HLA–B27–restricted CD8+ T cell response to cartilage‐derived self peptides in ankylosing spondylitis

Objective

Several hypotheses have been proposed to explain the strong association between HLA–B27 and ankylosing spondylitis (AS). Among these, the arthritogenic peptide theory proposes that certain B27 subtype alleles bind specific arthritogenic peptide(s) due to their unique amino acid anchor residues. Cartilage antigens have been discussed as candidate targets for the immune response in AS. The recognition of HLA–B27–peptide complexes by self‐reactive CD8+ T cells might contribute to joint‐specific tissue damage. Therefore, we investigated the presence of autoreactive CD8+ T cells specific for cartilage‐derived peptides in patients with AS.

Methods

An HLA–B27–binding prediction program and a proteasome‐cutting prediction program for the human 20S proteasome were used to screen 18 human cartilage proteins for potentially immunogenic nonamer peptides. The peptides identified were used to stimulate peripheral blood mononuclear cells from 20 HLA–B27–positive patients with AS and synovial fluid (SF) mononuclear cells from 7 HLA–B27–positive patients with AS. Activation of T cells was measured by antigen‐specific intracellular cytokine staining and quantified by flow cytometry.

Results

From the screening analysis, we identified 121 nonamer peptides. Of these, 1 peptide derived from type II collagen and 1 from type VI collagen were stimulatory for peripheral blood CD8+ T cells in only 1 of 20 patients. However, in 4 of 7 SF samples the same type VI collagen–derived nonamer peptide stimulated SF CD8+ T cells, but none of the other peptides was stimulatory. This CD8+ T cell response could be blocked by an anti–HLA–B27 antibody, confirming an HLA–B27–restricted immune response.

Conclusion

Our findings suggest that cartilage‐directed cellular autoimmunity might play an important role in joint‐specific tissue damage in patients with AS. Future research is necessary to determine whether the identified peptide is of pathogenetic relevance.

     

Antimicrobial activity of MHC class I-restricted CD8+ T cells in human tuberculosis

Studies of mouse models of tuberculosis (TB) infection have indicated a central role for MHC class I-restricted CD8+ T cells in protective immunity. To define antigens and epitopes of Mycobacterium tuberculosis (MTB) proteins that are presented by infected cells to CD8+ T cells, we screened 40 MTB proteins for HLA class I A*0201-binding motifs. Peptides that bound with high affinity to purified HLA molecules were subsequently analyzed for recognition by CD8+ cytotoxic T lymphocytes. We identified three epitopes recognized by CD8+ T cells from patients recovering from TB infection. Those three epitopes were derived from three different antigens: thymidylate synthase (ThyA(30-38)), RNA polymerase beta-subunit (RpoB(127-135)), and a putative phosphate transport system permease protein A-1 (PstA1(75-83)). In addition, CD8+ T cell lines specific for three peptides (ThyA(30-38), PstA1(75-83), and 85B(15-23)) were generated from peripheral blood mononuclear cells of normal HLA-A*0201 donors. These CD8+ T cell lines specifically recognized MTB-infected macrophages, as demonstrated by production of IFN-gamma and lysis of the infected target cells. Finally, CD8+ cytotoxic T lymphocytes reduced the viability of the intracellular MTB, providing evidence that CD8+ T cell recognition of MHC class I-restricted epitopes of these MTB antigens can contribute to effective immunity against the pathogen.     

HLA B27 in health and disease: a double-edged sword?

The strong association of the HLA class 1 allele HLA B27 with ankylosing spondylitis (AS) has been recognized for over 25 yr, however the pathogenic mechanism linking HLA B27 with AS and other spondyloarthropathies remains a mystery. We now know that the principal natural function of HLA B27 is an immunologic one, namely to bind antigenic peptides and then present them to T lymphocytes. I have shown that HLA B27 functions as an excellent antigen-presenting molecule in both spondyloarthropathy patients and healthy individuals. A working molecular model of how T cells recognize HLA B27 has been generated and tested. Evidence that T cells have a role in spondyloarthritis has also been found. First, expanded populations of T lymphocytes were found in both the blood and synovial fluid of patients with reactive arthritis (ReA). Secondly, a strong cytotoxic T-cell response to an HLA B27-restricted peptide epitope from Chlamydia trachomatis was found in a patient with ReA. This peptide, derived from a bacterium known to trigger ReA, is thus a candidate 'arthritogenic' peptide. We have also found evidence that HLA B27 has an unusual cell biology compared with other HLA molecules. HLA B27 demonstrates an unusual ability to form heavy chain homodimers in vitro. Dimerization is dependent upon disulphide bonding through an unpaired cysteine at position 67. Remarkably these dimers lack beta2 microglobulin, previously thought to be an essential component of all mature MHC class 1 molecules. HLA B27 homodimer formation has also been demonstrated in certain cell lines in vivo, and preliminary data suggest that significant numbers of T cells from patients with spondyloarthropathy express a ligand for HLA B27 homodimers. These findings have extended our understanding of the beneficial immunologic function of HLA B27, and have also led us to propose the testable new hypothesis that HLA B27 heavy chain dimerization may be involved in the pathogenesis of spondyloarthritis.     

