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HLA-B7–Restricted Islet Epitopes Are Differentially Recognized in Type 1 Diabetic Children and Adults and Form Weak Peptide-HLA Complexes

The cartography of β-cell epitopes targeted by CD8+ T cells in type 1 diabetic (T1D) patients remains largely confined to the common HLA-A2 restriction. We aimed to identify β-cell epitopes restricted by the HLA-B7 (B*07:02) molecule, which is associated with mild T1D protection. Using DNA immunization on HLA-B7–transgenic mice and prediction algorithms, we identified GAD and preproinsulin candidate epitopes. Interferon-γ (IFN-γ) enzyme-linked immunospot assays on peripheral blood mononuclear cells showed that most candidates were recognized by new-onset T1D patients, but not by type 2 diabetic and healthy subjects. Some epitopes were highly immunodominant and specific to either T1D children (GAD530–538; 44% T cell–positive patients) or adults (GAD311–320; 38%). All epitopes displayed weak binding affinity and stability for HLA-B7 compared with HLA-A2–restricted ones, a general feature of HLA-B7. Single-cell PCR analysis on β-cell–specific (HLA-B7 tetramer–positive) T cells revealed uniform IFN-γ and transforming growth factor-β (TGF-β) mRNA expression, different from HLA-A2–restricted T cells. We conclude that HLA-B7–restricted islet epitopes display weak HLA-binding profiles, are different in T1D children and adults, and are recognized by IFN-γ+TGF-β+CD8+ T cells. These features may explain the T1D-protective effect of HLA-B7. The novel epitopes identified should find valuable applications for immune staging of HLA-B7+ individuals.

The cartography of b-cell epitopes targeted by CD8 + T cells in type 1 diabetic (T1D) patients remains largely confined to the common HLA-A2 restriction. We aimed to identify b-cell epitopes restricted by the HLA-B7 (B*07:02) molecule, which is associated with mild T1D protection. Using DNA immunization on HLA-B7transgenic mice and prediction algorithms, we identified GAD and preproinsulin candidate epitopes. Interferon-g (IFN-g) enzymelinked immunospot assays on peripheral blood mononuclear cells showed that most candidates were recognized by new-onset T1D patients, but not by type 2 diabetic and healthy subjects. Some epitopes were highly immunodominant and specific to either T1D children (GAD 530-538 ; 44% T cell-positive patients) or adults (GAD 311-320 ; 38%). All epitopes displayed weak binding affinity and stability for HLA-B7 compared with HLA-A2-restricted ones, a general feature of HLA-B7. Single-cell PCR analysis on b-cellspecific (HLA-B7 tetramer-positive) T cells revealed uniform IFN-g and transforming growth factor-b (TGF-b) mRNA expression, different from HLA-A2-restricted T cells. We conclude that HLA-B7-restricted islet epitopes display weak HLA-binding profiles, are different in T1D children and adults, and are recognized by IFN-g + TGF-b + CD8 + T cells. These features may explain the T1D-protective effect of HLA-B7. The novel epitopes identified should find valuable applications for immune staging of HLA-B7 + individuals. Diabetes 61:2546-2555, 2012 I dentification of the epitope targets igniting b-cell autoimmunity provides molecular probes to detect their cognate T cells. Detection of these autoreactive T cells offers useful biomarkers to monitor autoimmune progression along the course of disease (1,2) and regression after immunotherapeutic interventions (3). Second, epitopes can be used as therapeutic agents to neutralize pathogenic T cells after tolerogenic administration (4).
A wealth of information has been harvested in recent years through identification of different epitopes targeted by autoreactive CD8 + T cells. This information is highly relevant to the understanding of type 1 diabetes (T1D) pathogenesis, as CD8 + T cells play a central role in the development (5) and probably the final effector phase of autoimmune b-cell destruction (6). Despite this steady progress, this catalog of b-cell epitopes remains incomplete. With few exceptions (7,8), epitope discovery has been limited to peptides restricted for the most common HLA-A2 allele. This allele allows coverage of ;40% of the T1D population, thus leaving another 60% not amenable to be monitored for T-cell responses. Another point of interest in achieving additional epitope coverage is that some HLA class I alleles have recently been recognized as additional T1D-susceptible or -protective alleles independently of the long-known HLA class II haplotypes (9,10). While HLA-B*39:06 is the strongest T1D-predisposing HLA class I allele (odds ratio [OR] 10.31), HLA-B*57:01 is the one conferring the highest protection (OR 0.19) (10). Thus, it is relevant to address the immune recognition mechanisms underlining these protective effects. However, HLA-B*57:01 is relatively infrequent in the general Caucasian population (;3.5%) and exceedingly rare in matched T1D patients (;0.5%) (10), thus making these studies difficult to perform and yielding marginal population coverage.
