich ELISA assays. Following the manufacturer’s protocol (Pharmingen, San Diego, CA) 96-well microtitre ELISA plates were coated with 50 ìl anti-IL-4 or anti-IFN-ã mAb diluted to 1 ìg/ml in coating buffer (0.1 M NaHCO3, pH 8.2) overnight at 4°C. The plates were washed twice in PBS-T (PBS, 0.5% Tween-20 (Sigma Chemical Co, St Louis, MO)) and 100 ìl blocking buffer was added for 2 h at room temperature. The plates were washed twice in PBS-T and 50 ìl of sample supernatant was added. All assays were performed in triplicate. After overnight incubation at 4°C, plates were washed five times in PBS-T and 50 ìl of biotinylated anti-IL-4 or anti-IFN-ã mAb

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Figure 2. T-cell responses to GAD65 peptides after priming with huGAD65 protein antigen. 10 days after footpad immunization with huGAD65 (50 ìg), draining lymph node cells from at least three mice were pooled and tested for proliferative responses to a panel of huGAD65 peptides (25 ìM) in a 4-day proliferation assay. (A) Peptides are numbered consecutively from the N- to C-terminus and were prepared as previously reported [3]. ( ), Peptides which lead to consistently significant stimulation indices in subsequent experiments; GAD65p6 (huGAD65 78–97), GAD65p14 (huGAD65 202–221) and GAD65p15 (huGAD65 217– 236). (B) GAD65 peptides previously reported as leading to T-cell responses were tested to confirm the results in (A). ( ), Background proliferation in the presence of media only; ( ), proliferation observed with GAD65p15; ( ), proliferation induced by previously reported immunodominant GAD65 peptide epitopes; GAD65p17 (huGAD65 247–266), GAD65p34 (huGAD65 509–528) and GAD65p35 (huGAD 524–543) [3]. Note that no significant proliferation was observed with any of the previously reported GAD65 epitopes. Data are expressed as mean cpm±SEM of [3H]-Tdr DNA incorporation.

diluted to 1 ìg/ml in PBS-T was added for 2 h at room temperature. Plates were washed eight times in PBS-T and 50 ìl streptavidin–alkaline phosphatase diluted to 1 ìg/ml in PBS-T was added for 45 min at room temperature. After 10 washes in PBS-T, plates were incubated with 50 ìl p-nitrophenyl phosphate substrate (pNPP (Sigma)). Absorbance was determined at

Figure 3. T-cell responses to GAD65 peptides after priming with huGAD65 peptides. Female NOD mice were immunized as described in Figure 2 with 50 ìg GAD65p6 (A), 14 (B), or 15 (C). After 10 days draining lymph node T cells were tested in a 3-day proliferation assay using titrations of GAD65p6, 14 and 15. Strong specific recall responses were observed in all cases. In addition, GAD65p15 was observed to give rise to a cross-reactive recall response in the group of mice immunized with GAD65p14. Data are expressed as mean cpm±SEM of [3H]-Tdr DNA incorporation. — —, GAD65p6; —.—, GAD65p14; — —, GAD65p15.

OD405 using a microplate reader (Bio-Rad, Hercules, CA).

Results T-cell responses of huGAD65 protein-primed NOD mice to huGAD65 peptides To determine which GAD65 peptide determinants were responsible for immunological responses, 6–8 week-old NOD mice were primed s.c. with recombinant huGAD65 in CFA. After 10 days, draining lymph nodes were removed and single cell suspensions were incubated with various human GAD65 peptides (50 ìg/ml) for 72 h. GAD peptides are numbered as previously described [3]. It should be emphasized that huGAD65 shares 95% amino-acid identity and 98% conservation with mGAD65 [3] and huGAD65 shares approx. 75% similarity with mGAD67, as determined by sequence alignment of