Autoreactive CD8+ cytotoxic T lymphocytes to major histocompatibility complex class I chain–related gene A in patients with Behçet's disease

Objective

To detect and characterize the autoreactive CD8+ T cells to major histocompatibility complex class I chain–related gene A (MICA), a stress‐inducible antigen preferentially expressed on the epithelium and endothelium, in patients with Behçet's disease (BD).

Methods

A candidate for the antigenic MICA peptide was selected based on its predicted binding affinity for HLA–B51 and proteasomal cleavage sites. Peripheral blood T cells from 14 patients with BD and 15 healthy controls were repeatedly stimulated with the MICA peptide, and the specific T cell response was measured by peptide‐induced interferon‐γ. Cytotoxic T lymphocyte activity was examined by chromium‐51 release from an HLA–B51–transfected B cell line in the presence of the MICA peptide.

Results

A 9‐mer peptide AAAAAIFVI (termed MICA transmembrane [MICA‐TM]) was selected as a candidate for the antigenic peptide presented by HLA–B51. A specific T cell response to MICA‐TM was detected in 4 patients with BD (29%) but in none of the 15 healthy donors. All 4 responders had HLA–B51 and active disease, and the specific T cell response was lost after the BD‐related symptoms disappeared. The MICA‐induced T cell response was specifically inhibited by anti–HLA class I antibody or by CD8+ cell depletion. MICA‐reactive T cells recognized an HLA–B51–transfected B cell line pulsed with MICA‐TM or a B cell line transfected with both HLA–B51 and MICA in the absence of exogenous peptides. Finally, MICA‐stimulated T cell lines lysed the HLA–B51–expressing B cell line in the presence of MICA‐TM.

Conclusion

HLA–B51–restricted cytotoxic T lymphocytes autoreactive to MICA may be involved in the pathogenesis of BD.

     

HLA-A*26, HLA-B*4002, HLA-B*4006, and HLA-B*4801 alleles predispose to adult T cell leukemia: the limited recognition of HTLV type 1 tax peptide anchor motifs and epitopes to generate anti-HTLV type 1 tax CD8(+) cytotoxic T lymphocytes

Genetic risk for adult T cell leukemia (ATL) has been implicated by ethnic and familial segregation of ATL patients from HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). To clarify the genetic risk for ATL, we characterized HLA class I alleles of ATL patients and analyzed the anchor motifs of HTLV-1 peptides binding to HLA class I molecules, using 291 lines of anti-HTLV-1 CD8(+) cytotoxic T lymphocytes (CTLs) generated in vitro with a total of 165 synthetic peptides for HTLV-1 Tax and Env proteins. Allele frequencies of HLA-A*26, B*4002, B*4006, and B*4801 were significantly higher in ATL patients than in HAM/TSP patients and asymptomatic HTLV-1 carriers in southern Japan. CD8(+) CTL analysis revealed the HTLV-1 Tax peptide sequence to completely lack anchor motifs of peptides binding to HLA-A*26,B*4002, and B*4006 molecules but to possess one anchor for HLA-B*4801, while the HTLV-1 Env peptide sequence had many anchor motifs for HLA-A*26, B*4002, B*4006, and B*4801 molecules. Most ATL patients featured heterozygous HLA class I alleles composed of HLA-A*26, B*4002, B*4006, and B*4801, with a lower number of HTLV-1 Tax peptide anchor motifs and epitopes generating anti-HTLV-1 Tax CD8(+) CTLs than individuals possessing other HLA alleles. The relationship between Tax epitope and ATL incidence was verified by the significantly decreased number of HTLV-1 Tax epitopes in ATL patients compared with asymptomatic HTLV-1 carriers (p < 0.01) as well as late onset ATL patients (p < 0.001). These results indicate that HLA-A*26, B*4002, B*4006, and B*4801 alleles predispose to ATL because of the limited recognition of HTLV-1 Tax peptide anchor motifs and epitopes capable of generating anti-HTLV-1 Tax CD8(+) CTLs.     

T cell reactivity to DR*0401- and DQ*0302-binding peptides of the putative autoantigen IA-2 in type 1 diabetes.

