Combined insulin B:9-23 self-peptide and polyinosinic-polycytidylic acid accelerate insulitis but inhibit development of diabetes by increasing the proportion of CD4+Foxp3+ regulatory T cells in the islets in non-obese diabetic mice

Insulin peptide B:9-23 is a major autoantigen in type 1 diabetes. Combined treatment with B:9-23 peptide and polyinosinic-polycytidylic acid (poly I:C), but neither alone, induce insulitis in normal BALB/c mice. In contrast, the combined treatment accelerated insulitis, but prevented diabetes in NOD mice. Our immunofluorescence study with anti-CD4/anti-Foxp3 revealed that the proportion of Foxp3 positive CD4⁺CD25⁺ regulatory T cells (Tregs) was elevated in the islets of NOD mice treated with B:9-23 peptide and poly I:C, as compared to non-treated mice. Depletion of Tregs by anti-CD25 antibody hastened spontaneous development of diabetes in non-treated NOD mice, and abolished the protective effect of the combined treatment and conversely accelerated the onset of diabetes in the treated mice. These results indicate that poly I:C combined with B:9-23 peptide promotes infiltration of both pathogenic T cells and predominantly Tregs into the islets, thereby inhibiting progression from insulitis to overt diabetes in NOD mice.     

One amino acid difference is critical for suppression of the development of experimental autoimmune diabetes (EAD) with intravenous injection of insulinB:9-23 peptide

InsulinB:9-23 peptide (insB:9-23) reactive T cells has been reported as crucial for type 1 diabetes. In this study, experimental autoimmune diabetes (EAD) mice, which subcutaneous immunization of ins1 or 2B:9-23 induced autoimmune diabetes in F1(B7.1B6 × BALB/c), was investigated for antigen specific therapy to delete pathogenic T cells. Intravenous injection of ins1 or 2B:9-23 significantly delayed the development of diabetes on the corresponding peptide-induced EAD (ins1EAD or ins2EAD) concomitant with reduced insulitis and insulin autoantibodies expression. Population of Foxp3⁺ CD4⁺ T cell was unchanged whereas the level of anti-insB:9-23 specific IgG_2a but not IgG₁ were specifically decreased, suggesting reduction of pathogenic insB:9-23 reactive T cells. Most interestingly, intravenous administration of ins2B:9-23, whose amino acid sequence had one amino acid difference at position 9 delayed the development of diabetes in both ins1EAD and ins2EAD whereas ins1B:9-23 administration delayed diabetes in the ins1EAD but not ins2EAD, suggesting that one amino acid difference gives critical influence on the effect of intravenous injection of antigenic peptide for type 1 diabetes.     

T cell autoreactivity to insulin in diabetic and related non-diabetic individuals

T cell autoreactivity to insulin in type I diabetic and related non-diabetic individuals was analyzed. Peripheral T lymphocytes from both insulin-treated diabetic and untreated non-diabetic members of four families were found to proliferate in vitro in response to human insulin. T cell autoreactivity to insulin therefore does not appear to be diagnostic of the onset of type I diabetes. Highest T cell responses to human insulin were usually detected in insulin-dependent type I diabetes patients treated with a mixture of beef and pork insulin than with self insulin, the greater the dose of animal insulin the higher the T cell response. The T cell repertoires for self insulin appear to be similar in diabetics and non-diabetics based on their patterns of T cell reactivity to beef insulin, port insulin, human insulin, and various peptide of human insulin. The autoreactive T cells analyzed recognize two conformational epitopes of human insulin formed by interactions between A chain and B chain residues. One epitope is associated with the A chain loop and is present in the A1-A14/B1-B16 peptide, and the other epitope consists mainly of B chain residues located in the A16-A21/B10-B25 peptide. These two epitopes are present in amphipathic alpha-helical regions of insulin. HLA-DR (DR3, DR4, and DR5) and HLA-DQ (DQw2/DQw3) Ag can restrict these T cell responses to human insulin epitopes. The ability to detect insulin-specific autoreactive T cells in healthy non-diabetic individuals supports the hypothesis that autoreactive lymphocytes do not necessarily elicit autoimmune disease if present in an environment in which their activity is immunoregulated.     