Characterization of T-cell epitopes on glutamic acid decarboxylase

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0

Media GAD65p14 GAD65p14a GAD65p14b GAD65p14c

– TNMFTYEIAPVFVLLEYVTL – TNMFTYEIAPVFVLL – APVFVLLEYVTL YEIAPVFVLLEYVTL –

87

s.c. with GAD65p6, 14, 15. After 10 days, the recall responses to titrations of GAD peptides were determined (Figure 3). Strong responses were observed in mice immunized with GAD65p14 (Figure 3B), challenged in vitro with GAD65p14, and good responses were seen when GAD65p6 (Figure 3A) and GAD65p15-primed mice (Figure 3C) were challenged with GAD65p6 or GAD65p15, respectively. Moreover, a significant response was observed in the GAD65p14primed group when challenged in vitro with GAD65p15, suggesting that there was immunological cross-reactivity between GAD65p14 and 15 (Figure 3B).

10,000 20,000 30,000 40,000 50,000 60,000 70,000 80,000 B

1,000

Media GAD65p15 GAD65p15a GAD65p15b GAD65p15c

1,500

– EYVTLKKMREIIGWPGGSGD – EYVTLKKMREIIG – YVTLKKMREIIG KKMREIIGWPGGSGD –

2,000

2,500

cpm

Figure 4. T-cell responses to GAD65p14 and GAD65p15 Nand C-terminal deletion peptides. Ten days after footpad immunization with GAD65p14 or GAD65p15 (50 ìg), draining lymph node cells were prepared and tested for proliferative responses to GAD65p14 (A) or GAD65p15 (B) N- and C-terminal deletion peptides respectively (25 ìM) as listed in the graph legends in a 3-day proliferation assay. Recall response to the original priming antigen is shown as ( ). Preservation of the terminal residues EYVTL appears to be essential for peptide-induced T-cell responses and, as demonstrated in (B), preservation of the Glu residue is critical for T-cell responses.

huGAD65 with mGAD67 (Figure 1). Significant proliferative responses were initially seen with several peptides (Figure 2A). However, after subsequent repetition of these experiments, reproducibly significant responses were seen only to three peptides with respect to highest immune reactivity and this was the selection criterion by which these regions were studied further (Figure 2A, solid bars). These peptides (GAD65p6, 14, 15) are distinct from the previously reported epitopes: GAD65p17, 34, 35 [3]. It should be noted that we did not observe significant proliferative responses to any of the previously reported determinants [3] in NOD mice primed with huGAD65 (Figure 2B].

T-cell responses of huGAD65 peptide-primed NOD mice to huGAD65 peptides To confirm the results obtained from immunizing mice with huGAD65, NOD mice were immunized

Analysis of GAD65p14 and 15 epitopes by amino-acid deletion As proliferation was observed to two overlapping peptides, GAD65p14 and GAD65p15, it was necessary to ascertain the region(s) responsible for eliciting this immunological response. Initially, amino-acid deletions were generated in the GAD65p14 sequence (Table 1, GAD65p14a, b, c). These deletions were generated to give a starting point for the region of GAD65p14 responsible for the responses observed. NOD mice were immunized s.c. with GAD65p14 and the in vitro recall responses to the original priming antigen (GAD65p14) and deletion peptides GAD65p14a, b, and c were determined (Figure 4A). T cells from NOD mice immunized with GADp14 responded only to the peptides containing the EYVTL motif (GAD65p14b, c), since the removal of the C-terminal EYVTL in GAD65p14a resulted in an associated loss of immune reactivity. The N-terminal residues are not essential as their deletion still allowed for immune responses to this peptide. Responses to GAD65p15 were also observed, which made it necessary to determine if the region responsible for this was shared with GAD65p14. To this end, amino-acid deletions were generated removing either regions from the N- or C-terminus of GAD65p15 (Table 1). After immunizing NOD mice with GAD65p15 and recalling the response in vitro with the original priming antigen (GAD65p15), or the deletion peptides, significant responses were observed only when the EYVTL motif was present (Figure 4B). Moreover, removal of the Glu residue from the amino-terminal of GAD65p15b reduced the response, suggesting this was a critical residue for immunogenicity.