Type 1 diabetes is thought to be an autoimmune disease mediated by T lymphocytes recognizing critical islet cell antigens. Recently, the tyrosine phosphatase like protein IA-2 was suggested as a putative autoantigen in type 1 diabetes since autoantibodies are detected in sera of diabetic patients and prediabetic subjects. Similarly, T cell responses of peripheral blood lymphocytes of type 1 diabetic patients to this protein have been described. Only very few data is available about immunodominant epitopes of IA-2 recognized by T cells. We have studied T cell responses in type 1 diabetic patients and age and partly HLA matched controls to IA-2 peptides designed to bind HLA risk alleles of IDDM as DR*0401 and DQ*0302. Both diabetic patients and controls responded to IA-2ic and some of the peptides. Three peptides of the C-terminal region of IA-2 were recognised by T cells of a fraction of diabetic patients but at least two of these peptides triggered also T cell responses in DR*0401/DQ*0302-matched controls. Most peptides bound to different HLA alleles ("promiscous binders"). The identification of autoantigenic epitopes may offer clues to related sequences e.g. of viral origin what relates to models of diabetes pathogenesis ("molecular mimicry"). Secondly, the design of antigen- or even epitope-specific immune intervention strategies aiming at tolerization of disease specific T cells in type 1 diabetes may profit from the knowledge of immunodominant T cell epitopes of a putative autoantigen.     

T-cell epitopes within the complementarity-determining and framework regions of the tumor-derived immunoglobulin heavy chain in multiple myeloma

The idiotypic structure of the monoclonal immunoglobulin (Ig) in multiple myeloma (MM) might be regarded as a tumor-specific antigen. The present study was designed to identify T-cell epitopes of the variable region of the Ig heavy chain (VH) in MM (n = 5) using bioinformatics and analyze the presence of naturally occurring T cells against idiotype-derived peptides. A large number of human-leukocyte-antigen (HLA)-binding (class I and II) peptides were identified. The frequency of predicted epitopes depended on the database used: 245 in bioinformatics and molecular analysis section (BIMAS) and 601 in SYFPEITHI. Most of the peptides displayed a binding half-life or score in the low or intermediate affinity range. The majority of the predicted peptides were complementarity-determining region (CDR)-rather than framework region (FR)-derived (52%-60% vs 40%-48%, respectively). Most of the predicted peptides were confined to the CDR2-FR3-CDR3 "geographic" region of the Ig-VH region (70%), and significantly fewer peptides were found within the flanking (FR1-CDR1-FR2 and FR4) regions (P <.01). There were 8- to 10-amino acid (aa) long peptides corresponding to the CDRs and fitting to the actual HLA-A/B haplotypes that spontaneously recognized, albeit with a low magnitude, type I T cells (interferon gamma), indicating an ongoing major histocompatibility complex (MHC) class I-restricted T-cell response. Most of those peptides had a low binding half-life (BIMAS) and a low/intermediate score (SYFPEITHI). Furthermore, 15- to 20-aa long CDR1-3-derived peptides also spontaneously recognized type I T cells, indicating the presence of MHC class II-restricted T cells as well. This study demonstrates that a large number of HLA-binding idiotypic peptides can be identified in patients with MM. Such peptides may spontaneously induce a type I MHC class I- as well as class II-restricted memory T-cell response.     

Physical association between the CD8 and HLA class I molecules on the surface of activated human T lymphocytes

Immune recognition by cytotoxic effector T cells requires participation of the CD8 and major histocompatibility complex class I antigens. We found that the CD8 molecule is noncovalently associated with the HLA class I heavy chain on the surface of human T cells activated by Con A. Accordingly, anti-CD8 monoclonal antibodies precipitated a heterodimer containing polypeptides of 32 and 43 kDa from the lysates of activated T cells. The 43-kDa chain of this heterodimer can be adsorbed from cell lysates with anti-HLA-A, -B, and -C antibodies. Endoglycosidase F treatment and chymotryptic peptide mapping identified a structural similarity between this 43-kDa molecule and the HLA class I heavy chain precipitated by the anti-HLA-A, -B, and -C antibody W6/32. Analysis of anti-CD8 precipitates under nonreducing and reducing conditions indicated a lack of interchain disulfide bonding between the CD8 and HLA heavy chain molecules. The CD8-HLA heavy chain complex was also detected in mixed lymphocyte cultures and a cloned cytotoxic T-lymphocyte line but not in purified natural killer cells. The present study indicates that CD8 is complexed with HLA heavy chain on the same cells, and the complex may have functional relevance in the T-cell recognition process.     