Production of a recombinant cholera toxin B subunit-insulin B chain peptide hybrid protein by Brevibacillus choshinensis expression system as a nasal vaccine against autoimmune diabetes

Mucosally induced tolerance is an attractive strategy for preventing or reducing autoimmune diseases. Here, we produced a recombinant CTB fusion protein linked with autoantigen T cell epitope of insulin B chain peptide 9-23 (C19S) at levels up to 200 mg/L culture media in Brevibacillus choshinensis secretion-expression system. Receptor-competitive assay showed that the CTB-insulin peptide binds to GM1 receptor almost equivalent degree as the native form of CTB. Non-obese diabetes (NOD) mice that spontaneously develop an insulin-dependent diabetes were nasally immunized with CTB-insulin peptide (5 microg) for three times. The nasal treatment significantly reduced the development of insulin-dependent diabetes and peptide specific DTH responses after systemic immunization with the insulin peptide B 9-23(C19S) in CFA. Nasal administration of as high as 50 microg of the peptide alone demonstrated a similar level of the disease inhibition. In contrast, all mice given 5 microg of the insulin peptide alone or 5 microg of insulin peptide with 25 microg of the free form of CTB did not lead to the suppression of diabetes development and DTH responses. Because molecular weight of the insulin peptide is about one tenth of that of the CTB-insulin peptide, the results demonstrate that the recombinant hybrid of autoantigen and CTB increased its tolerogenic potential for nasal administration by up 100-fold on molar base of autoantigen peptide. Taken together, nasally-induced tolerance by administration of the recombinant B. choshinensis-derived hybrid protein of CTB and autoantigen T cell-epitope peptide could be useful mucosal immunetherapy for the control of T cell-mediated autoimmune diseases.     

Effect of point mutations in the native simian virus 40 tumor antigen, and in synthetic peptides corresponding to the H-2Db-restricted epitopes, on antigen presentation and recognition by cytotoxic T lymphocyte clones.

The effects on CTL recognition of individual amino acid substitutions within epitopes I, II, and III of SV40 tumor Ag (T Ag) were examined. Epitope I spans amino acids 207 to 215, and epitope II/III is within residues 223 to 231 of SV40 T Ag. An amino acid substitution at position 207 (Ala----Val) or 214 (Lys----Glu) of SV40 T Ag expressed in transformed cells resulted in loss of epitope I, recognized by CTL clone Y-1. The amino acid substitution at residue 214 in the corresponding synthetic peptide, LT207-215(214-Lys----Glu), also led to loss of recognition by CTL clone Y-1. The recognition, by CTL clone Y-1, of peptides LT207-215 and LT207-217 with an Ala----Val substitution at position 207 was severely affected. Peptides LT205-215 and LT205-219 with the Ala----Val substitution at residue 207 were, however, recognized by CTL clone Y-1, suggesting that residues 205 and 206 may be involved in presentation of site I. Alteration of residue 224 (Lys----Glu) in the native T Ag resulted in loss of recognition by both CTL clones Y-2 and Y-3. However, a peptide corresponding to epitope II/III with an identical amino acid substitution at residue 224 provided a target for CTL clone Y-3 but not clone Y-2. A change of Lys----Gln at residue 224 in both the native protein and a synthetic peptide caused loss of recognition by CTL clone Y-2 but not CTL clone Y-3. Further, an amino acid substitution of Lys----Arg at position 224 of the native T Ag decreased recognition of epitope II/III by CTL clones Y-2 and Y-3 but had no effect on recognition of a synthetic peptide bearing the same substitution. These results indicate that the mutagenesis approach, resulting in identical amino acid substitutions in the native protein and in the synthetic peptides, may provide insight into the role of individual residues in the processing, presentation, and recognition of CTL recognition epitopes.     

T cell response to preproinsulin I and II in the nonobese diabetic mouse

Immunization against insulin, insulin B chain, or B chain peptide B(9-23) (preproinsulin peptide II(33-47)) prevents diabetes in the nonobese diabetic (NOD) mouse. Whether or not peptide II(33-47) is the only proinsulin determinant recognized by CD4 T cells remains unclear. Using two peptide libraries spanning the entire sequence of preproinsulin I and preproinsulin II, respectively, we identified T cells specific for four proinsulin epitopes within the islet cell infiltrate of prediabetic female NOD mice. These epitopes were among immunogenic epitopes to which a T cell response was detected after immunization of NOD mice with individual peptides in CFA. Immunogenic epitopes were found on both isoforms of insulin, especially proinsulin II, which is the isoform expressed in the thymus. The autoimmune response to proinsulin represented only part of the immune response to islet cells within the islet cell infiltrate in 15-wk-old NOD mice. This is the first systematic study of preproinsulin T cell epitopes in the NOD mouse model.     

