CD4+ and CD8+ T-cell clones from congenital rubella syndrome patients with IDDM recognize overlapping GAD65 protein epitopes. Implications for HLA class I and II allelic linkage to disease susceptibility.

To fully characterize human glutamic acid decarboxylase (GAD)65 protein T-cell epitopes associated with insulin-dependent diabetes mellitus (IDDM), CTL clones specific to GAD65 protein antigens were isolated from two congenital rubella syndrome (CRS)-associated IDDM patients. Overlapping nonamer T-cell epitopes recognized by both CD4+ or CD8+ CTL clones within peptides GAD65(252-266) and GAD65(274-286) were identified as sequences bounded by GAD65(255-266) with 6/9 overlapping residues, and GAD65(276-285) with 8/9 overlapping residues, respectively, using two panels of overlapping peptide analogs in cytotoxicity assays. HLA restrictive elements of the T-cell clones were also identified using a panel of B cell lines with different HLA phenotypes as targets in cytotoxicity assays. The antigenic GAD65 peptides elicited cytotoxic responses of peptide-specific CD4+ T-cell clones in the context of HLA DRB1*0404. The CD8+ T-cell clone specific to GAD65(255-263) was found to be restricted by HLA A3 and A11. Similarly, the CD8+ T-cell clone specific to GAD65(277-285) killed peptide-sensitized target cells expressing HLA B35 and B15. The observed HLA restriction of these overlapping epitopes implies that a tandem of [DRB1*0404-A11(3)] and/or a tandem of [DRB1*0404-B35(15)] might predispose CRS patients to development of IDDM.     

In Vitro Stimulation with Glutamic Acid Decarboxylase (GAD65) Leads to an Oligoclonal Response of Peripheral T-Cells in an IDDM Patient

The enzyme glutamic acid decarboxylase (GAD65) is a major autoantigen in insulin-dependent diabetes mellitus (IDDM). To study T-cell reactivity towards GAD, peripheral blood leucocytes from seven patients with IDDM and five control subjects were stimulated in vitro with recombinant GAD. All diabetics studied were heterozygous for diabetes-associated HLA alleles, i. e. HLA-DRB1*03, *04-DQBl*0302, *0201. A single IDDM subject (no. GAD65.05) revealed a strong response against GAD65. After stimulation, his T-cell receptor β (TCRBV) usage was found to be oligoclonal. The sequence analysis of the putative peptide binding region of the T-cell receptor (CDR3 region) of 37 GAD-reactive T-cell clones revealed no common CDR3 motif. The stimulation of GAD-reactive T-cells could be inhibited with anti-class II monoclonal antibodies, indicating a class II restricted T-cell response. In addition, GAD65-responsive T-cells revealed a Thl cytokine response pattern. The author's data suggest that GAD-reactive T-cells of Thl phenotype can be obtained after in vitro stimulation of peripheral blood leucocytes from an HLA-DRBl*03/*04 heterozygous IDDM patient. The lack of a common CDR3 motif suggests the absence of an immunodominant T-cell epitope in that patient, or may indicate receptor repertoire spreading of peripheral T-lymphocytes.     

Glutamic acid decarboxylase T lymphocyte responses associated with susceptibility or resistance to type I diabetes: analysis in disease discordant human twins, non-obese diabetic mice and HLA-DQ transgenic mice