Fine analysis of critical residues in GAD65p14 by amino-acid substitution Since the observations from peptide-deletion analysis suggested that specific amino acid(s) could be critical for immunogenicity, Ala substitutions in GAD65p14 were synthesized. NOD mice were immunized s.c. with GAD65p14 and, after 10 days,

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0.025 Peptide (µM)

TNMFTYEIAPVFVLLEYVTL TNMFTYEIAPVFVLLEYVAL TNMFTYEIAPVFVLLEYATL TNMFTYEIAPVFVLLEAVTL TNMFTYEIAPVFVLLAYVTL

250

25

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TNMFTYEIAPVFVLLEYVTA TNMFTYEIAPVFALLEYATL TNMFTYEIAPVAVLLEYVTL TNMFTYEIAPAFVLLEYVTL TNMFTYEIAAVFVLLEYVTL TNMFTYEAAPVFVLLEYVTL

B GAD65p14 T220

A

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219

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218

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217

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214

A

F213

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212

A

211

A

209

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20 40 60 80 Original proliferative response to GAD65p14 (%)

100

Figure 5. T-cell responses to scanning alanine-substitutions in GAD65p14. (A) Ten days after footpad immunization with GAD65p14 (50 ìg), draining lymph node cells were prepared and tested for proliferative responses in a 3-day assay to titrations of GAD65p14 scanning alanine substitution peptides from the C-terminus to the N-terminus, as listed in the insets where emboldened A residues indicate the position of the Ala substitution. Recall responses to the original priming antigen (GAD65p14) are shown as ( ). Data are expressed as the mean cpm±SEM of [3H]-Tdr DNA incorporation. (B) Cumulative data from (A) are expressed as % of original response from the original priming antigen (GAD65p14)±SEM. ( ), 100% baseline response when recalling the response with the original priming antigen (GAD65p14). At least two residues, Glu217 and Phe213, appear to be essential for immune reactivity.

the in vitro proliferative response to the original priming antigen (GAD65p14) and Ala substitution peptides was determined (Figure 5A). Consistent with the data observed from the deletion peptide GAD65p15b, that removal of the Glu residue inhibited response, the GAD65p14 Glu217→Ala217 substitution peptide gave rise to a substantially ablated response. Further substitution analysis revealed that at least one other amino acid, Phe213, was critical to immunogenicity in GAD65p14. These results are summarized in Figure 5B.

Age-dependent spontaneous (unprimed) responses to huGAD65 peptides responses in NOD mice GAD65 and peptide-primed responses could be recalled in vitro with GAD65p6, 14, 15, so it was essential to determine if these peptide determinants could elicit unprimed responses in NOD mice. To achieve this, spleens from NOD mice of various ages (4, 8, 12 and 16 (diabetic) weeks) were harvested, grouped, and single cell suspensions were

Characterization of T-cell epitopes on glutamic acid decarboxylase

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Figure 6. Spontaneous proliferative responses to GAD65p6, 14 and 15 as a function of age. Pooled splenocytes from at least three NOD mice aged 4, 8 and 12 weeks, and diabetic (16 weeks) were incubated with the GAD65 peptides as indicated in the graph legends (25 ìM) in a 5-day proliferation assay. Responses were observed in all mice until 12 weeks, when responses to GAD65p6 were not observed (P<0.05). Data are expressed as the mean cpm±SEM of [3H]-Tdr DNA incorporation. , Media; , GAD65p6; , GAD65p14; , GAD65p15.

incubated in the presence of either GAD65p6, 14, or 15 (Figure 6) for 5 days. Maximal proliferative responses were initially seen to GAD65p14 in 4-week-old mice. Responses to all these peptides were observed at least up to 8 weeks of age. Interestingly, by 12 weeks, no significant response (P<0.05) to GAD65p6 remained while responses to GAD65p14 and 15 remained high. This may be due to a lack of homology between the mouse GAD65 and GAD67 peptide 6 sequence (Figure 1). No reproducible spontaneous responses to GAD65p17, 34, or 35 were observed in NOD mice at any age. As a control, unprimed BALB/c splenocytes were incubated with titrations of GAD65p6, 14 and 15. In this case, no significant responses were observed (data not shown).