HLA-B7 (B*07:02, accounting for ;98% of Caucasian HLA-B7 + subjects) (10,11) has also been associated with T1D protection (10). This association persists once adjusted for linkage disequilibrium with class II alleles (OR 0.58 [95% CI 0.47-0.70]) (10). HLA-B7 is expressed in ;15% of the healthy population and in ;7% of T1D patients of Caucasian descent (10). Identification of HLA-B7-restricted b-cell epitopes would thus provide coverage of a significant subgroup of patients. Moreover, the relative T1D protection of HLA-B7 + patients may allow using epitope identification to generate hypotheses for this protective effect. We therefore set forth to identify HLA-B7-restricted epitopes derived from the two b-cell antigens (Ags) GAD and preproinsulin (PPI).
T-cell reactivities to these epitopes were further validated for their T1D specificity in T2D and healthy controls (Fig. 3A). Although most (5/7, 71%) epitopes (GAD 3-11 , GAD 100-108 , GAD 311-320 , GAD 498-506 , and GAD 530-538 ) were only recognized by new-onset T1D patients, PPI [8][9][10][11][12][13][14][15][16] and GAD 175-184 were less T1D specific, being recognized by 11 and 6% of healthy subjects, respectively. Moreover, PPI [8][9][10][11][12][13][14][15][16] was also recognized by a significant fraction (4/12, 33%) of T2D patients. Further controls included four new-onset T1D patients (one adult and three children) who were positive for the B7-related HLA-B27 restriction element. None of these subjects tested positive to any of the epitopes considered (data not shown). Different epitopes are targeted in T1D children and adults. For those epitopes that were T1D specific, different profiles of T-cell reactivity were observed in T1D children and adults (Fig. 3A). Although some epitopes (GAD 3-11 and GAD 100-108 ) were recognized in both patient groups with FIG. 1. HLA-B7-restricted GAD candidate epitopes were identified. HLA-B7-transgenic mice (n = 9) were immunized with plasmid DNA encoding full-length human GAD. Their splenocytes were subsequently recalled with the 37 GAD peptides listed in Table 1  similar frequencies (11-14%), others were specific for either T1D children (GAD 498-506 and GAD 530-538 ) or adults (GAD 311-320 ). This was evident not only when analyzing the prevalence of T-cell responses but also when considering the relative immunodominance of these epitopes among the total b-cell epitope-reactive T cells detected (Fig. 3B). Indeed, GAD 530-538 was highly immunodominant in T1D children, as it was targeted by 40% of the total autoreactive T-cell responses and recognized by 44% of pediatric patients (P = 0.008), but in none of the T1D adults. Conversely, GAD 311-320 was the major T1D-specific epitope in T1D adults, as it accounted for 34% of the total T-cell responses and was recognized in 38% of T1D adults (P = 0.022), but in none of the T1D children. Most CD8 + T-cell IFN-g responses rapidly wane after T1D onset. We previously reported that IFN-g-producing CD8 + T-cell responses against different HLA-A2-restricted b-cell epitopes rapidly wane after diagnosis (2). This was the case also for HLA-B7-restricted epitopes. Testing a subgroup of five patients (one adult and four children) near diagnosis and 7 days to 14 months thereafter (Fig. 4), most (6/10, 60%) responses that were positive at diagnosis became undetectable. There was only one instance of a GAD 175-184 -specific response that was absent at diagnosis and became detectable 14 days after (patient A03). As an internal control, T-cell responses against a viral epitope pool remained stable in four of the five patients studied and disappeared in one child (C05), perhaps witnessing the resolution of an acute viral infection. Moreover, the same T-cell responses tested in six long-standing HLA-B7 + T1D adults were infrequent, with only two patients (33%) scoring positive for a single epitope (PPI [8][9][10][11][12][13][14][15][16] , which was also targeted in T2D patients, and GAD 311-320 ) (Supplementary Fig. 3) compared with 63% (5/8) of new-onset T1D adults testing positive, most frequently for two different epitopes (Supplementary Fig. 1). HLA-B7-restricted epitopes show lower HLA binding affinity and stability than HLA-A2-restricted ones. Next, we analyzed the HLA binding affinity and stability of the identified HLA-B7-restricted epitopes and compared vs. 12.1 h, 1.1-49.1; P = 0.0002). Moreover, the lower HLA binding affinity and stability frequently reported for