Evidence that HLA class II-restricted human CD4+ T cells specific to p53 self peptides respond to p53 proteins of both wild and mutant forms

By stimulating peripheral blood mononuclear cells of four healthy donors with a mixture of overlapping peptides representing the core domain of p53, we established two CD4+ α β T cell clones and four lines that recognized wild-type and mutant p53 proteins as well as p53 self peptides in an HLA class II-restricted fashion. Two T cell lines established from two unrelated donors reacted to the p53 peptide (p)153 – 166 and p108 – 122, respectively, in the context of DP5 molecules. Two T cell clones established from two other unrelated donors were specific for p193 – 204 in the context of DRB1*1401 and for p153 – 165 in the context of DP5, respectively. These two T cell clones responded almost equally to both wild-type and four mutant recombinant p53 proteins. The proliferative responses of these T cell clones to p53 recombinant proteins were augmented by heat denaturing, thereby suggesting that altered conformation of the protein facilitates proteolytic processing to produce antigenic peptides. The DRB1*1401-restricted T cell clone specific for p193 – 204 killed a B lymphoblastoid cell line homozygous for HLA-DRB1*1401 when the cell line was pre-pulsed with p53 protein as well as peptide. These results indicate that CD4+ T cells reactive to p53 do exist in healthy individuals and the epitopes are probably ignored by the immune system under physiological conditions. It is suggested that such epitopes stimulate T cells to induce anti-p53 antibody production in cancer patients as previously reported by others. The possible involvement of p53-reactive T cells in anti-tumor immunity is discussed.     

Interaction among human leucocyte antigen–peptide–T cell receptor complexes in cow''s milk allergy: the significance of human leucocyte antigen and T cell receptor–complementarity determining region 3 loops

BACKGROUND: Allergic individuals respond to only a few specific antigens, therefore allergic diseases are characterized by antigen specificity. Clarification of the mechanism of antigen specificity will lead to progress in the therapy of allergic diseases. OBJECTIVES: The purpose of this study is to determine the specific association among T cell epitopes, antigen-presenting molecules and T cell receptor (TCR), and to determine the TCR usage in the pathogenesis of allergies using antigen-specific T cell clones (TCCs). The results can clarify the mechanism of the antigen specificity of allergic diseases, and provide new therapeutic possibilities using analogue peptides. METHODS: Short-term T cell clones specific to beta-lactoglobulin (BLG) were established from peripheral blood mononuclear cells (PBMCs) collected from five patients allergic to cow's milk. We then identified the T cell epitopes and antigen-presenting molecules, and examined TCR usage. We also determined the sequence of the TCR-complementarity-determining region 3 (CDR3). RESULTS: Six TCCs established from the five patients recognized three different peptides, and BLGp97-117 was recognized by four of the six TCCs. BLGp101-112 (KYLLFCMENSAE) was the core sequence in the fragment. Sequence analysis of TCR by the RT-PCR method revealed a marked heterogeneity in TCR usage, and similar amino acid sequences were recognized in the CDR3 region. Four of the six TCCs recognized BLG in association with human leucocyte antigen (HLA)-DRB1*0405 as antigen-presenting molecules. CONCLUSION: We proposed the motif of the interaction between the HLA-DRB1*0405 allele and antigen peptide, and suggested that HLA-DRB1*0405 is an immunoregulatory gene product for T cell responses to BLG.     

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.     

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.     

Cervical cancer cell lines expressing NKG2D-ligands are able to down-modulate the NKG2D receptor on NKL cells with functional implications

Background

Molecular mimicry between microbial antigens and host-proteins is one of the etiological enigmas for the occurrence of autoimmune diseases. T cells that recognize cross-reactive epitopes may trigger autoimmune reactions. Intriguingly, autoimmune diseases have been reported to be prevalent in tuberculosis endemic populations. Further, association of Mycobacterium tuberculosis (M. tuberculosis) has been implicated in different autoimmune diseases, including rheumatoid arthritis and multiple sclerosis. Although, in silico analyses have identified a number of M. tuberculosis specific vaccine candidates, the analysis on prospective cross-reactive epitopes, that may elicit autoimmune response, has not been yet attempted. Here, we have employed bioinformatics tools to determine T cell epitopes of homologous antigenic regions between M. tuberculosis and human proteomes.