Cytotoxic T lymphocyte clone specific for autologous human hepatocellular carcinoma cell line SUHC-1

We established a cytotoxic T lymphocyte (CTL) clone directed against an autologous hepatocellular carcinoma (HCC) cell line SUHC-1 which had been established in our department from a patient with HCC associated with hepatitis C virus infection. The CTL clone lysed autologous SUHC-1 cells but did not lyse autologous Epstein-Barr (EB) virus-transformed B cells, natural killer (NK) cell-sensitive erythroleukaemia cell line K562, the NK-resistant B cell line Daudi, or allogeneic HCC cell lines, Hep-G2, Hep-3B, Mahlavu and PLC/PRF/5. The CTL clone expressed CD3 and CD8 molecules. The cytotoxic activity of the clone was inhibited by anti-CD3, anti-CD8 and anti-histocompatibility antigen (HLA) class I monoclonal antibodies. These results indicated that the CTL clone recognized HCC tumour antigen in an HLA class I-restricted manner. Furthermore, we investigated the T cell receptor (TCR) gene usage of the CTL clone. The CTL clone expressed TCRαβ. We searched for expression of TCR variable (V) α and β regions and sequenced complementary determining region (CDR) 3 of the clone. The clone expressed Vα14, junctional (J) region α9.7 and Vβ7, Jβ2.1. The amino acid sequence of the N region of the α chain was S-P-G-G-G-G-A-D-G-L-T and of the N-D-N region of the β chain was S-W-T-G-A-S-T-D-T-Q-Y. These results suggested that HLA class I-restricted CTL play an important role in the elimination of human HCC cells.     

Identification of citrullinated vimentin peptides as T cell epitopes in HLA–DR4–positive patients with rheumatoid arthritis

Objective

Antibodies directed against citrullinated proteins (ACPAs) are highly specific for rheumatoid arthritis (RA). The production of ACPAs is most likely dependent on the presence of T cells, since ACPAs undergo isotype switching and are associated with the shared epitope (SE)–containing HLA–DRB1 alleles. Vimentin is a likely candidate protein for T cell recognition, since >90% of patients positive for ACPAs that are reactive with (peptides derived from) citrullinated vimentin carry SE‐containing HLA–DRB1 alleles. The aim of this study was to identify citrullinated vimentin peptides that are presented to HLA–DRB1*0401–restricted T cells.

Methods

HLA–DR4–transgenic mice were immunized with all possible citrulline‐containing peptides derived from vimentin, and T cell reactivity was analyzed. Peptides recognized in a citrulline‐specific manner by T cells were selected and analyzed for their ability to be processed from the entire vimentin protein. A first inventory of the selected epitopes recognized by T cells was performed using peripheral blood mononuclear cells (PBMCs) from ACPA+, HLA–DR4+ patients with RA.

Results

A citrulline‐specific response was observed for 2 of the peptides analyzed in DR4‐transgenic mice. These peptides were found to be naturally processed from the vimentin protein, since citrullinated vimentin was recognized by peptide‐specific T cells. T cell reactivity against these peptides was also observed in cultures of PBMCs from RA patients.

Conclusion

This study identifies, for the first time, 2 naturally processed peptides from vimentin that are recognized by HLA–DRB1*0401–restricted T cells in a citrulline‐specific manner. These peptides can be recognized by T cells in ACPA+, HLA–DR4+ patients with RA, as shown in a first inventory.

     

Influence of HLA–DR genes on the production of rheumatoid arthritis–specific autoantibodies to citrullinated fibrinogen

Objective

Antibodies directed against citrullinated fibrinogen are highly specific for rheumatoid arthritis (RA). This study was undertaken to test whether RA‐associated HLA–DR alleles are associated with anti–citrullinated fibrinogen in RA patient sera and whether replacement of arginyl by citrullyl residues on fibrinogen peptides modifies their binding to HLA–DR molecules and their recognition by T cells.

Methods

Antikeratin, antifilaggrin, and anti–citrullinated fibrinogen antibodies were assayed in RA patients who had undergone HLA–DR typing. Direct assays were performed to investigate binding of citrullinated or native fibrinogen peptides (encompassing the entire α‐ and β‐chains of fibrinogen) to purified HLA–DR molecules. T cell proliferative responses to citrullinated or native fibrinogen peptides were measured in RA patients and controls.

Results

HLA–DRB1*0404 was associated with anti–citrullinated fibrinogen in RA sera (P = 0.002). For the RA‐associated alleles HLA–DRB1*0401 and HLA–DR1, there was a nonsignificant trend toward association (P = 0.07). Multiple peptides from the α‐ and β‐chains of fibrinogen bound many HLA–DR alleles; DRB1*0404 was the best fibrinogen peptide binder. Citrullination did not influence fibrinogen peptide binding to HLA–DR or fibrinogen peptide recognition by T cells. Peripheral blood T cells that recognized native or citrullinated fibrinogen peptides were common in RA patients but not in healthy controls.