Hierarchy among multiple H-2b-restricted cytotoxic T-lymphocyte epitopes within simian virus 40 T antigen.

Simian virus 40 large tumor (T) antigen contains three H-2Db-restricted (I, II/III, and V) and one H-2Kb-restricted (IV) cytotoxic T lymphocyte (CTL) epitopes. We demonstrate that a hierarchy exists among these CTL epitopes, since vigorous CTL responses against epitopes I, II/III, and IV are detected following immunization of H-2b mice with syngeneic, T-antigen-expressing cells. By contrast, a weak CTL response against the H-2Db-restricted epitope V was detected only following immunization of H-2b mice with epitope loss variant B6/K-3,1,4 cells, which have lost expression of CTL epitopes I, II/III, and IV. Limiting-dilution analysis confirmed that the lack of epitope V-specific CTL activity in bulk culture splenocytes correlated with inefficient expansion and priming of epitope V-specific CTL precursors in vivo. We examined whether defined genetic alterations of T antigen might improve processing and presentation of epitope V to the epitope V-specific CTL clone Y-5 in vitro and/or overcome the recessive nature of epitope V in vivo. Deletion of the H-2Db-restricted epitopes I and II/III from T antigen did not increase target cell lysis by epitope V-specific CTL clones in vitro. The amino acid sequence SMIKNLEYM, which species an optimized H-2Db binding motif and was found to induce CTL in H-2b mice, did not further reduce epitope V presentation in vitro when inserted within T antigen. Epitope V-containing T-antigen derivatives which retained epitopes I and II/III or epitope IV did not induce epitope V-specific CTL in vivo: T-antigen derivatives in which epitope V replaced epitope I failed to induce epitope V-specific CTL. Recognition of epitope V-H-2Db complexes by multiple independently derived epitope V-specific CTL clones was rapidly and dramatically reduced by incubation of target cells in the presence of brefeldin A compared with the recognition of the other T-antigen CTL epitopes by epitope specific CTL, suggesting that the epitope V-H-2Db complexes either are labile or are present at the cell surface at reduced levels. Our results suggest that processing and presentation of epitope V is not dramatically altered (reduced) by the presence of immunodominant CTL epitopes in T antigen and that the immunorecessive nature of epitope V is not determined by amino acids which flank its native location within simian virus 40 T antigen.     

Identification of a novel HLA-A2-restricted cytotoxic T lymphocyte epitope from cancer-testis antigen PLAC1 in breast cancer

Identification of cytotoxic T lymphocyte (CTL) epitopes from tumor antigens is essential for the development of peptide vaccines against tumor immunotherapy. Among all the tumor antigens, the caner-testis (CT) antigens are the most widely studied and promising targets. PLAC1 (placenta-specific 1, CT92) was considered as a novel member of caner-testis antigen, which expressed in a wide range of human malignancies, most frequently in breast cancer. In this study, three native peptides and their analogues derived from PLAC1 were predicted by T cell epitope prediction programs including SYFPEITHI, BIMAS and NetCTL 1.2. Binding affinity and stability assays in T2 cells showed that two native peptides, p28 and p31, and their analogues (p28-1Y9?V, p31-1Y2L) had more potent binding activity towards HLA-A*0201 molecule. In ELISPOT assay, the CTLs induced by these four peptides could release IFN-γ. The CTLs induced by these four peptides from the peripheral blood mononuclear cells (PBMCs) of HLA-A*02⁺ healthy donor could lyse MCF-7 breast cancer cells (HLA-A*0201⁺, PLAC1⁺) in vitro. When immunized in HLA-A2.1/K^b transgenic mice, the peptide p28 could induce the most potent peptide-specific CTLs among these peptides. Therefore, our results indicated that the peptide p28 (VLCSIDWFM) could serve as a novel candidate epitope for the development of peptide vaccines against PLAC1-positive breast cancer.     