Results

Employing bioinformatics tools, we have identified potentially cross-reactive T cell epitopes restricted to predominant class I and II alleles of human leukocyte antigens (HLA). These are similar to peptides of mycobacterial proteins and considerable numbers of them are promiscuous. Some of the identified antigens corroborated with established autoimmune diseases linked with mycobacterial infection.

Conclusions

The present study reveals many target proteins and their putative T cell epitopes that might have significant application in understanding the molecular basis of possible T cell autoimmune reactions during M. tuberculosis infections.     

Identification of mimicry peptides based on sequential motifs of epitopes derived from 65-kDa glutamic acid decarboxylase

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease with a predominantly non-hereditary etiology that results in a destruction of pancreatic beta cells by autoaggressive T lymphocytes. Neither the mechanism of initial stimulation of these T cells nor the nature of the environmental factors implicated in the disease have so far been identified. However, both issues are taken into account by the hypothesis of initial T cell activation by viral or bacterial mimicry peptides with sequence similarities to pancreatic self antigens. We determined sequential epitope motifs to search for mimicry peptides stimulating T cell lines specific for two epitopes derived from the IDDM autoantigen 65-kDa glutamic acid decarboxylase (GAD65). These were GAD65 (88 – 99), presented by HLA-DRB1*0101, and GAD65 (248 – 257), presented by HLA-DRB5*0101. T cell stimulation by peptides with substitutions in HLA anchor or T cell contact positions was analyzed to establish degenerate epitope motifs for database searching. Out of 28 tested candidate mimicry peptides derived from bacterial, viral and human proteins, 3 stimulated T cell lines and a T cell clone specific for epitope GAD65 (248 – 257). Our results demonstrate that mono- and polyclonal GAD65-specific T cells from IDDM patients can be stimulated by viral and bacterial peptides with little apparent sequence homology with autoantigenic epitopes. Moreover, in a synopsis with related published studies, our findings suggest that simple degenerate search motifs comprising principal T cell contacts plus HLA class II binding motifs may suffice to identify most mimicry peptides.     

Epitope Analysis of Birch Pollen Allergen in Japanese Subjects

Birch pollen is a very common cause of nasal allergy (pollinosis) not only in Scandinavia, Europe, Canada, and the northern part of the United States but also in Hokkaido, Japan. We have previously reported a positive association between the HLA-DR9 phenotype and the development of birch pollen allergy in Japanese subjects. However, there is little information about T cell epitopes of birch pollen which are presented by HLA class II molecules other than HLA-DR9. Therefore, we analyzed the difference in T cell epitope usage in patients who had HLA-DR9 versus those who did not. Seven Japanese patients with birch pollinosis were studied. Some groups of peptides representing T cell epitopes (Betula verrucosa; Bet VI peptides, p7–33, p23–46, p138–160) appeared to be shared by the majority, while another peptide (Bet VI p72–95) was recognized predominantly by patients who expressed HLA-DR9 and/or HLA-DQ3 molecules. Moreover, seven T cell clones and eight T cell lines were generated from two patients who did not have HLA-DR9 or HLA-DQ3. Using some of these T cell clones/lines, we investigated the relationship between HLA class II molecules and antigenic peptides. One of these T cell clones recognized antigenic peptides in the context of the HLA-DQ1 molecule. To our knowledge, this is the first indication that the epitope on Bet VI can be presented by the HLA-DQ molecule.     

Human B cells secrete migration inhibition factor (MIF) and present a naturally processed MIF peptide on HLA-DRB1*0405 by a FXXL motif

A better knowledge of peptide structures interacting with major histocompatibility complex (MHC) molecules is of great interest for better understanding of the molecular basis of immune recognition. We have isolated naturally processed peptides from a continuously growing antigen-presenting Epstein-Barr virus-transformed human B-cell line. HLA-DR complexes were purified by specific affinity chromatography and complexed peptides were released by acid treatment. The isolated peptides were separated by reversed phase chromatography and fractions were analysed by Edman degradation at picomolar ranges. From 30 fractions that were examined seven peptides bound to the HLA-DRB1*0405 and two peptides from the human leucocyte antigen (HLA) class II associated invariant chain bound to HLA-DRB1*1302. In addition, a N-terminal beta-chain peptide of the 0405 allele was identified. Evaluation of amino acid sequences revealed a refined FXXL motif for the 0405 allele, in which F (phenylalanine) stands for any aromatic amino acid and L (leucine) can be exchanged by either I (isoleucine) or V (valine). In total, three fractions contained a peptide derived from the human migration inhibition factor (MIF), a pro-inflammatory cytokine that is normally produced by activated T lymphocytes and monocytes/macrophages. Indeed, cytokine analysis revealed high amounts of MIF secreted by the B-cell line, confirming that MHC class II expressing cells can present any intrinsic peptide that contains the distinct motif for HLA-binding. For MIF, the amino acid sequence Y36IAV39 represents the required binding motif for HLA-DRB1*0405. Nevertheless, it is the first time that cytokine fragments were found to bind to HLA molecules on human B cells.     