Glutamic acid decarboxylase (GAD65) has been implicated as a targeted self antigen in the immune destruction of pancreatic beta cells. T cell responses to GAD65 peptides have been detected in both patients with type I diabetes and in the non-obese diabetic (NOD) mouse. To establish which GAD65 epitopes are important in the immunopathogenesis of disease we initially compared T cell responses to GAD65 epitopes in conditions of disease susceptibility and protection. T cell responses to GAD65 peptides were measured in monozygotic twin pairs selected on the basis of disease discordance and T cell recognition of immunogenic regions of GAD65. Peptides of interest were then used to immunize susceptible NOD mice and H2-E transgenic NOD mice which are protected from diabetes. A differential response to the epitope GAD65 521-535 discriminated diabetic from non-diabetic human twins as well as susceptible from protected mice. This epitope as well as GAD 505-519 induces T cell responses despite binding the type I diabetes associated HLA- DQA1*0301/DQB1*0302 product with low affinity. Since DQ-restricted T cell responses are difficult to study in humans, HLA-DQ8 transgenic mice were then used: GAD epitopes 521-535 and 505-519 induced responses in DQ8 transgenic mice and T cell lines were established. Long-term T cell lines against GAD 505-519 were HLA-DQ restricted, and responded to peptide with a strong IFN-gamma and IL-10 response. The findings implicate GAD 521-535 as a possible target peptide in pathogenesis and are compatible with a model whereby self-reactive T cells specific for low-affinity peptide-MHC complexes may escape thymic negative selection.      

An HLA-binding-motif-aided peptide epitope library: A novel library design for the screening of HLA-DR4-restricted antigenic peptides recognized by CD4+T cells

Susceptibility to a series of autoimmune diseases is strongly associated with particular HLA class II alleles. Identification of T cell clones and antigenic epitopes bound by HLA class II molecules involved in autoimmune diseases is critical to understanding the etiology of these HLA class II-associated diseases. However, establishment of T cell clones in autoimmune diseases is difficult because the antigenic peptides are unknown. Peptide library methods which include all possible peptide sequences offer a potentially powerful tool for the detection of cross-reactive antigenic peptides recognized by T cells. Here, we reduced the number of peptides per mixture by utilizing the known binding motifs of peptides for the HLA-DRB1*0405 molecule and evaluated the effectiveness of this library design. Each library mixture evoked a strong proliferative response in the unprimed peripheral blood lymphocytes (PBL) from HLA-DRB1*0405-positive donors but little or no response in the PBL from HLA-DRB1*0405-negative donors. The library also detected antigenic peptides that activated three antigen-specific T cell lines restricted by HLA-DRB1*0405, with different specificities. The motif-based approach thus presents a powerful method for monitoring T cells in large, heterogeneous T cell populations and is useful for the identification of the mimic peptide epitopes of T cell lines and clones. Received: October 3, 1997 / Accepted: October 23, 1997     

Complexity of Human Immune Response Profiles for CD4+ T Cell Epitopes from the Diabetes Autoantigen GAD65

Complex protein antigens contain multiple potential T cell recognition epitopes, which are generated through a processing pathway involving partial antigen degradation via proteases, binding to MHC molecules, and display on the APC surface, followed by recognition via the T cell receptor. We have investigated recognition of the GAD65 protein, one of the well-characterized autoantigens in type I diabetes, among individuals carrying the HLA-DR4 haplo-types characteristic of susceptibility to IDDM. Using sets of 20-mer peptides spanning the GAD65 molecule, multiple immunostimulatory epitopes were identified, with diverse class II DR molecules functioning as the restriction element. The majority of T cell responses were restricted by DRB1 molecules; however, DRB4 restricted responses were also observed. Antigen-specific T cell clones and lines were derived from peripheral blood samples of pre-diabetic and IDDM patients and T cell recognition and response were measured. Highly variable proliferative and cytokine release profiles were observed, even among T cells specific for a single GAD65 epitope.     

Complexity of human immune response profiles for CD4+ T cell epitopes from the diabetes autoantigen GAD65

Complex protein antigens contain multiple potential T cell recognition epitopes, which are generated through a processing pathway involving partial antigen degradation via proteases, binding to MHC molecules, and display on the APC surface, followed by recognition via the T cell receptor. We have investigated recognition of the GAD65 protein, one of the well-characterized autoantigens in type I diabetes, among individuals carrying the HLA-DR4 haplotypes characteristic of susceptibility to IDDM. Using sets of 20-mer peptides spanning the GAD65 molecule, multiple immunostimulatory epitopes were identified, with diverse class II DR molecules functioning as the restriction element. The majority of T cell responses were restricted by DRB1 molecules; however, DRB4 restricted responses were also observed. Antigen-specific T cell clones and lines were derived from peripheral blood samples of pre-diabetic and IDDM patients and T cell recognition and response were measured. Highly variable proliferative and cytokine release profiles were observed, even among T cells specific for a single GAD65 epitope.     