Identification of a ras oncogene peptide that contains both CD4(+) and CD8(+) T cell epitopes in a nested configuration and elicits both T cell subset responses by peptide or DNA immunization

Mutations in ras proto-oncogenes are commonly found in a diversity of malignancies and may encode unique, non-self epitopes for T cell-mediated antitumor activity. In a BALB/c (H-2(d)) murine model, we have identified a single peptide sequence derived from the ras oncogenes that contained both CD8(+) and CD4(+) T cell epitopes in a nested configuration. This peptide reflected ras sequence 4-16, and contained the substitution of Gly to Val at position 12 ?i.e., 4-16(Val12)?. Mice immunized with this 13-mer peptide induced a strong antigen (Ag)-specific CD4(+) proliferative response in vitro. In contrast, mice inoculated with the wild-type ras sequence failed to generate a peptide-specific T cell response. Additionally, mice immunized with the ras 4-16(Val12) peptide concomitantly displayed an Ag-specific CD8(+) cytotoxic T lymphocyte (CTL) response, as determined by lysis of syngeneic tumor target cells incubated with the nominal 9-mer nested epitope peptide ?i.e., 4-12(Val12)?, as well as lysis of tumor target cells expressing the corresponding ras codon 12 mutation. Analysis of the Valpha- and Vbeta-chains of the T cell receptor (TCR) expressed by these CTL revealed usage of the Valpha1 and Vbeta9 subunits, consistent with the TCR phenotype of anti-ras Val12 CTL lines produced by in vivo immunization with the nominal peptide epitope alone. Moreover, immunization with the nested epitope peptide, as compared to immunization with either the 9-mer CTL peptide alone or an admixture of the 9-mer CTL peptide with an overlapping 13-mer CD4(+) T cell helper peptide ?i.e., 5-17(Val12)? lacking the class I N-terminus anchor site, enhanced the production of the CD8(+) T cell response. Finally, immunization with plasmid DNA encoding the ras 4-16(Val12) sequence led to the induction of both Ag-specific proliferative and cytotoxic responses. Overall, these results suggested that a single peptide immunogen containing nested mutant ras-specific CD4(+) and CD8(+) T cell epitopes: (1) can be processed in vivo to induce both subset-specific T lymphocyte responses; and (2) leads to the generation of a quantitatively enhanced CD8(+) CTL response, likely due to the intimate coexistence of CD4(+) help, which may have implications in peptide- or DNA-based immunotherapies.     

The hypervariable immunodominant NP418-426 epitope from the influenza A virus nucleoprotein is recognized by cytotoxic T lymphocytes with high functional avidity

Recently it was shown that influenza A viruses can accumulate mutations in epitopes associated with escape from recognition by human virus-specific cytotoxic T lymphocytes (CTL). It is unclear what drives diversification of CTL epitopes and why certain epitopes are variable and others remain conserved. It has been shown that simian immunodeficiency virus-specific CTL that recognize their epitope with high functional avidity eliminate virus-infected cells efficiently and drive diversification of CTL epitopes. T-cell functional avidity is defined by the density of major histocompatibility complex class I peptide complexes required to activate specific CTL. We hypothesized that functional avidity of CTL contributes to epitope diversification and escape from CTL also for influenza viruses. To test this hypothesis, the functional avidity of polyclonal CTL populations specific for nine individual epitopes was determined. To this end, peripheral blood mononuclear cells from HLA-A- and -B-genotyped individuals were stimulated in vitro with influenza virus-infected cells to allow expansion of virus-specific CTL, which were used to determine the functional avidity of CTL specific for nine individual epitopes in enzyme-linked immunospot assays. We found that the functional avidity for the respective epitopes varied widely. Furthermore, the functional avidity of CTL specific for the hypervariable NP(418-426) epitope was significantly higher than that of CTL recognizing other epitopes (P < 0.01). It was speculated that the high functional avidity of NP(418-426)-specific CTL was responsible for the diversification of this influenza A virus CTL epitope.     