autoimmune epitopes (25) compared with nonautoimmune ones was observed for HLA-A2 (median affinity = 12 vs. 2 nmol/L, P = 0.001; stability = 7.1 vs. 12.1 h, P = 0.043), but not for HLA-B7 (median affinity = 95 vs. 35 nmol/L, P = 0.78; stability = 2.4 vs. 2.4 h, P = 1), further suggesting a general feature of weak binding for HLA-B7. HLA-B7-restricted autoreactive CD8 + T cells show higher IFNG and TGFB expression than HLA-A2restricted ones. The lower binding affinity and stability imposed by HLA-B7 compared with HLA-A2 would lead to more transient availability of epitope/HLA-B7 versus epitope/HLA-A2 complexes, which may deliver weaker signals to T cells. To gain insight into the functional consequences of this weak epitope binding to HLA-B7, we used singlecell PCR to functionally characterize epitope-specific CD8 + T cells. PBMCs from the T1D patient C09, who tested positive for PPI [8][9][10][11][12][13][14][15][16] and GAD 530-538 , and A07, who tested positive for GAD 311-320 ( Supplementary Fig. 1), were further assayed using HLA-B7 TMrs loaded with the corresponding peptides. Results were concordant with those of IFN-g ELISpot, yielding significant fractions of TMr + CD8 + cells compared with samples stained with TMrs loaded with a control peptide (Fig. 6A). As expected, this concordance was observed relative to the absence or presence of epitope-specific T cells and not to their frequency, given the different readout of the two assays (functional vs. structural) and the lower threshold needed for T-cell activation compared with stable TMr binding (26), especially in the presence of weak peptide/HLA-B7 complexes.

DISCUSSION
Identification of b-cell epitopes allows tracking of autoreactive T cells during disease progression and after immunotherapeutic interventions. Although T-cell assays bypassing this need for HLA-specific epitope identification have been previously described (3,27), it remains important to draw a comprehensive epitope cartography not only for the most prevalent HLA-A2 allele but also for other common restriction elements. HLA-B7 fits these requirements, being expressed by ;15% of the Caucasian general population and ;7% of T1D patients (10). This difference in frequency has suggested a relative protection conferred by this allele, which was confirmed in genetic studies correcting for linkage disequilibrium (10). This feature of HLA-B7 makes it additionally relevant to explore immune mechanisms underlying this protection.
We elected to identify HLA-B7-restricted epitopes derived from GAD and PPI, as these two Ags yielded most of the immunodominant epitopes described for HLA-A2 (1,13). The epitopes thus obtained should prove useful for immune staging of HLA-B7 + individuals. One lesson learned is that epitope panels need to be tailored for the T1D population under study, i.e., pediatric versus adult. It has long been postulated that T1D children harbor more aggressive b-cell autoimmunity, which may explain their earlier age at onset compared with adults. The immunological bases for this difference are unclear, and previous T-cell studies have not compared these two groups. We show that the b-cell epitopes targeted are only partially shared. Indeed, some epitopes were exclusively targeted in T1D children (i.e., GAD 530-538 ), whereas others were typical of T1D adults (i.e., GAD 311-320 ). Although the size of our study groups was relatively small due to the rarity of HLA-B7 + patients, this agerelated epitope difference proved significant. Furthermore, age-specific epitopes were highly immunodominant in the corresponding population. Why these epitopes are differentially selected and whether they are more immunogenic or give rise to more aggressive cytotoxic T lymphocytes in children remains to be elucidated. There was also one epitope (PPI [8][9][10][11][12][13][14][15][16] ) that was found to not be T1D specific, being similarly recognized in T2D patients and healthy controls. Similar observations have been made for some HLA-A2restricted b-cell epitopes (8,28) and may reflect a high number of naïve precursors that succeed in activating upon epitope-driven stimulation. As previously observed in HLA-A2 + T1D patients (1,2) and even in inbred NOD mice (29,30), there was considerable heterogeneity in the epitopes targeted in different individuals. These repeated findings may explain the disappointing results of vaccination trials using one single islet Ag such as insulin or GAD (4).