Conclusion

The RA‐associated HLA–DRB1*0404 allele is also associated with production of antibodies to citrullinated fibrinogen. DRB1*0401 and DRB1*01 tend to be associated with anti–citrullinated fibrinogen, but this is not statistically significant. Citrullination of fibrinogen peptide does not influence peptide–DR–T cell interaction. Finally, T cell proliferation in response to citrullinated or uncitrullinated fibrinogen peptides is frequent in RA patients and very infrequent in controls.

     

Promiscuous binding of proinsulin peptides to Type 1 diabetes-permissive and -protective HLA class II molecules

AIMS/HYPOTHESIS: Presentation of peptide epitopes derived from beta-cell autoantigens, such as insulin and its precursor molecules, by MHC class II molecules to autoreactive T-cells is believed to play a role in the development of Type 1 diabetes. However, little is known about the interaction between peptides of (prepro)insulin and MHC class II molecules permissive and protective for Type 1 diabetes. In this study therefore, peptides spanning the human preproinsulin sequence were assessed for their binding characteristics to Type 1 diabetes-protective and -permissive HLA molecules. METHODS: HLA-DR2, -DQ6.2 (Type 1 diabetes-protective) and HLA-DR4, -DQ8 (Type 1 diabetes permissive) molecule binding affinity for overlapping synthetic 20mer peptides spanning human preproinsulin was measured in a direct competition binding assay against a biotinylated indicator peptide. RESULTS: All HLA molecules tested showed similarity in their binding characteristics across the preproinsulin molecule, with regions of the insulin A-chain showing the highest affinity and C-peptide regions the lowest affinity for all HLA molecules tested. Furthermore, an insulin peptide implicated as a major CD4+ T-cell target in disease pathogenesis (B9-23) had high affinity binding to both protective and permissive HLA molecules but did not represent the highest affinity region of (prepro)insulin identified in either case. CONCLUSION/INTERPRETATION: The results suggest that peptide binding affinity alone is unlikely to be the major determinant of disease susceptibility in relation to interactions between (prepro)insulin epitopes and HLA molecules. The identification of epitopes derived from beta-cell autoantigens that bind promiscuously to diabetes-permissive HLA molecules could be important in the design of peptide-based immunotherapeutic strategies for the prevention of Type 1 diabetes.     

Autoreactive T cells specific for insulin B:11-23 recognize a low-affinity peptide register in human subjects with autoimmune diabetes