CD4+ T cells recognize diverse epitopes within GAD65: implications for repertoire development and diabetes monitoring

Type 1 diabetes is associated with T‐cell responses to β‐cell antigens such as GAD65. Single T‐cell epitopes have been investigated for immune monitoring with some success, but multiple epitopes may be required to fully characterize responses in all subjects. We used a systematic approach to examine the diversity of the GAD65‐specific T‐cell repertoire in subjects with DRB1*04:01 haplotypes. Using class II tetramers, we observed responses to 15 GAD65 epitopes, including five novel epitopes. The majority were confirmed to be processed and presented. Upon stimulation with peptides, GAD‐specific responses were equally broad in subjects with diabetes and healthy controls in the presence or absence of CD25⁺ T cells, suggesting that a susceptible HLA is sufficient to generate a potentially autoreactive repertoire. Without depleting CD25⁺ cells, GAD_113–132 and GAD_265–284 responses were significantly stronger in subjects with diabetes. Although nearly every individual responded to at least one GAD65 epitope, most were seen in less than half of the subjects tested, suggesting that multiple epitopes are recommended for immune monitoring.     

Characterization of the T cell recognition of hepatitis B surface antigen (HBsAg) by good and poor responders to hepatitis B vaccines

To study the regulation of the human cellular immune response to HBsAg we produced a series of HBsAg-specific T cell lines from good and poor responders to the hepatitis B vaccine. All T cell lines expressed CD4 on their membrane and could therefore be considered of the helper/inducer phenotype. The different HBsAg-specific T cell lines were restricted by HLA-DRB5*0101, DRB1*1201, -DRB1*0701, -DRB1*0301, -DPB1*0201, -DPB1*0402, and -DPB1*0901. In good responders to the hepatitis B vaccine different HLA molecules could act as restricting element. In poor responders the diversity of HLA class II restriction determinants was more limited. This leads us to conclude that the immune response to HBsAg is multispecific and polyclonal in good responders and paucispecific and oligoclonal in poor responders to the hepatitis B vaccine. By using a panel of synthetic peptides representing selected sequences of the HBsAg, the fine specificities of each of these T cell lines could be determined. Strikingly, the majority of the identified T cell epitopes was located in and around the first hydrophobic transmembranous region of the HBsAg. This was observed in T cell lines from good and poor vaccine responders, without distinction. The remarkable T cell immunogenicity of this region may reside in its richness in binding motifs for a variety of HLA class II determinants.     

Influence of HLA-DRB1 alleles on lymphoproliferative responses to a naturally processed and presented measles virus phosphoprotein in measles immunized individuals

Identification of stimulatory T-cell epitopes recognized by CD4+ T lymphocytes is important for vaccine development. Our previous studies using mass spectrometry identified a naturally processed HLA class II restricted DRB1*0301 T cell epitope in the measles virus phosphoprotein, MV-P1 (residues 179-197). Here we provide lymphocyte proliferation data from peripheral blood mononuclear cells (PBMC) obtained from 131 HLA-DRB1*0301-positive and HLA-DRB1*0301-negative (HLA discordant) individuals previously immunized against measles and report that a single amino acid substitution in the MV-P1 T cell epitope can reduce T cell proliferation and CD4+ T-cell recognition. Measles virus and measles peptide-specific lymphoproliferative responses and HLA-DRB1 allele associations reveal that the DRB1*0701 allele provided suggestive evidence of association with both measles virus (p = 0.03) and MV-P1 peptide (p = 0.06) lymphoproliferation. A marginally significant increase in the frequency of the *0301 allele (p = 0.10) was found among subjects who demonstrated low cellular responses to the measles virus. We found no associations between proliferation levels to the MV-P1 and MV-P2 peptides with *0301 alleles. We speculate that the glutamic acid at position 192 of the measles phosphoprotein is a critical immunogenicity factor and may influence the antigenicity of the naturally processed HLA class II MV-P1 epitope.     