Development of an epitope panel for consistent identification of antigen‐specific T‐cells in humans

We aimed to establish a panel of MHC–peptide multimers suitable as a positive control in the detection of HLA A*0201 restricted antigen specific T cells (ASTC) by flow cytometry. MHC Dextramers were loaded with HLA A*0201 binding peptides from viral antigens and melanoma targets identified from a literature search and in silico prediction. Peripheral blood mononuclear cells (PBMC) from healthy donors were analysed with the MHC Dextramers using flow cytometry. The best performing epitopes were tested on PBMC from patients undergoing testing for Mycobacterium tuberculosis infection to assess the coverage of this epitope panel. Of 21 candidate epitopes, ASTC could be detected against 12 (57·1%) in at least one of 18 healthy blood donors. Reactivity to two or more epitopes was seen in 17 of the 18 donors (94·4%). We selected the six best‐performing epitopes and demonstrated a positive response in 42 (97·7%) of 43 patient samples (healthy, latent and active M. tuberculosis infection). The selected panel of six antigenic epitopes sufficed as a positive control in the detection of ASTC in HLA A*0201. Performance was robust in different stages of latent and active M. tuberculosis infection, indicating reliability also during infection.     

The genetic basis of autoantibody production

Many autoimmune diseases are characterized by autoantibody subsets that are associated with specific clinical manifestations. The primary genetic associations of these autoantibodies are with MHC genes, most specifically HLA class II, which in many instances better explain the HLA association of the disease per se. It is noteworthy that certain genes and haplotypes, notably HLA-DRB1*0301, DQA1*0501, DQB1*0201 in Caucasians and DRB1*0405, DQA1*03, DQB1*0401 in Asians, as well as PTPN22, seem to be associated with a variety of autoimmune diseases. On the other hand, others are more disease specific (HLA-DRB1*11 for systemic sclerosis and HLA-DRB1 alleles encoding the "shared epitope" in RA) as well as non MHC genes, such as FcyRIIa and IIIa in SLE, the beta2 glycoprotein I gene in the aPL syndrome, and the TSHR gene in Graves' disease). Autoantibody responses also are influenced by the presence of specific MHC and non-MHC genes which may not be associated with the disease per se. These novel associations offer new clues not only to pathogenesis but also to potential therapeutic targets.     

Extensive major histocompatibility complex class I binding promiscuity for Mycobacterium tuberculosis TB10.4 peptides and immune dominance of human leucocyte antigen (HLA)‐B*0702 and HLA‐B*0801 alleles in TB10.4 CD8+ T‐cell responses

The molecular definition of major histocompatibility complex (MHC) class I‐presented CD8⁺ T‐cell epitopes from clinically relevant Mycobacterium tuberculosis (Mtb) target proteins will aid in the rational design of T‐cell‐based diagnostics of tuberculosis (TB) and the measurement of TB vaccine‐take. We used an epitope discovery system, based on recombinant MHC class I molecules that cover the most frequent Caucasian alleles [human leucocyte antigen (HLA)‐A*0101, A*0201, A*0301, A*1101, A*2402, B*0702, B*0801 and B*1501], to identify MHC class I‐binding peptides from overlapping 9‐mer peptides representing the Mtb protein TB10.4. A total of 33 MHC class I‐binding epitopes were identified, spread across the entire amino acid sequence, with some clustering at the N‐ and C‐termini of the protein. Binding of individual peptides or closely related peptide species to different MHC class I alleles was frequently observed. For instance, the common motif of xIMYNYPAMx bound to six of eight alleles. Affinity (50% effective dose) and off‐rate (half life) analysis of candidate Mtb peptides will help to define the conditions for CD8⁺ T‐cell interaction with their nominal MHC class I‐peptide ligands. Subsequent construction of tetramers allowed us to confirm the recognition of some of the epitopes by CD8⁺ T cells from patients with active pulmonary TB. HLA‐B alleles served as the dominant MHC class I restricting molecules for anti‐Mtb TB10.4‐specific CD8⁺ T‐cell responses measured in CD8⁺ T cells from patients with pulmonary TB.     