A new unconventional HLA-A2-restricted epitope from HBV core protein elicits antiviral cytotoxic T lymphocytes

Cytotoxic T cells (CTLs) play a key role in the control of Hepatitis B virus (HBV) infection and viral clearance. However, most of identified CTL epitopes are derived from HBV of genotypes A and D, and few have been defined in virus of genotypes B and C which are more prevalent in Asia. As HBV core protein (HBc) is the most conservative and immunogenic component, in this study we used an overlapping 9-mer peptide pool covering HBc to screen and identify specific CTL epitopes. An unconventional HLA-A2-restricted epitope HBc141–149 was discovered and structurally characterized by crystallization analysis. The immunogenicity and anti-HBV activity were further determined in HBV and HLAA2 transgenic mice. Finally, we show that mutations in HBc141–149 epitope are associated with viral parameters and disease progression in HBV infected patients. Our data therefore provide insights into the structure characteristics of this unconventional epitope binding to MHC-I molecules, as well as epitope specific CTL activity that orchestrate T cell response and immune evasion in HBV infected patients.     

Two distinctly HLA-associated contiguous linear epitopes uniquely expressed within the islet antigen 2 molecule are major autoantibody epitopes of the diabetes-specific tyrosine phosphatase-like protein autoantigens

The related tyrosine phosphatase-like proteins islet Ag (IA)-2 and IA-2beta are autoantigens of type 1 diabetes in humans. Autoantibodies are predominantly against IA-2, and IA-2-specific epitopes are major autoantibody targets. We used the close homology of IA-2 and IA-2beta to design chimeras and mutants to identify humoral IA-2-specific epitopes. Two major IA-2 epitopes that are absent from the related autoantigens IA-2beta and IA-2Delta 13 splice variant ICA512.bdc were found contiguous to each other within IA-2 juxtamembrane amino acids 611-620 (epitope JM1) and 621-630 (epitope JM2). JM1 and JM2 are recognized by sera from 67% of patients with IA-2 Abs, and relatives of patients with type 1 diabetes having Abs to either JM epitope had a >50% risk for developing type 1 diabetes within 6 years, even in the absence of diabetes-associated HLA genotypes. Remarkably, the presence of Abs to one of these two epitopes was mutually exclusive of the other; JM2 Abs and not JM1 Abs were found in relatives with HLA DR3/4, DR4/13, or DR1/4 genotypes; and the binding of autoantibodies to the JM2 epitope, but not the JM1 epitope, markedly affected proteolysis of IA-2. This is a unique demonstration of HLA-associated B cell responses to epitopes within a single autoantigen in humans and is consistent with modification of Ag processing by specific Ab-influencing peptide presentation by HLA molecules.     

Immunobiology of cytotoxic T-cell escape mutants of lymphocytic choriomeningitis virus.

Infection with virus variants exhibiting changes in the peptide sequences defining immunodominant determinants that abolish recognition by antiviral cytotoxic T cells (CTL) presents a considerable challenge to the antiviral T-cell immune system and may enable some viruses to persist in hosts. The potential importance of such variants with respect to mechanisms of viral persistence and disease pathogenesis was assessed by infecting adult mice with variants of lymphocytic choriomeningitis virus (LCMV) strain WE. These variants were selected in vivo or in vitro for resistance to lysis by CD8+ H-2b-restricted antiviral CTL. The majority of anti-LCMV CTL in infected H-2b mice recognize epitopes defined by residues 32 to 42 and 275 to 289 (epitopes 32-42 and 275-289) of the LCMV glycoprotein or 397 to 407 of the viral nucleoprotein. The 8.7 variant exhibits a change in the epitope 32-42 (Val-35-->Leu), and variant CL1.2 exhibits a change in the epitope 275-289 (Asn-280-->Asp) of the wild-type LCMV-WE. The double-mutated 8.7-B23 variant had the variation of 8.7 and an additional change located in the epitope 275-289 (Asn-280-->Ser). The 8.7 variant peptide with unchanged anchor positions bound efficiently to H-2Db and H-2Kb molecules but induced only a very weak CTL response. CTL epitope 275-289 of CL1.2 and 8.7-B23 altered at predicted anchor residues were unable to bind Db molecules and were also not recognized by antiviral CTL. Infection of C57BL/6 mice (H-2b) with the variants exhibiting mutations of one of the CTL epitopes, i.e., 8.7 or CL1.2, induced CTL responses specific for the unmutated epitopes comparable with those induced by infection with WE, and these responses were sufficient to eliminate virus from the host. In contrast, infection with the double-mutated variant 8.7-B23 induced CTL activity that was reduced by a factor of about 50-fold compared with wild-type LCMV. Consequently, high doses (10(7) PFU intravenously) of this virus were eliminated slowly and only by about day 100 after infection. 8.7-B23 failed to cause lethal lymphocytic choriomeningitis after intracerebral infection with a dose of > 10(4) PFU in C57BL/6 mice (but not in mice of nonselecting H-2d haplotype); with the other variants or wild-type LCMV, doses greater than 10(6) to 10(7) PFU were necessary to avoid lethal choriomeningitis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Human insulin autoantibody fine specificity and H and L chain use