T cell response pattern to glutamic acid decarboxylase 65 (GAD65) peptides of newly diagnosed type 1 diabetic patients sharing susceptible HLA haplotypes.

Autoantibodies and autoreactive T lymphocytes directed against several pancreatic beta cell proteins such as GAD65 have been identified in the circulation before and at the onset of clinical type 1 (insulin-dependent) diabetes. Using GAD65 synthetic peptides, we studied the proliferative response of peripheral blood mononuclear cells (PBMC) either from recently diagnosed type 1 diabetic patients, of whom the majority share the disease-associated HLA class II haplotype (DR4-DQB1*0201 or DR3-DQB1*0302), or from HLA-matched control subjects. We found that 67% (14/21) of the type 1 diabetic patients and 39% (9/23) of the control subjects exhibited a positive proliferative response. Compared with control subjects, however, PBMC from diabetic patients proliferated more frequently (P < 0.05) in the presence of peptide pools from the C-terminal region of GAD65 (amino acids 379-585). Diabetic patients with the same HLA-DQ or HLA-DR alleles showed partially identical T cell reactivity, but no clear correlation could be made between MHC class II specificity and T cell epitopes because of multiple combinations of class II alleles. In addition, by flow cytometry, we studied the direct binding of GAD65 peptides to MHC class II molecules of Epstein-Barr virus (EBV)-transformed B (EBV-B) cells obtained from a diabetic patient. We found that 11 GAD peptides were able to bind to the highly susceptible haplotype DRB1*0301/0401-DQA1*0301/0501-DQB1*0302/0201 on the surface of EBV-B cells in partial correlation with the results obtained in the proliferation assays.     

Induction of CTL response by a minimal epitope vaccine in HLA A*0201/DR1 transgenic mice: dependence on HLA class II restricted T(H) response

CTL play a pivotal role in the immune response during viral infections. In this study, the HLA class II restricted T(H) requirement for optimal in vivo induction of HLA class I restricted CTL responses has been investigated. Towards this goal, transgenic mice expressing both HLA class I (A*0201 or A2.1) and class II (DRB1*0101 or DR1) molecules have been derived. Immunization of these mice with an HLA A*0201-restricted and CMV-specific CTL epitope (pp65(495-503)), and either of three different tetanus toxin-derived MHC class II-binding T(H) epitopes, resulted in a vigorous CTL response. CTL specific for the pp65(495-503) epitope were dramatically enhanced in mice expressing both the HLA-DR1 and HLA-A*0201 transgenes. Notably, preinjection of three TT peptides (TT(639-652), TT(830-843), and TT(947-967)) increased the capability of HLA A*0201/DR1 Tg mice to respond to subsequent immunization with the T(H) + CTL peptide mixture. These results indicate that the use of HLA A*0201/DR1 Tg mice constitute a versatile model system (in lieu of immunizing humans) for the study of both HLA class I and class II restricted T-cell responses. These studies provide a rational model for the design and assessment of new minimal-epitope vaccines based on their in vivo induction of a pathogen-specific CTL response.     

Epitope analysis of HLA-DR-restricted helper T-cell responses to Der p II, a major allergen molecule of Dermatophagoides pteronyssinus