The current study also gives further validation of our DNA immunization technique on HLA-transgenic mice for epitope identification (13). We were not able to apply the same strategy to PPI due to inefficient in vivo expression of the corresponding plasmid. All the GAD epitopes preselected in immunized mice proved to be recognized by patient T cells, as was previously the case for HLA-A2restricted islet epitopes (13,28). Although the reactivities detected in mice are induced by exogenous immunization, they prove relevant once transferred to human T1D. The major advantage of this approach is its rapidity and ease of use, as panels of candidate epitopes can be generated within weeks with minimal hands-on work. Moreover, candidates are selected based not only on immunogenicity but also on natural processing, as DNA immunization ensures in vivo Ag translation and processing (24). Selection of the initial peptides to test based on a wide range of predicted HLA-binding affinities also yields weak binding epitopes, which are particularly relevant in autoimmunity. Indeed, most of the epitopes identified here were weak and unstable HLA-B7 binders, and would thus be missed if initially selected based on strong binding. HLA-B7restricted epitopes were uniformly weak binders irrespective of their Ag source (islet or nonautoimmune), pointing to a general feature of HLA-B7, as recently described (31). This feature is not shared by all HLA-B alleles, as it is not observed for HLA-B*35:01 (31). Apart from the special case of HLA-B7, the HLA-A2-restricted b-cell epitopes previously characterized displayed significantly weaker HLA binding than epitopes derived from nonautoimmune Ags. Thus, autoreactive epitope-HLA complexes are not only characterized by weaker recognition (T-cell receptor affinity) from cognate T cells (32), but also by weaker peptide-HLA interactions, probably leading to decreased epitope presentation. These two features may explain failure of thymic negative selection.
The study of HLA-B7 + T1D patients also allows a comparison with patients carrying the T1D-indifferent allele HLA-A2 (9,10). We show that, despite the mild T1D protection conferred, HLA-B7 can function as a restriction element for b-cell-reactive CD8 + T cells. These cells are present in the blood of T1D patients at frequencies (0.003% of PBMCs) similar to those of HLA-A2-restricted T cells (1,24). However, their functional profile is different, being characterized by higher and uniform IFN-g and TGF-b mRNA expression. These cells might represent a regulatory subset, which may explain the HLA-B7 protective effect. Indeed, Weiner and colleagues (33) described a regulatory population of murine CD8 + T cells that are LAP + (latencyassociated peptide) and produce high amounts of IFN-g and TGF-b. These cells are regulatory both in vitro and in vivo FIG. 6. mRNA expression profiles of epitope-specific HLA-B7-and HLA-A2-restricted TMr + CD8 + T cells. A: Comparison of IFN-g ELISpot and HLA-B7 TMr readouts for different epitopes in patients C09 and A07, as indicated at the top of each column. Top row displays representative ELISpot wells out of triplicate measurements. Bottom row shows the corresponding fraction of TMr + CD8 + cells detected in the same individual. B: TMr + CD8 + cells were single-cell sorted, analyzed by reverse-transcriptase PCR, and compared with similar measurements previously performed on HLA-A2 + , new-onset T1D patients (16). Percent numbers of HLA-B7-restricted PPI 8-16 -(black round symbols), GAD 530-538 -(black diamond symbols), GAD 311-320 -(back triangle symbols), and CMV 417-426 -specific (gray round symbols) T cells (n = 20/each) expressing each mRNA are depicted. White and gray squares show frequencies of gene-expressing single HLA-A2-restricted PPI 6-14 -and CMV 495-503 -specific T cells sorted from 7 HLA-A2 + , new-onset T1D patients. neg, negative; +++, positive response ‡5 SDs above background. and suppress experimental autoimmune encephalomyelitis in an IFN-g-and TGF-b-dependent fashion (33). An Ag-specific CD8 + T-regulatory subset with similar characteristics has been reported after peptide immunization (34). The observed IFN-g + TGF-b + HLA-B7-restricted T cells may be generated after low-density epitope-HLA-B7 stimulation, either in the thymus or in the periphery. Nonetheless, the subjects analyzed here developed T1D, suggesting that HLA-B7-mediated protection is fallible, as is the case for that conferred by HLA class II alleles (35).
HLA-B7-restricted CD8 + T-cell responses producing IFN-g rapidly waned after disease onset, as previously reported for HLA-A2 (2). This rapid disappearance may be due to loss of residual b-cells, which limits Ag availability, or to the effects of insulin therapy, which may induce tolerance to insulin-derived epitopes and relieve metabolic stress from b-cells, possibly making them less immunogenic (36). This decrease in autoreactive T cells, however, seems specific to IFN-g-producing cells, as it is not observed in studies using TMrs, including our own (16). The fact that TMr + CD8 + T cells in long-standing patients are predominantly central memory (16), i.e., they secrete little IFN-g (37), may explain these findings. TMrs may thus allow the use of the identified epitopes for immune staging of both the prediabetic phase, to stratify risk, and after T1D onset, to monitor response to immunotherapies.
In conclusion, the novel epitopes identified in this study demonstrate that the T1D-protective HLA-B7 molecule is capable of restricting the response of b-cell-reactive T cells. These responses target different epitopes in T1D children and adults and are imprinted by the weak peptidebinding features of HLA-B7, possibly conditioning their phenotype. Further studies are warranted to verify whether an opposite phenomenon takes place for HLA class I alleles associated with T1D susceptibility.