Previous studies in type 1 diabetes (T1D) in the nonobese diabetic mouse demonstrated that a crucial insulin epitope (B:9-23) is presented to diabetogenic CD4 T cells by IA^g7 in a weakly bound register. The importance of antigenic peptides with low-affinity HLA binding in human autoimmune disease remains less clear. The objective of this study was to investigate T-cell responses to a low-affinity self-epitope in subjects with T1D. HLA-DQ8 tetramers loaded with a modified insulin peptide designed to improve binding the low-affinity register were used to visualize T-cell responses following in vitro stimulation. Positive responses were only detectable in T1D patients. Because the immunogenic register of B:9-23 presented by DQ8 has not been conclusively demonstrated, T-cell assays using substituted peptides and DQ8 constructs engineered to express and present B:9-23 in fixed binding registers were used to determine the immunogenic register of this peptide. Tetramer-positive T-cell clones isolated from T1D subjects that responded to stimulation by B:11-23 peptide and denatured insulin protein were conclusively shown to recognize B:11-23 bound to HLA-DQ8 in the low-affinity register 3. These T cells also responded to homologous peptides derived from microbial antigens, suggesting that their initial priming could occur via molecular mimicry. These results are in accord with prior observations from the nonobese diabetic mouse model, suggesting a mechanism shared by mouse and man through which T cells that recognize a weakly bound peptide can circumvent tolerance mechanisms and play a role in the initiation of autoimmune diseases, such as T1D.Type 1 diabetes (T1D) is a polygenic T-cell–mediated autoimmune disease with strong genetic linkages to the MHC class II and insulin promoter regions (1). Class II molecules are fundamental for CD4⁺ T-cell activation, whereas the insulin promoter polymorphism can modulate insulin levels in the thymus thereby influencing the threshold of selection for insulin-specific autoreactive T cells (2, 3). In the nonobese diabetic (NOD) mouse model of autoimmune mediated diabetes, insulin-specific IA^g7-restricted CD4⁺ T cells have been strongly implicated in β-cell destruction. In prediabetic NOD mice, 50% of the T-cell clones established from islet-infiltrating lymphocytes were insulin-specific and the majority of these clones recognized the insulin B:9-23 (B:9-23 SHLVEALYLVCGERG) epitope (4, 5). Moreover, substitution of a single residue in the B:9-23 region abrogated development of diabetes in a transgenic mouse model (6, 7). Thus, in the murine NOD model, the IA^g7-restricted B:9-23 epitope is considered to be pivotal in the development of diabetes.The position or “register” that B:9-23 occupies within the IA^g7 binding groove has been a controversial but important question. At least three binding registers (R) have been considered, defined here by which B:9-23 amino acid occupies the first binding pocket (p1) position in the IA^g7 binding groove: R1, p1 V12; R2, p1 E13; and R3, p1 A14. Each binding register creates a unique set of upward pointing amino acid side-chains for T-cell recognition and also exhibits a distinct binding affinity for MHC class II conferred by the downward-facing amino acids that occupy its binding pockets. The Unanue group first reported that B:9-23 can be presented by IA^g7 in either R1 or R2 and that each register is recognized by a different set of B:9-23–spepcifc CD4⁺ T cells (8). In contrast, a separate study (9, 10) reported that T cells recognize the B:9-23 peptide bound to IA^g7 in R3, the weakest binding of the three registers. Mutation of R22 to the highly p9 favorable E greatly improved the binding of the peptide to IA^g7 and its presentation to CD4 T cells. Disulfides introduced between the peptide and IA^g7 confirmed the R3 binding. These data support the relevance of antigenic peptides with low-affinity HLA binding in the NOD model of diabetes.In humans, the HLA-DQB1*0302 allele confers high disease risk. The DQ8 molecule, which is comprised of the DQA1*0301 and DQB1*0302 chains, is the human homolog for IA^g7. In particular, both IA^g7 and DQ8 have a basic pocket 9 (p9, because of having a non-Asp β57 residue in the class II β-chain) that accommodates and is stabilized by peptides with acidic residues at p9 (11). Insulin autoreactive antibodies precede the onset of disease and have been used for risk stratification in susceptible individuals (12, 13). CD8⁺ T cells that are specific for insulin have also been detected (14, 15), and it has been documented that they are capable of lysing islets (16, 17) and there is one report of human CD4⁺ T cells responsive to an insulin A-chain peptide presented by HLA-DR4 (18). These data all suggest that insulin is also an important autoantigen in human T1D.In the light of the accumulated data from both murine and human studies, it is logical to postulate that DQ8-restricted B:9-23–specific CD4⁺ T cells are present in human subjects with T1D and that this peptide may also be recognized in a weakly bound register. Although recognition of B:9-23 has long been suspected, direct evidence for the presence and importance of DQ8-restricted B:9-23–specific CD4⁺ T cells in human subjects with T1D is limited and no study has definitively established the immunogenic register of B:9-23 as presented by DQ8. Alleva et al. showed that T cells from the peripheral blood of DR4-DQ8 T1D subjects had measurable proliferative and IFN responses to the B:9-23 peptide (19). In addition, the proliferation of a B:9-23–specific T-cell line could be blocked through addition of an anti-DQ Ab (19). Eerligh et al. also reported the isolation of a DQ8 restricted Ins B:6-22 T-cell clone from a T1D subject (20). However, more detailed analysis of these responses, for example by direct visualization and cloning of multiple DQ8/B:9-23–specific cells, has remained a desirable but elusive goal at least in part because of the technical challenge of interrogating DQ8 restricted autoreactive T cells.In the current study, our objective was to extend these previous observations by visualizing DQ8-restricted T-cell responses to B:9-23 in human subjects with T1D and determining the immunogenic register of the DQ8/B:9-23 complex. In particular, we wished to investigate responses to B:9-23 as a possible example of T-cell responses to a self-epitope with low-affinity MHC binding. We used DQ8/insulin tetramers to detect B:11-23–specific T cells in peripheral blood samples from T1D subjects and compared the frequency of these responses in T1D subjects and healthy controls with DQ8 haplotypes. After directly cloning these T cells, we investigated their responsiveness to stimulation with insulin peptides, denatured insulin protein, and homologous peptides derived from bacterial antigens. By assessing proliferation in response to peptides with alanine or phenylalanine substitutions, we identified a clear immunogenic register within B:11-23 that is bound by DQ8 with low affinity and recognized by human T cells.     