Spontaneous (unprimed) responses to huGAD65 peptides in CFA-protected NOD mice It has been demonstrated that NOD mice are protected from diabetes by immunization with CFA [9]. As such, it was reasonable to ascertain the immunological responses to GAD65 peptides. It is noteworthy that CFA-immunized NOD mice, though protected from diabetes, are observed to maintain immunological responses to presumptive autoantigens, including GAD [10]. Spleens from 20-week-old CFA-protected mice or age-matched diabetic mice were grouped

and single cell suspensions incubated with either GAD65p6, 14, or 15 for 5 days. A strong response to GAD65p6 and a substantially weaker response to GAD65p14 was observed in CFA-protected mice (Figure 7A). In the age-matched diabetic NOD mice, a strong response to GAD65p14 was observed compared to a weak response to GAD65p6 (Figure 7B).

GAD67 peptide T-cell responses in unimmunized NOD mice As illustrated in Figure 1, there is substantial homology between human GAD65 and mouse GAD67, except in the amino-terminal 100 amino acids. To resolve the potential presence of additional determinants in mouse GAD67, seven overlapping peptide epitopes were synthesized covering the region GAD67 1–121. Pooled splenocytes from mice of different ages (4, 8, 12 and 16 (diabetic) weeks) were incubated in the presence of titrating concentrations of GAD67 peptides. As shown in Figure 8, T-cell responses were consistently observed in all age groups to two GAD67 peptides, GAD67 (28–47), and GAD67 (42– 61)—GAD67p2 and GAD67p3 respectively. T-cell responses to GAD67p2 were not observed in diabetic mice.

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suspensions were incubated with the priming antigen. Supernatants were harvested at optimal time points for cytokine production, 24 h (IL-4) and 72 h (IFN-ã), and subsequently analysed for IL-4 and IFN-ã by ELISA (Figure 10). High levels of IFN-ã relative to IL-4 were observed in mice primed with GAD65p6, and GAD65p14 and IFN-ã were substantially elevated in the GAD65p14 group. GAD65p15-primed cells were observed to give rise to relatively higher levels of IL-4 relative to IFN-ã.

cpm

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Figure 7. Spontaneous proliferative responses to GAD65p6, 14 and 15 in CFA-protected (A) or age-matched control diabetic (B) NOD mice. Pooled splenocytes from three diabetic NOD mice or 3 CFA-protected NOD mice were incubated with the indicated GAD peptide (25 ìM) in a 5-day proliferation assay. Strong proliferation was observed to GAD65p14 in diabetic mice but not in CFA-protected mice and the reverse was true of GAD65p6. Responses to GAD65p15 were consistently observed in all cases. Data are expressed as the mean cpm±SEM of [3H]-Tdr DNA incorporation. Key as in Figure 6.

GAD67 peptide T-cell responses in GAD67 peptide-immunized mice In order to confirm that immunological responses to GAD67p2 and 3 could be elicited, NOD mice were immunized s.c. with peptides. After 10 days, in vitro recall responses to titrating quantities of GAD67p2 and 3 were determined (Figure 9). T-cell responses were observed in animals to their respective priming peptides. Also, a significant response was observed in the GAD67p2-primed group when challenged in vitro with GAD67p3 and vice versa, suggesting there is immunological cross-reactivity between GAD67p2 and 3.