T-cell epitopes of Der p II, a major allergen of Dermatophagoides pteronyssinus , were analyzed by using human T-cell clones. We tested 38 cloned T cells from two Japanese patients with allergic rhinitis, and identified at least two peptides (K33-T47 and 158-C73) as helper T-cell epitopes. The former epitope was shown to be restricted by HLA-DRB1* 1502, and the latter by HLA-DRB1* 0405, both of which are typical Japanese HLA-DR alleles, suggesting that those T-cell epitopes might be important for the onset of house-dust mite allergy in the Japanese population. We prepared 15 analog peptides of the HLA-DRB1* 1502-restricted 15-mer peptide. Of those 15 residues, five (F35, L37, A39, F41, and E42) were critical for the epitope activity, and three residues (F35, A39, and E42) seemed to be included in anchor motifs for HLA-DRB1* 1502. The epitope peptide was also recognized by HLA-DRB1* 1502-positive healthy donors; however, only allergic T cells showed Th2 functions. Antigen-presenting cells of nonallergic donors were able to activate allergic T cells to express Th2 function. This seemed to suggest that antigen recognition of T cells, as well as additional unknown factors which promote Th2, rather than Th1, responses, might be important for the onset of house-dust mite allergy.     

Identification of potential human respiratory syncytial virus and metapneumovirus T cell epitopes using computational prediction and MHC binding assays

Human respiratory syncytial virus (RSV) and human metapneumovirus (MPV) are two of the most common causes of serious viral lower respiratory tract illness in humans. CD8+ T cells have been shown to be important in animal models and human clinical studies for the clearance of viral infection, and they may contribute in part to protection against severe disease during reinfections. Precise enumeration and accurate phenotyping of RSV- or MPV-specific CD8+ T cells in humans is currently limited by the relatively small number of T cell epitopes that have been mapped with accompanying identification of MHC restriction patterns. We sought to expand the number of potential RSV and MPV epitopes for use in clinical and translational studies by identifying an expanded set of MHC-binding peptides based on RSV and MPV wild-type virus strain protein sequences. We interrogated the full protein sequences of all 9 or 11 proteins of MPV or RSV respectively using four established epitope prediction algorithms for human HLA A*0101, A*0201, or B*0702 binding and attempted to synthesize the top-scoring 150-152 peptides for each of the two viruses. Synthesis resulted in 442 synthesized and soluble peptides of the 452 predicted epitopes for MPV or RSV. We then determined the binding of the synthetic peptides to recombinant human HLA A*0101, A*0201 or B*0702 molecules with the predicted restriction using a commercially available plate-based assay, iTopia. A total of 230 of the 442 peptides tested exhibited binding to the appropriate MHC molecule. The binding results suggested that existing algorithms for prediction of MHC A*0201 binding are particularly robust. The binding results also provided a large benchmarking data collection for comparison of new prediction algorithms.     

HLA, molecular mimicry and multiple sclerosis

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system in which an autoimmune response most probably contributes to pathogenesis. To date, the best characterized susceptibility-associated gene has been mapped to the HLA complex. The HLA-DRB1*1501 - DRB5*0101 - DQA1*0102 - DQB1*0602 haplotype is both associated and linked to MS in different ethnic groups. The locus within the HLA class II region encoding the MS-susceptibility gene is under intensive investigation. Epidemiological studies, however, have suggested that environmental antigens also play a critical role in MS pathogenesis. One of the ways a pathogen could trigger autoimmune disease is via immunological cross-reactivity or molecular mimicry. This concept argues that a microbial peptide with certain degree of homology to a self peptide can stimulate pathogenic self-reactive specific T cells to cause an autoimmune disease. Many microbial agents have regions of sequences that may serve as binding motifs for HLA-DR2. HLA genetics and molecular mimicry may therefore be intimately interlinked in the disease process. In the present review, we focus on the HLA association with MS and the role of microbial antigens in MS, with special reference to the molecular mimicry hypothesis.     

In silico prediction of binding of putative antigenic peptides to HLA-DRB1 alleles in Vogt-Koyanagi-Harada disease

We evaluated peptides demonstrated to be immunogenic in studies of HLA-DRB1*0405-restricted T cells from patients with Vogt-Koyanagi-Harada (VKH) disease for likelihood of binding to HLA-DRB1*0405 using internet-accessible algorithms. Immunogenic peptides were more likely to overlap with fragments predicted to have a high likelihood of binding to HLA-DRB1*0405 and were more likely to bind to HLA-DRB1*0405 than the negative control, HLA-DRB1*0701. We also determined the likelihood of binding to other HLA molecules associated with VKH disease. The peptide immunogenic in the most patients was predicted to bind to HLA-DRB1*0101, 0404, and 0405, but not to the negative control, HLA-DRB1*0701. In silico methods hold promise for high-throughput evaluation of putative auto-antigens, for testing and generating hypotheses, and suggest a correlation between HLA binding and immunogenicity.     