Fine specificity and H and L chain isotypes of insulin autoantibodies in sera from 11 subjects were examined. None of these 11 subjects was treated with exogenous insulin. Two patterns of fine specificity were found. In one, the autoantibodies were specific for human insulin, with a requirement for threonine at B30. The conservative substitution in pork insulin (threonine to alanine) abrogated IgG binding by these sera. Insulin autoantibodies in other sera cross-reacted with beef, pork, and human insulin; not requiring threonine at B30. Reciprocal competitive inhibition experiments showed that epitopes recognized by the human specific insulin autoantibodies were exclusively on the B chain, whereas the cross-reactive sera contain autoantibodies that recognize both the B chain and combinatorial (A and B chain) epitopes. The fine specificity of cross-reactive insulin autoantibodies are thus similar to insulin antibodies from insulin-treated subjects. When IgG subclasses and L chains of insulin autoantibodies were examined, however, restricted C region usage was found. The hierarchy was IgG3 greater than G1 greater than G2 greater than G4; with one subclass dominant in each serum, although others were used. L chain use was similarly restricted. There was no correlation between isotype and fine specificity or between H and L chain type. It is concluded that heterogeneity of insulin autoantibodies is restricted. The response is probably more oligo- or pauciclonal than insulin antibody from insulin-treated subjects.     

Generation of T cells specific for the wild-type sequence p53(264-272) peptide in cancer patients: implications for immunoselection of epitope loss variants

Alterations in the p53 gene occur frequently and can lead to accumulation of p53 protein in squamous cell carcinomas of the head and neck (SCCHN). Since accumulation of p53 is associated with enhanced presentation of wild-type sequence (wt) p53 peptides to immune cells, the development of pan vaccines against SCCHN has focused on wt p53 epitopes. We used the HLA-A2.1-restricted wt p53(264-272) epitope to generate CTL from circulating precursor T cells of HLA-A2.1(+) healthy donors and patients with SCCHN. Autologous peptide-pulsed dendritic cells were used for in vitro sensitization. CTL specific for the wt p53(264-272) peptide were generated from PBMC obtained from two of seven normal donors and three of seven patients with SCCHN. These CTL were HLA class I restricted and responded to T2 cells pulsed with p53(264-272) peptide as well as HLA-A2-matched SCCHN cell lines naturally presenting the epitope. Paradoxically, none of the tumors in the three patients who generated CTL could adequately present the epitope; two had a wt p53 genotype and no p53 protein accumulation, while the third tumor expressed a point mutation (R to H) in codon 273 that prevents presentation of the p53(264-272) epitope. In contrast, patients who did not generate CTL had tumors that accumulated altered p53 and potentially could present the p53(264-272) epitope. These findings suggest that in vivo, CTL specific for the wt p53(264-272) peptide might play a role in the elimination of tumor cells expressing this epitope and in immunoselection of epitope-loss tumor cells. Immunoselection of tumors that become resistant to anti-p53 immune responses has important implications for future p53-based vaccination strategies.     