Analysis of cytokines in GAD65p6, 14 and 15-reactive T cells To characterize phenotypically the functional subset of Th cells responding to the GAD65 peptides, NOD mice were primed s.c. with either GAD65p6, GAD65p14, or GAD65p15. After 10 days, popliteal draining lymph nodes were removed and single cell

In this study, we sought to identify GAD epitopes which lead to T-cell mediated autoimmune responses in NOD mice. Although T-cell epitopes of GAD65 have been previously reported [3], recent studies [6, 7] suggest that additional epitopes may, in fact, be responsible for eliciting immune responses. Significantly, it has been demonstrated that responses to the GAD epitope, GAD65p35 could only be induced in peptide-immunized mice, whereas no significant spontaneous proliferation was observed in unimmunized mice [6, 7]. Moreover, two recent studies did not find a protective effect of GAD65p34 and 35 on diabetes in NOD mice [11, 12]. NOD mice primarily express GAD67 in islets and no epitopes of GAD67 have been identified in these mice. By immunizing mice with huGAD65 and examining recall responses with peptides, we have successfully revealed epitopes which lead to significant T-cell responses in NOD mice. A series of peptides previously described [3] were used initially to screen NOD mice for T-cell autoreactivity. The peptide epitopes we found (Figure 2A) were unique and different from those previously reported [3]. Importantly, we could not detect significant proliferative responses to GAD65p17 and 35 and detected only weak responses to GAD65p34 after immunization with huGAD65 (Figure 2). Moreover, it had been previously reported that the majority of T-cell responses were confined to the carboxy-terminal of the GAD protein, whereas our data have identified the central region of the protein to be more relevant in autoreactivity in pre-diabetic, diabetic and GADimmunized NOD mice. To confirm the immunogenicity of the peptides GAD65p6, 14 and 15, mice were immunized s.c. with each of the peptides and the recall responses determined 10 days later in vitro (Figure 3). Although good responses were elicited in all mice, exceptionally strong proliferative responses were observed in mice primed and challenged with GAD65p14. This suggests that this particular determinant may be considerably immunodominant. Apparent inhibition of proliferation was observed at times during in vitro challenge with high peptide concentrations (Figures 3 & 5; >200 ìM). There are a number of conceivable explanations for this observation: (1) peptides may be immediately cytotoxic to lymphocytes at such a high dose range; (2) T cells may have proliferated to the

Characterization of T-cell epitopes on glutamic acid decarboxylase

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67 A p1 D 6 G 7p A D 2 6 G 7p A D 3 6 G 7p A D 4 6 G 7p A D 5 6 G 7p A D 6 67 p7 p7

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Challenge peptide

Figure 8. Spontaneous proliferative responses to GAD67 peptides as a function of age. Pooled splenocytes from at least three NOD mice aged 4, 8 and 12 weeks, and diabetic were incubated with GAD67 peptides (25 ìM) as indicated in the axis legends in a 5-day proliferation assay. Responses were observed in all mice until diabetic, whereas responses to GAD67p2 were not observed. Data are expressed as the mean cpm±SEM of [3H]-Tdr DNA incorporation.

maximum allowed by the tissue culture conditions by the time tritiated-thymidine was added to assess proliferation; and (3) anergy, induced by repeated cross-linking of TCR/peptide–MHC, possibly in the absence of a costimulatory signal [13] may have occurred. This phenomenon occurs particularly in Th1-cell primary cultures when repeatedly stimulated by antigen in the context of APCs [14]. The specific region(s) of GAD65p14 and 15 responsible for immune responses in NOD mice were determined as these epitopes are overlapping in sequence (Figure 4). By immunizing mice with either peptide and recalling the immune response with peptides containing various N- and C-terminal deletions of GAD65p14 and 15, it was found that the presence of the EYVTL motif was essential for immune responses to these peptides. More specifically, the reactivity of this motif appeared to be dependent on the presence of the Glu residue at position E217, as its deletion in a GAD65p15 peptide resulted in inhibited recall responses to the priming antigen, GAD65p15 (Figure 4B). Thus, it seems likely that the observed immune responses to both GAD65p14 and GAD65p15 were a consequence of the sharing of the EYVTL motif. As is