Prediction of HLA class I-restricted T-cell epitopes of islet autoantigen combined with binding and dissociation assays

Identification of cognate peptides recognized by human leucocyte antigen (HLA)/T cell receptor (TCR) complex provides insight into the pathogenic process of type 1 diabetes (T1D). We hypothesize that HLA-binding assays alone are inadequate metrics for the affinity of peptides. Zinc transporter-8 (ZnT8) has emerged in recent years as a novel, major, human autoantigen. Therefore, we aim to identify the HLA-A2-restricted ZnT8 epitopes using both binding and dissociation assays. HLA class I peptide affinity algorithms were used to predict candidate ZnT8 peptides that bind to HLA-A2. We analyzed 15 reported epitopes of seven β-cell candidate autoantigens and eight predicted candidate ZnT8 peptides using binding and dissociation assays. Using IFN-γ ELISpot assay, we tested peripheral blood mononuclear cells (PBMCs) from recent-onset T1D patients and healthy controls for reactivity to seven reported epitopes and eight candidate ZnT8 peptides directly ex vivo. We found five of seven recently reported epitopes in Chinese T1D patients. Of the eight predicted ZnT8 peptides, ZnT8_153–161 had a strong binding affinity and the lowest dissociation rate to HLA-A*0201. We identified it as a novel HLA-A*0201-restricted T-cell epitope in three of eight T1D patients. We conclude that ZnT8_153–161 is a novel HLA-A*0201-restricted T-cell epitope. We did not observe a significant correlation (P = 0.3, R = ? 0.5) between cytotoxic T cell (CTL) response and peptide/HLA*0201 complex stability. However, selection of peptides based on affinity and their dissociation rate may be helpful for the identification of candidate CTL epitopes. Thus, we can minimize the number of experiments for the identification of T-cell epitopes from interesting antigens.     

Strong association of HLA class II sequences in Mexicans with Vogt-Koyanagi-Harada's disease.

Vogt-Koyanagi-Harada's syndrome (VKH) is an autoimmune disease prevalent in Mongoloids with evident participation of HLA. The aim of this study was to identify the class II DNA sequences involved in the etiopathogenesis of VKH in Mexican Mestizos. This study included 46 VKH patients and 170 controls. 75% were females (mean age at onset of 33.5 years). The disease evolved to chronicity (68%) and 25% of the patients were unresponsive to corticotherapy. DNA typing of HLA-DRB1, DQA1 and DQB1 was done following the 12th International Histocompatibility protocols. VKH was strongly dependent of DRB1 gene; DRB1*04 was found in 78.2% of the patients vs. 50.6% of the controls (p = 0.001). No particular DRB*04 subtype was significantly increased, suggesting that residues E-9 V-11; H-13; H-33 and Y-37 shared by all DR4s are implicated in susceptibility to VKH. However DRB1*0101 (p = 0.009, OR = 4.2) was clearly associated. This allele shares the motif LLEQRRAAG located at position 67-74 and 86 of DRB1 with *0405 associated in Japanese. Two HLA associated mechanisms may be triggering the autoimmune phenomena. One involving critical polymorphic residues expressed in different alleles. Secondly, some peptides may anchor to the conserved residues leaving other sequences to bind to the T cell receptor.     

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 epitopes of type II collagen in HLA-DRB1*0101 or DRB1*0405-positive Japanese patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is a T cell-mediated autoimmune disease, but target antigens (autoantigens) responsible for T cell activation remain unclear. Type II collagen (CII) is a candidate autoantigen that is largely confined to the articular cartilage. To investigate whether CII is an important antigen in patients with RA, we examined peripheral blood T cell reactivity to CII in HLA-DRB1*0101 and DRB1*0405-positive RA patients. Reactivities to candidate T cell epitopes of CII were also examined. Peripheral blood T cell reactivity to CII and CII peptides (256-271, 429-442, 593-610, 1064-1081) were detected by measurement of IL-2, IFN-gamma, and IL-4 in culture supernatant of PBMC after in vitro antigen stimulation. Cytokine concentration was measured by ELISA. In DRB1*0101-positive patients, T cell reactivity to CII as detected by measurement of IL-2 production in culture supernatant, was present in 4 out of 9 patients. IL-2 production upon stimulation with CII 256-271 peptide was found in all of these 4 patients. In DRB1*0405-positive patients, high frequency of positive T cell response to CII was detected in 9 out of 11 patients. IFN-gamma production was also detected in 4 out of 6 patients producing IL-2 by stimulation with CII. T cell response to CII 256-271 and/or CII 1064-1081 was detected in these patients. In DRB1*0101-positive RA patients, CII 256-271 peptide might function as a T cell epitope, whereas either CII 256-271 or CII 1064-1081 peptide may be a major T cell epitope in DRB1*0405-positive RA patients. In DRB1*0405-positive RA patients, CII reactive T cells might play a crucial role in the development of RA through IFN-gamma production.     