Differential selection pressure exerted on HIV by CTL targeting identical epitopes but restricted by distinct HLA alleles from the same HLA supertype

HLA diversity is seen as a major challenge to CTL vaccines against HIV. One current approach focuses on "promiscuous" epitopes, presented by multiple HLA alleles from within the same HLA supertype. However, the effectiveness of such supertype vaccines depends upon the functional equivalence of CTL targeting a particular epitope, irrespective of the restricting HLA. In this study, we describe the promiscuous HIV-specific CTL epitopes presented by alleles within the B7 supertype. Substantial differences were observed in the ability of CTL to select for escape mutation when targeting the same epitope but restricted by different HLA. This observation was common to all six promiscuous B7 epitopes identified. Moreover, with one exception, there were no significant differences in the frequency, magnitude, or immunodominance of the CTL responses restricted by different HLA alleles to explain these discrepancies. This suggests that the unique peptide/MHC complexes generated by even closely related HLA induce CTL responses that are qualitatively different. This hypothesis is supported by additional differences observed between CTL targeting identical epitopes but restricted by different HLA: first, the occurrence of distinct, HLA-specific escape mutation; second, the recruitment of distinct TCR repertoires by particular peptide/MHC complexes; and, third, significant differences in the functional avidity of CTL. Taken together, these data indicate that significant functional differences exist between CTL targeting identical epitopes but restricted by different, albeit closely related HLA. These findings are of relevance to vaccine approaches that seek to exploit HLA supertypes to overcome the problem of HLA diversity.     

Identification of insulin intermediates and sites of cleavage of native insulin by insulin protease from human fibroblasts

We have studied the time sequence degradation of native insulin by insulin protease from human fibroblast using multiple steps involving purification of the products by high performance liquid chromatography, determination of peak composition by amino acid sequence analysis, and confirmation of structure by mass spectrometry and thus elucidated the sites of cleavage of insulin by human insulin protease. We observed that as early as 0.5 min of incubation, three major new peptide peaks, intact insulin, and four smaller peptide peaks can be detected. The major peptides are portions of the insulin molecule, with the amino ends of the A and B chains or the carboxyl ends of the A and B chains still connected by disulfide bonds. Peptide peak I is A1-13-B1-9. Peptide peak II is A1-14-B1-9. Peptide peak III is A14-21-B14-30. The smaller peptide peaks are A14-21-B17-30, A15-21-B14-30, A15-21-B10-30, and A14-21-B10-30. The major peptide bond cleavage sites therefore consist of A13-14, A14-15, B9-10, B13-14, and B10-17. With longer incubation times, peptide peak II appears to lose the A14 tyrosine to form peptide peak I. This peptide I, which is the amino end of the A and B chains, is not further degraded even after 1.5 h of incubation. With longer incubation times, the peptides containing the carboxyl ends of the A and B chains are further degraded to form products from cleavage at the A18-19, B14-15, B25-26, and a small amount of A19-20, B10-11, and B24-25 cleavage and the emergence of 2-5-amino acid peptide chains, tyrosine, alanine, histidine, and leucine-tyrosine. We conclude, based on the three-dimensional structure of insulin, that human insulin protease recognizes the alpha-helical regions around leucine-tyrosine bonds and that final degradation steps to small peptides do not require lysosomal involvement.     

Identification of five new HLA-B*3501-restricted epitopes derived from common melanoma-associated antigens, spontaneously recognized by tumor-infiltrating lymphocytes

We previously described HLA-B35-restricted melanoma tumor-infiltrating lymphocyte responses to frequently expressed melanoma-associated Ags: tyrosinase, Melan-A/MART-1, gp100, MAGE-A3/MAGE-A6, and NY-ESO-1. Using clones derived from these TIL, we identified in this study the corresponding epitopes. We show that five of these epitopes are new and that melanoma cells naturally present all the six epitopes. Interestingly, five of these epitopes correspond to or encompass melanoma-associated Ag epitopes presented in other HLA contexts, such as A2, A1, B51, and Cw3. In particular, the HLA-B35-restricted Melan-A epitope is mimicked by the peptide 26-35, already known as the most immunodominant melanoma epitope in the HLA-A*0201 context. Because this peptide lacked adequate anchor amino acid residues for efficient binding to HLA-B35, modified peptides were designed. Two of these analogues were found to induce higher PBL- and tumor-infiltrating lymphocyte-specific responses than the parental peptide, suggesting that they could be more immunogenic in HLA-B*3501 melanoma patients. These data have important implications for the formulation of polypeptide-based vaccines as well as for the monitoring of melanoma-specific CTL response in HLA-B*3501 melanoma patients.