shown in Figure 4A, peptide GAD65p14c elicited a stronger response than GAD65p14b and it may be that the addition of three amino acids improved MHC class II binding and immunoreactivity. Nonetheless, the data cannot be explained on the sole basis of peptide size since GAD65p14a did not give rise to an immune response while GAD65p14c, a peptide of identical length, lent to a strong T-cell response. In an attempt to address further the molecular basis of the interaction of GAD65p14 with I-Ag7, peptides with alanine substitutions from the carboxy-terminal of GAD65p14 were synthesized (Figure 5). NOD mice were immunized and the in vitro recall response was determined to the substituted peptides. Two peptides, GAD65p14-Ala217 and GAD65p14–Ala213, in which Glu217 and Phe213 were respectively substituted, stood out as being particularly incapable of eliciting immune responses. The identification of the critical presence of a Glu residue in GAD65p14 is consistent with studies on the peptide binding characteristics of I-Ag7 in which a negatively charged residue at position 9 was necessary for binding [15]. In Reizis et al. [15], the authors report that the presence of a Leu residue at position p7, which GAD65p14 contains,

M. A. Zechel et al.

92

35,000

2,000

A

30,000

IFN-γ (U/ml)

cpm

25,000 20,000 15,000 10,000

400 300

0

0.25 2.5 Peptide (µM)

200

25

Figure 9. T-cell responses to GAD67 peptides after priming with GAD67 peptides. Female NOD mice were immunized as described in Figure 2 with GAD67p2 and 3 (50 ìg). After 10 days, draining lymph node T cells were tested in a 3-day proliferation assay using titrations of GAD67p2 and 3. Cross-reactive recall responses to GAD67p2 ( , ) and 3 ( , ) were observed in mice primed either with GAD67p2 (closed symbols), or GAD67p3 (open symbols). Data are expressed as means cpm±SEM of [3H]-Tdr DNA incorporation.

also greatly enhances peptide binding. In other respects, our finding that F213 was critical for reactivity is consistent with results from another study which maintains that a Phe residue at position 9 is critical for I-Ag7 binding [16]. Moreover, when considering two of the reported binding motifs [15, 16], GAD65p14 aligns well with other I-Ag7-binding epitopes (Table 2). We believe that EYVTL epitope is critical for both GAD65 and GAD67 responses. The YV residues in EYVTL epitope in GAD65, which are replaced by QI residues in GAD67 sequence, do not alter the recognition of this epitope. Moreover the replacement of YV residues by A did not influence its recognition by GAD65 peptides in primed T cells. The spontaneous (i.e. non-immunized) responses to these peptides in NOD mice were further investigated (Figures 6 & 7). Since responses to various epitopes in autoimmune diseases may not remain constant throughout the course of the disease [3] we determined the responses of NOD mice to GAD peptides as a function of age (Figure 6). As demonstrated, the early predominant response was to GAD65p6 and GAD65p14. Interestingly, by 12 weeks a significant response to GAD65p6 was no longer observed, whereas responses to GAD65p14 and 15 remained. Additionally, it was found in diabetic mice that the immune response to GAD65p6 was almost abolished. Therefore, a split response was observed to GAD65p6 and GAD65p14. Because immunization of juvenile NOD mice with CFA results in long-term disease protection [9], the T-cell responses to GAD65 peptides 6, 14 and 15 were examined in these animals (Figure 7). Indeed, CFA-protected animals that were agematched with diabetic NOD mice had a T-cell repertoire that exhibited a differential reactivity towards the GAD peptides (i.e. they responded strongly to

100

B

80 IL-4 (U/ml)

5,000

1,000 900 800 700 600 500

60 40 20 0

Figure 10. Levels of IL-4 and IFN-ã produced by GAD peptide-primed NOD T cells. Ten days after footpad immunization with GAD65p6 ( ), 14 ( ) or 15 ( ) (50 ìg), draining lymph node cells were incubated with the priming antigen (25 ìM). Supernatants were subsequently tested for the presence of the cytokines IFN-ã (A) and IL-4 (B) by sandwich ELISA. Data are expressed as mean enzymatic units of activity per ml (U/ml)±SEM.