Serum proinsulin levels at fasting and after oral glucose load in patients with Type 2 (non-insulin-dependent) diabetes mellitus

Summary A simple and sensitive human proinsulin radioimmunoassay system was developed using guinea pig antiproinsulin serum, which cross-reacted neither with human insulin nor C-peptide. The recognition site of the antiserum seems to be located near the junction between the B chain and C-peptide. With this assay system, we studied the serum proinsulin concentration at fasting and after an oral 100 g glucose load in 25 healthy subjects, 21 subjects with impaired glucose tolerance and 40 patients with Type 2 (non-insulin-dependent) diabetes mellitus. At fasting, serum proinsulin was 5.8±3.3 pmol/l in normal subjects as compared to 9.5±6.9 pmol/l (p<0.05) in subjects with impaired glucose tolerance and 12.6±7.5 pmol/l (p<0.001) in diabetic patients. The molar ratio of proinsulin to insulin was also increased in subjects with impaired glucose tolerance or diabetes compared to control subjects. After a 100 g oral glucose load, serum proinsulin increased more slowly than insulin. The proinsulin response after an oral glucose load was augmented in subjects with impaired glucose tolerance and diabetes, while the insulin response decreased with the elevation of fasting plasma glucose. Diabetic patients with high fasting plasma glucose had a very poor insulin response, but the proinsulin response was similar to control subjects. There was a linear correlation between summed proinsulin values and summed insulin values, but the slope of the regression line was steeper in diabetic patients than in control subjects. There was a relative increase in serum proinsulin both in subjects with impaired glucose tolerance and diabetic patients. We suggest that B cells may release ‘immature’ granules richer in proinsulin content as well as mature granules in the over-stimulated state.     

Molecular and cellular analyses of HLA class II-associated susceptibility to autoimmune diseases in the Japanese population

It is well known that individuals who are positive for particular HLA class II alleles show a high risk of developing autoimmune diseases. HLA class II molecules expressed on antigen-presenting cells present antigenic peptides to CD4⁺ T cells. Their extensive polymorphism affects the structures of peptides bound to HLA class II molecules to create individual differences in immune responses to antigenic peptides. In order to gain a better understanding of mechanisms of the association between HLA class II alleles and susceptibility to autoimmune diseases, it is important to identify self-peptides presented by disease-susceptible HLA class II molecules and triggering disease-causative T cells. Many of the autoimmune diseases are observed in all ethnic groups, whereas the incidence of diseases, clinical manifestations and disease-susceptible HLA class II alleles are different among various ethnic groups for some autoimmune diseases. These phenomena suggest that differences in autoimmune self-peptide(s) in the context of disease-susceptible HLA class II molecules may cause these differences. Therefore, comparisons among disease-susceptible HLA class II alleles, autoantigenic peptides, and clinical manifestations of autoimmune diseases in different ethnic groups would be helpful in elucidating the pathogenesis of the diseases. In this review, we describe our recent findings on (1) the uniqueness of both clinical manifestations and the HLA-linked genetic background of Asian-type (opticospinal form) multiple sclerosis, (2) the characteristics of glutamic acid decarboxylase 65 (GAD65) or β₂-glycoprotein I (β₂-GPI) autoreactive T cells in Japanese patients with insulin-dependent diabetes mellitus (IDDM) or anti-β₂-GPI antibody-associated autoimmunity, respectively, and (3) the generation of an efficient delivery system of peptides to the HLA class II-restricted antigen presentation path-way by utilizing a class II-associated invariant chain peptide (CLIP)-substituted invariant chain, which may be applicable to an evaluation of the "molecular mimicry hypothesis" for the activation of autoreactive T cells.     

Identification of novel hepatitis C virus-specific cytotoxic T lymphocyte epiotpe in NS3 region

Hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTL) are thought to be effective in limiting viral spread and in clearing virus during infection. Therefore, we attempted to establish HCV-specific CTL and identify novel HCV-specific CTL epitopes in a patient with acute hepatitis C by a novel screening method using recombinant vaccinia viruses (rVV) and synthetic peptides. CD8(+)CD45RA(-) T cells (memory T cells) were isolated from peripheral blood mononuclear cells (PBMC) of a patient with acute hepatitis C. HCV-specific CTL were cloned at limited dilutions and tested for HCV-specific CTL activity using a standard (51)Cr release assay. CTL assay was performed using rVV expressing regions of HCV-J, and overlapping and truncated synthetic peptides from HCV-J. CTL recognizing the NS3 region were isolated by (51)Cr release assay with rVV-HCV. Isolated CTL were restricted by HLA class I molecules B(*)5603. We confirmed that isolated CTL recognized 8-mer amino acids in the NS3 region of HCV-J by (51)Cr release assay with overlapping and truncated synthetic peptides. In conclusion, we isolated HCV-specific CTL restricted by HLA-B(*)5603 and identified a novel HCV-specific CTL epitope (IPFYGKAI, amino acids 1373-1380) in the NS3 region. The identified HCV-specific CTL epitope might be useful for HCV therapy.     

IgE antibodies to insulin and related peptides, a result of insulin treatment?