Analysis of memory T lymphocyte activity following stimulation with overlapping HLA-A*2402, A*0101 and Cw*0402 restricted CMV pp65 peptides

The continuous efforts aimed at the identification of new immune epitopes across the MHC system has led to the discovery that more than one peptide may be restricted to the same HLA antigen and function as an immune determinant for that association. The aim of this study was to compare the ability of two overlapping peptides, the nonamer (9-mer) cytomegalovirus (CMV) pp65_341–349 (QYDPVAALF) and the decamer (10-mer) CMV pp65_341–350 (QYDPVAALFF), and the esadecamer (16-mer) peptide containing both the 9-mer and 10-mer sequences, CMV pp65_340–355 (RQYDPVAALFFFDIDL), to stimulate and maintain over time a T cell immune reactivation by HLA-A*2402, A*0101, and Cw*0402 cells from CMV-seropositive subjects. The 9-mer, 10-mer, and 16-mer peptides effectively stimulated CTLs from HLA-A*2402, HLA-A*0101, and HLA-Cw*0402 CMV seropositive donors. This data confirms that both the 9-mer and the 10-mer peptides are promiscuous and are not restricted to a single HLA antigen. CMV pp65_341–349 and CMV pp65_341–350 have the ability to produce CMV-specific CTLs in subjects with several different HLA types, presenting a practical advantage over other peptides that are restricted only to a single HLA antigen, and thus being optimal for CMV adoptive immune therapy. Moreover, since the 16-mer peptide encompasses both the 9-mer and 10-mer peptides, it may be better than either of these peptides for CMV adoptive immune therapy.     

Motif analysis of HLA class II molecules that determine the HPV associated risk of cervical carcinogenesis

In previous studies, the HLA class II haplotype HLA DRB1*0401-DQB1*0301 was shown to correlate with susceptibility to HPV infection, CIN and cervical cancer while DRB1*0101-DQB1*0501 indicated protection. The present study was designed to identify naturally processed peptide sequences bound to the susceptibility and protective HLA DR-DQ molecules, and use this for T-helper epitope prediction from HPV 16. The HLA class II molecules were obtained by immuno-affinity purification of Epstein-Barr virus B lymphoblastoid cell lines (BCL) homozygous for HLA DQA1*0301-DQB1*0301 and HLA DQA1*0101-DQB1*0501. Peptide pools eluted from the HLA molecules were sequenced by Edman degradation. On the basis of the peptide sequence data obtained, the E6, E7, L1 and L2 proteins of HPV 16 were examined to identify sequences which are likely to bind to HLA DQB1*0301 and DQB1*0501. In addition, motif prediction as well as the binding affinity of predicted peptide motifs for HLA DRB1*0401 and DRB1*0101, the DR alleles associated with susceptibility and protection respectively, was accomplished using published data and a prediction algorithm for the naturally processed peptide sequences bound to these molecules. The HLA DQB1*0501 peptide ligand sequence showed that proline gives an outstanding signal at position 2, Asn/Arg at P1, aliphatic/aromatic amino acids in the central portion, a hydrophobic cluster at P5 with a small contribution by small polar residues and another cluster of aromatic residues towards the C-terminus. The HLA DQB1*0301 sequence also showed that proline gives an outstanding signal at position 2, Thr/Arg at P1, aliphatic/aromatic amino acids in the central portion and an aliphatic cluster with a small contribution by small polar residues at P5. There were no differences in the number of HPV peptides that were predicted as being capable of binding to HLA DQB1*0301 and HLA DQB1*0501, but more HPV peptide motifs were predicted to bind with high affinity to HLA DRB1*0101 than DRB1*0401. The results suggest that HPV 16 peptide epitopes bind with higher affinity to the protective than to susceptible HLA DR-DQ molecules which may lead to a more effective immune response.     