GAD65p6). This may also be related to a lack of homology between GAD65 and GAD67p6 (Figure 1). It is important to note that extensive similarity exists between both the human and mouse isoforms of GAD65p14 and 15 (Figure 1). However, there is appreciably little sequence homology between huGAD65p6 and mGAD67p6, though substantial homology does exist between huGAD65 and mGAD65 isoforms with respect to GADp6 (Figure 1). In fact, this may be an explanation for our observations, in that the reduced T-cell responses to GAD65p6 may be a function of a lack of homology between the tested peptide and mGAD67. Therefore, this region may be important in T-cell response to GAD65 and GAD67 in NOD mice at the onset of disease. In mouse islets, GAD67 is the predominant isoform of GAD expressed [17, 18] and, as such, is probably the most relevant isoform of GAD in investigations into autoimmunity in the NOD mouse. Also, since CFA treatment preserved the â-cell mass, it may be responsible for maintaining levels of GAD65 and hence the responses to the non-GAD67-homologous GAD65 peptide. Responses to GAD65p6 may be more dependent on GAD65 antigen as a result of the conserved amount of islet â-cell mass in CFAprotected mice versus diseased animals. In other

Characterization of T-cell epitopes on glutamic acid decarboxylase

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Table 2. Alignment of GAD65 peptides with known I-Ag7 binding motifs and peptides Protein Relative position Motif [15]

GAD65 GAD65 GAD65 M. tub. HSP60 Insulin Relative position Motif [16]

Epitope

Sequence 1

4 L A T V Y

—

78–97(p6) 202–21(p14) 217–36(p15) 430–46 B9–23

67 9 TL E A D V G

KPCSCSKVDVNYAFLHATDL TNMFTYEIAPVFVLLEYVTL EYVTLKKMREIIGWPGGSGD EGDEATGANIVKVALEA SHLVEALYLVCGERG 1

—

3 E F

Binding

Presumed Presumed Presumed Yes Yes

6 89 I WY M YF V K L R

GAD65 GAD65 GAD65

78–97(p6) 202–21(p14) 217–36(p15)

KPCSCSKVDVNYAFLHATDL TNMFTYEIAPVFVLLEYVTL EYVTLKKMREIIGWPGGSGD

Presumed Presumed Presumed

HEL* Insulin

9–29 B9–23

AAAMKRHGLDNYRGYSLGNW SHLVEALYLVCGERG

Yes Yes

Peptides known to interact/bind to I-Ag7 are aligned according to the binding motif. Single-letter amino acids in bold denote major anchor consensus residues. Amino acids in italics have been shown to increase binding affinity for I-Ag7 [15]. Underlined amino acids are not tolerated in this position [16]. *Hen egg lysozyme.

words, since GAD65 has been shown to be present in minute quantities in the NOD pancreas to begin with [17, 18], the autoimmune processes would ultimately result in the GAD65 antigen being all but extinguished, whereas GAD67 may remain at high levels for substantially longer. This does not, however, explain the fact that responses to GAD65p14 were substantially reduced in CFA-protected mice. Hypothetically, responses to GAD65p14 may be associated with disease induction and/or progression, whereas responses to GAD65p6 may be associated with disease suppression and/or protection. However, this interpretation would seem to contradict the observation that both GAD65p6 and GAD65p14 presented strong levels of IFN-ã in tissue culture supernatants, as disease protection is commonly associated with the Th2 phenotype. This would also conflict with our preliminary data which suggest that immunization with GAD65p14 protects NOD mice from disease onset whereas immunization with GAD65p6 seems to have no appreciable effect (data not shown). Indeed, the precise role of the GAD65 peptides identified in this study is unclear with respect to diabetogenesis. Until now, experiments have centred around GAD65 peptide epitopes. However, as reported previously, GAD67 is the predominant isoform of GAD expressed in mouse islets [17, 18]. As such, it was important to determine the autoimmunogenic