Invariant chain as a vehicle to load antigenic peptides on human MHC class I for cytotoxic T‐cell activation

Protective T‐cell responses depend on efficient presentation of antigen (Ag) in the context of major histocompatibility complex class I (MHCI) and class II (MHCII) molecules. Invariant chain (Ii) serves as a chaperone for MHCII molecules and mediates trafficking to the endosomal pathway. The genetic exchange of the class II‐associated Ii peptide (CLIP) with antigenic peptides has proven efficient for loading of MHCII and activation of specific CD4⁺ T cells. Here, we investigated if Ii could similarly activate human CD8⁺ T cells when used as a vehicle for cytotoxic T‐cell (CTL) epitopes. The results show that wild type Ii, and Ii in which CLIP was replaced by known CTL epitopes from the cancer targets MART‐1 or CD20, coprecipitated with HLA‐A*02:01 and mediated colocalization in the endosomal pathway. Furthermore, HLA‐A*02:01‐positive cells expressing CLIP‐replaced Ii efficiently activated Ag‐specific CD8⁺ T cells in a TAP‐ and proteasome‐independent manner. Finally, dendritic cells transfected with mRNA encoding IiMART‐1 or IiCD20 primed naïve CD8⁺ T cells. The results show that Ii carrying antigenic peptides in the CLIP region can promote efficient presentation of the epitopes to CTLs independently of the classical MHCI peptide loading machinery, facilitating novel vaccination strategies against cancer.     

Identification of HLA-DRB1-bound self-peptides following measles virus infection

We developed a B-lymphocyte cell line derived from a measles seropositive individual who was homozygous for the HLA-DRB1*0301 allele. Peptides associated with the HLA-DRB1*0301 protein were purified from this lymphoblastoid cell line after infection with the Attenuvax measles vaccine virus (Merck Research Laboratories, West Point, PA) and with "sham" infection. More than 40 peptide sequences were obtained by nano-scale reversed phase-high performance liquid chromatography coupled to tandem mass spectrometry (nano-LC/MS/MS). These peptides originated from 21 different source proteins--the majority from membrane-bound proteins. Most of the peptides (>73%) bound to HLA-DRB1*0301 appeared to be in lower abundance on measles-infected cells compared to the "sham-infected" cells. However, 26% of the identified peptides seem to have increased expression after measles infection. Measles vaccine virus infection did not change the level of HLA-DR expression. We demonstrate the power of nano-LC/MS/MS in the rapid determination of changes in the spectrum and expression of HLA-DRB1*0301-bound peptides after infection with measles virus. This provides further knowledge of the changes in peptide expression after viral infection and provides a powerful tool for identifying HLA-presented host and viral epitopes in the context of class II HLA molecules.     

Peptide-Based Molecular Analyses of HLA class II-Associated Susceptibility to Autoimmune Diseases

Recent advances in knowledge of crystal structures of MHC class II molecules has advanced understanding of the molecular basis for interactions between peptides and HLA class II molecules. Polymorphism of HLA class II molecules influences structures of peptides bound to HLA class II molecules. To better understand mechanisms related to particular HLA class II alleles and autoimmune diseases, it is important to identify self-peptides presented by disease-susceptible HLA class II molecules and triggering disease-causative autoreactive T cells. Autoimmune diseases occur in Caucasians, Blacks and Asians, albeit with a different incidence. In some autoimmune diseases, disease-susceptible HLA class II alleles are closely related but different, and clinical manifestations of diseases differ among ethnic groups. These phenomena strongly suggest that difference in autoimmune self-peptide(s) in the context of disease-susceptible HLA class II molecules may explain the different clinical manifestations of diseases. Therefore, a comparison among disease-susceptible HLA class II alleles, autoimmune self-peptides and clinical manifestations of autoimmune diseases in different ethnic groups would be instructive. We directed efforts to determining: (1) HLA-class II alleles specific to Asian populations and which are associated with susceptibility to autoimmune diseases, (2) binding-peptide motifs for these HLA class II molecules, and (3) self-peptides presented by susceptible HLA class II molecules to stimulate autoreactive T cells related to the development of autoimmune diseases in Asians. In this review, our related recent investigations are described and the uniqueness of HLA class II-associated autoimmune diseases in Asians is given emphasis.     