Epitopes and motifs of the HLA‐B*14 allele family products and related HLA‐B14 cross‐reactive specificities

The split specificities of HLA‐B14 (B64, B65) are assigned to the B*14:01 (B64) and B*14:02 (B65) products only. Of the further 50 B*14 expressed products, only B*14:03 and B*14:06 are officially designated as HLA‐B14. The B*14:08 product differs from B64 by a single amino acid substitution of W97R, while the B*14:53 specificity (which is a “short” B14 and neither B64 nor B65) differs from B64 by three residues (W97S, Y113H and F116Y). Comprehensive testing of B*14:08:01 cells (using 49 alloantisera with B64 or B64, B65 specificities, and five monoclonal antibodies with B65 or B64, B65 activity) showed that the B*14:08 specificity is, like the B*14:53 product, neither B64 nor B65 and appears as a “short” B14 specificity. To help understand the serological reactivity of the B*14:08 and B*14:53 products, and B64 and B65, we identified seven published epitopes (11AV, 97W, 61ICT, 116F, 131S+163T, 170RH and 420) and, by inspection, 29 motifs, that encompass one or more of B64, B65 and various HLA‐B14 cross‐reactive group specificities. We then considered the possession of these epitopes and motifs by the products of B*14:01 to B*14:06, B*14:08 and B*14:53. Seventeen of the 29 motifs fully complied with the one‐/two‐patch functional epitope concept for amino acid proximity, as determined by Cn3D software, the remainder partially complied. The nature and patterns of epitopes and motifs possessed by both B*14:08 and B*14:53 specificities supported their designation as HLA‐B14 but non‐B64/B65. Also that epitope 97W, with 11S or 11A, is critical for serological B64 and B65 reactivity. And conversely, that epitope 116F, and several identified motifs, are probably unimportant for HLA‐B14 antibody reactivity. The previous submission that the B*14:03 specificity is HLA‐B65 was compatible with its epitope/motif pattern. B*14:04 cells would also be expected to react as B65, based on its epitope/motif pattern, and not as B64 as previously implied. Also, from their epitope/motif patterns, and external serological information, it is probable that the B*14:05 and B*14:06 specificities will both appear as “short” HLA‐B14, non‐B64/B65. Several epitopes and motifs encompassed a range of HLA‐B specificities included in the serological HLA‐B14 cross‐reactive group, thus supporting these original serological findings.     

Responses of coxsackievirus B4-specific T-cell lines to 2C protein-characterization of epitopes with special reference to the GAD65 homology region

Coxsackie B viruses (CBV) have been indicated as environmental triggers initiating autoimmune destruction of insulin-producing pancreatic beta-cells, and molecular mimicry might be the mechanism. A prime candidate for inducing cross-reactive immune responses is a homology sequence, PEVKEK, found both in CBV4 2C protein and in GAD65. To characterize the CBV4-specific T-cell epitopes, overlapping peptides covering the 2C protein were synthesized and CBV4-specific T-cell lines were established from healthy and diabetic subjects. The T-cell epitopes were dependent on the HLA-DR genotype of the T-cell donor, but no difference between diabetic and healthy subjects could be detected. Peptide p4, which included the PEVKEK sequence, contained an HLA-DR1-restricted T-cell epitope. Three randomly selected CBV4-specific T-cell lines, which responded to peptide p4, failed to recognize GAD65 protein or GAD65 peptides containing the PEVKEK sequence. We conclude that the CBV4 2C protein is strongly immunogenic for T-cells and PEVKEK is included in a T-cell epitope. However, presentation of this epitope in the context of neutral HLA-DR1 allele does not support its role in pathogenesis of type 1 diabetes.