peptides in GAD67 (if any). As Figure 1 suggests, immunological homology is probably sufficient to allow one to extrapolate results obtained from GAD65 to GAD67 in the majority of the protein, including GAD65p14 and 15, with the exception of the 100 N-terminal amino acids. Overlapping peptides were synthesized to investigate this incompletely homologous region. To test for immunoreactivity, splenocytes from NOD mice of various ages were incubated in the presence of the GAD67 peptides (Figure 8). Significant proliferation was consistently observed with two peptides, GAD67p2 and 3. There is also appreciably little homology between GAD65 and GAD67 in these regions. In order to confirm the immunogenicity of GAD67p2, three mice were immunized and good in vitro recall responses were observed (Figure 9). These data suggest that at least two more epitopes may play critical role in the cell-mediated autoimmune responses to GAD in NOD mice. Supernatants from cell cultures were analysed for IL-4 and IFN-ã to determine the nature of the T-cell responses (Figure 10). It was determined that supernatants from both GAD65p6 and GAD65p14 were predominantly Th1-like, as evidenced by their strongly skewed IFN-ã cytokine profiles. Interestingly, supernatants from GAD65p15 mice were proportionately higher in IL-4 than in the other peptide samples. This may be consistent with the observation that

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GAD65p15-primed splenocytes do not proliferate as strongly as do the Th1-like GAD65p6 and 14 responding T cells. High quantities of IFN-ã were consistently observed in GAD65p14 supernatants, suggesting that these cells are particularly biased towards the Th1 subset. Preliminary data suggest that immunization with GAD65p14 is capable of reducing the frequency of onset of overt diabetes (data not shown). This is significant since immune responses to GAD65p14 are the highest of those observed in unprimed NOD mice. It was also found that GAD65p14 was highly immunogenic and gave rise to a Th1-like cytokine profile—a seemingly disease-favouring immune condition. It has been reported that immunization of animals with immunogenic proteins and peptides results in protection from diabetes [3, 12]. Consequently, it was suggested that this immunization may, in effect, shift the dominant immune response from a diseasefavouring Th1-like response to a disease-protective Th2-like response [10, 19]. The induction of such a Th2-like response was not observed after immunization with GAD65p14. However, an eventual switch to a Th2-like response in vivo cannot be dismissed. Another possible explanation lies in the results from a recently reported NOD-derived T-cell clone which phenotypically expresses the cytokines IL-10, IFN-ã and TGF-â, but not IL-4 or IL-2 [20]. Adoptive transfer of these cells into juvenile NOD mice prevented the onset of diabetes and enabled grafted islets to be tolerated in acutely diabetic mice. It is critical, therefore, to examine further the cytokine profiles of mice primed with GAD peptides. The data obtained in this study are important in elucidating the immunological mechanism(s) leading to the development of IDDM. Once identified, epitopes of one of the critical autoantigens in the autoimmune diabetes disease process will serve as important reagents in further studies. Certainly it will be important to investigate the development of T-cell clones with specificity to these GAD determinants, ultimately allowing determination of the specific role of GAD antigens in disease induction/ protection.

Acknowledgements We acknowledge support from the Juvenile Diabetes Foundation International, The Canadian Diabetes Association, and the Medical Research Council of Canada for this work. We would also like to thank Edwin Lee-Chan for the synthesis of peptides, Jane Cator for animal care, and Queendy Yu for technical assistance.

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