Identification of ricin A-chain HLA class II-restricted epitopes by human T-cell clones

The identification of ricin toxin A-chain (RTA) epitopes and the molecular context in which they are recognized will allow strategies to be devised that prevent/suppress an anti-RTA immune response in patients treated with RTA-based immunotoxins. RTA-specific human T-cell lines and T-cell clones were produced by in vitro priming of PBMC. The T-cell clones used a limited set of Vbeta chains (Vbeta1, Vbeta2 and Vbeta8) to recognize RTA epitopes. The use of RTA deletion mutants demonstrated that T-cell lines and T-cell clones from three out of four donors responded to RTA epitopes within the domain D124-Q223, whereas one donor recognized the region I1-D124. The response to RTA peptides of T-cell lines and T-cell clones from two donors allowed the identification of immunogenic segments (D124-G140 and L161-T190) recognized in the context of different HLA-DRB1 alleles (HLA-DRB1*0801, and HLA-DRB1*11011 and B1*03011, respectively). The response to L161-T190 was investigated in greater detail. We found that the HLA-DRB1*03011 allele presents a minimal epitope represented by the sequence I175-Y183 of RTA, whereas the HLA-DRB1*11011 allele presents the minimal epitope M174-I184. RTA peptides and an I175A RTA point mutant allowed us to identify I175 as a crucial residue for the epitope(s) recognized by the two HLA-DRB1 alleles. Failure of T-cell clones to recognize ribosome inactivating proteins (RIPs) showing sequences similar but not identical to RTA further confirmed the role of I175 as a key residue for the epitope recognized in the context of HLA-DRB1*11011/03011 alleles.     

DQCAR 113 and DQCAR 115 in combination with HLA-DRB1 alleles are significant markers of susceptibility to rheumatoid arthritis in the Korean population

We have investigated HLA region microsatellite polymorphisms in rheumatoid arthritis (RA) which are known to be associated with HLA class II alleles in the Korean population. Ninety patients with RA and 106 controls were employed for this study, in which TAP1CA, DQCAR, D6S273, HLA-DRB1, -DQA1 and -DQB1 allele typing were performed. DQCAR 113 (RR = 3.2, P<0.0002), DQCAR 115 (RR = 3.6, P<0.0001) and heterozygous DQCAR 113/115 (RR = 11.2, P<0.0001) frequencies were significantly increased in the RA group compared with the control group. The HLA-DRB1 genotypes of patients who had DQCAR 113/115 alleles were defined as DRB1*04 and/or DRB1*09. There was no significant difference between RA and controls in D6S273 and TAP1CA allele frequencies. We demonstrated that HLA-DRB1*0405 (RR = 6.6, P<10(-6)), DQA1*03 (RR = 5.2, P<10(-6)), DQB1*04 (RR = 3.5, P<0.002) alleles were useful markers of susceptibility to RA in Koreans. The frequency of HLA-DRB1*0405 was higher in DQCAR 113 allele-positive RA (68.1%) than in DQCAR 113 allele-negative (16.3%) and total RA (43.3%) groups, and the susceptibility risk of DQCAR 113 allele to RA was more increased in the DRB1*0405-positive group (RR = 5.5, P<0.04). On the other hand, DQCAR 115 allele was more significantly associated with susceptibility to RA in HLA-DRB1*0405-negative patients (RR = 5.1, P<0.0005), and the association between RA and HLA-DRB1*0405 was also significantly associated with DQCAR 115 allele-negative patients (RR = 13.2, P<0.00001) as compared with DQCAR 115 allele-negative control groups. HLA-DRB1*0405-DQA1*03-DQCAR113-DQB1*03 haplotype showed high relative risk value (RR= 17.7, P<0.0002). In conclusion, the DQCAR allele in combination with HLA class II, especially DR, is probably a useful risk marker for RA susceptibility in the Korean population.