Anti-insulin immunoglobulin E (IgE) antibodies in sera of insulin-treated diabetic patients were characterized by affinity and concentration, and by binding to proinsulin and insulin A and B chains. The affinities of IgE to insulin scattered between less than 10(7) and 0.6 x 10(9) l/mol, the concentrations between 0.3 and 3.5 ng/ml. All positive sera of type 1 and few of type 2 patients recognized proinsulin, A chain, and B chain with comparable affinities and concentrations. Elevated concentrations of total serum IgE in 12 of the 16 anti-insulin IgE-positive sera from insulin-treated patients indicate that these patients are predisposed to allergies. The incidence of elevated levels of total serum IgE in type 1 (17.7%) and in type 2 (7.8%) diabetic patients did not differ from the general population. Reagins to insulin and related antigens were also observed in sera of non-diabetic allergic persons without previous contact with exogenous insulin. The natural occurrence of insulin-specific reagins makes the use of IgE as a marker of antigenicity of insulin questionable. Discrepancies between the insulin-positive radioallergosorbent test (RAST) or skin test and clinical manifestations of insulin allergy exist, because IgE antibodies with low affinities require high concentrations of insulin for binding. Such amounts of insulin may occur at the injection site, but not in the circulation.     

HLA class I antigen display on hepatocyte membrane in chronic hepatitis B virus infection: its role in the pathogenesis of chronic type B hepatitis

It has been suggested that cytotoxic T cells are involved in the recognition and lysis of the infected hepatocytes in chronic hepatitis B virus infection, and that the target antigen is probably HBcAg which is displayed on the hepatocyte membrane during active viral replication. However, studies in other viral infection have demonstrated that cytotoxic T cells recognize viral antigen on the infected cells only in the context of HLA class I antigens. To test whether this mechanism is also operative in chronic hepatitis B virus infection, we studied the expression of HLA class I antigens in livers from 35 patients with chronic hepatitis B virus infection by indirect immunofluorescence using monoclonal antibody against HLA class I antigens. The blocking effect of monoclonal antibody against HLA class I antigens on the in vitro T cell cytotoxicity to autologous hepatocytes was also studied. The results revealed that HLA class I antigen was undetectable on the hepatocyte membrane in all of 10 HBeAg-positive carriers with minor hepatitic activity, whereas it was demonstrated in 15 (88%) of the 17 HbeAg-positive patients with chronic active liver disease and in 7 (87%) of the 8 anti-HBe-positive "normal" carriers. The in vitro T cell cytotoxicity to autologous hepatocytes in six HBeAg-positive patients with chronic active liver disease was significantly inhibited by preincubation of hepatocytes with monoclonal antibody (10 to 40 micrograms per ml) against HLA class I antigen, but not by monoclonal antibody against HLA class II antigens and non-HLA-associated surface molecules (Leu 11).(ABSTRACT TRUNCATED AT 250 WORDS)     

CDR1 T-cell receptor beta-chain peptide induces major histocompatibility complex class II-restricted T-T cell interactions.

T-T cell interactions have been proposed in postulated network theories of immunoregulation and autoimmunity. Despite previous reports of protection induced by T-cell receptor (TcR)-derived peptides in experimental autoimmunity, no evidence for T-T cell interactions by direct recognition of processed TcRs on native T cells was obtained. Here we report that immunization of rats with overlapping sets of peptides of the TcR alpha or beta chain allowed us to detect immunogenic TcR peptides. Remarkably enough, these TcR peptides appeared to cluster within the hypervariable complementarity-determining regions of the TcR. Immunization of rats with these TcR peptides induced CD4+ TcR peptide-specific T cells, which recognized both rDNA TcR proteins and the original, arthritogenic T cell in a major histocompatibility complex class II-restricted way. These findings indicate that activated T cells can process and present their own TcR in the context of major histocompatibility complex class II molecules and, furthermore, that such peptides can be recognized by TcR variable gene-specific T cells.     

Breast and ovarian cancer-specific cytotoxic T lymphocytes recognize the same HER2/neu-derived peptide.

The identification of antigenic peptides presented on the tumor cell surface by HLA class I molecules and recognized by tumor-specific cytotoxic T lymphocytes may lead to a peptide vaccine capable of inducing protective cellular immunity. We demonstrate that both HLA-A2-restricted breast and ovarian tumor-specific cytotoxic T lymphocytes recognize shared antigenic peptides. At least one of these peptides is derived from the oncogene product of HER2/neu, which is overexpressed in 30-40% of all breast and ovarian cancers. T cells sensitized against this nine-amino acid sequence demonstrate significant recognition of HLA-A2+, HER2/neu+ tumors. Since 50% of the tumor-cell population is HLA-A2+ and many different tumors express HER2/neu, this peptide may be widely recognized and have many clinical applications.