Characterization of self-glutamic acid decarboxylase 65-reactive CD4+ T-cell clones established from Japanese patients with insulin-dependent diabetes mellitus

To investigate autoimmunity to glutamic acid decarboxylase (GAD) 65 in Japanese patients with insulin-dependent diabetes mellitus (IDDM, type I diabetes), we established seven CD4+ T-cell clones, by stimulating peripheral blood mononuclear cells (PBMC) of six IDDM patients, using a mixture of overlapping human GAD65 peptides. No GAD65 autoreactive T-cell clones were evidenced in four healthy controls. Specificities of T-cell clones were as follows: (a) two clones specific to GAD65 p111-131 (residue 111 to 131) + DR53 (DRB4*0103); (b) one clone specific to GAD65 p413-433 + DR1 (DRB1*0101); (c) two clones specific to GAD65 p200-217 + either DR9 (DRB1*0901) or DR8 (DRB1*0802); and (d) two clones specific to GAD65 p368-388 + DP2 (DPA1*01 or 0201-DPB1*0201). Two DR53-restricted and one DR1-restricted T-cell clones, responded to a recombinant human GAD65 protein, and showed cytotoxicity against B lymphoblastoid cell lines pre-pulsed with the peptides. Six T-cell clones exhibited the Th1-like phenotype. Interestingly, two DR53-restricted T-cell clones killed a Fas-deficient B lymphoblastoid cell line, thereby indicating that cytotoxicity was not completely dependent on a Fas-Fas ligand interaction. Thus, the T-cell epitopes were mapped in a limited portion of GAD65 protein, with a tendency to be restricted by disease-associated HLA-DR, but not DQ molecules.     

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+ alphabeta 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.     

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.     

HIV-1 variation diminishes CD4 T lymphocyte recognition

Effective long-term antiviral immunity requires specific cytotoxic T lymphocytes and CD4(+) T lymphocyte help. Failure of these helper responses can be a principle cause of viral persistence. We sought evidence that variation in HIV-1 CD4(+) T helper epitopes might contribute to this phenomenon. To determine this, we assayed fresh peripheral blood mononuclear cells from 43 asymptomatic HIV-1(+) patients for proliferative responses to HIV-1 antigens. 12 (28%) showed a positive response, and we went on to map dominant epitopes in two individuals, to p24 Gag restricted by human histocompatibility leukocyte antigen (HLA)-DR1 and to p17 Gag restricted by HLA-DRB52c. Nine naturally occurring variants of the p24 Gag epitope were found in the proviral DNA of the individual in whom this response was detected. All variants bound to HLA-DR1, but three of these peptides failed to stimulate a CD4(+) T lymphocyte line which recognized the index sequence. Antigenic variation was also detected in the p17 Gag epitope; a dominant viral variant present in the patient was well recognized by a specific CD4(+) T lymphocyte line, whereas several natural mutants were not. Importantly, variants detected at both epitopes also failed to stimulate fresh uncultured cells while index peptide stimulated successfully. These results demonstrate that variant antigens arise in HIV-1(+) patients which fail to stimulate the T cell antigen receptor of HLA class II-restricted lymphocytes, although the peptide epitopes are capable of being presented on the cell surface. In HIV-1 infection, naturally occurring HLA class II-restricted altered peptide ligands that fail to stimulate the circulating T lymphocyte repertoire may curtail helper responses at sites where variant viruses predominate.     

T-cell epitope analysis using subtracted expression libraries (TEASEL): application to a 38-kDA autoantigen recognized by T cells from an insulin-dependent diabetic patient

Studies on circulating T cells and antibodies in newly diagnosed type 1 diabetic patients and rodent models of autoimmune diabetes suggest that beta-cell membrane proteins of 38 kDa may be important molecular targets of autoimmune attack. Biochemical approaches to the isolation and identification of the 38-kDa autoantigen have been hampered by the restricted availability of islet tissue and the low abundance of the protein. A procedure of epitope analysis for CD4+ T cells using subtracted expression libraries (TEASEL) was developed and used to clone a 70-amino acid pancreatic beta-cell peptide incorporating an epitope recognized by a 38-kDa-reactive CD4+ T-cell clone (1C6) isolated from a human diabetic patient. The minimal epitope was mapped to a 10-amino acid synthetic peptide containing a DR1 consensus binding motif. Data base searches did not reveal the identity of the protein, though a weak homology to the bacterial superantigens SEA (Streptococcus pyogenes exotoxin A) and SEB (Staphylococcus aureus enterotoxin B) (23% identity) was evident. The TEASEL procedure might be used to identify epitopes of other autoantigens recognized by CD4+ T cells in diabetes as well as be more generally applicable to the study low-abundance autoantigens in other tissue-specific autoimmune diseases.     

Immunopotency of a viral peptide assembled on the carbohydrate moieties of self immunoglobulins

The T-cell receptor recognizes peptides bound to the major histocompatibility complex antigens. Synthetic peptides corresponding to microbial epitopes can efficiently stimulate the in vitro proliferation of T-cell hybridoma or in vivo primed T cells. However, the in vivo immune responses elicited by synthetic peptides are weak because of their short half-life and poor immunogenicity. We previously showed that a genetically engineered immunoglobulin (Ig-HA), in which the CDR3 region of VH gene was replaced with a viral peptide recognized by CD4+ T cells, was able to deliver this epitope in the correct frame to antigen-processing cells that efficiently presented the peptide to T cells. Recently, we developed an enzymatic method to assemble viral peptides on the sugar moieties of immunoglobulins without alteration of the biological functions of either molecule. The viral peptide carried by these conjugates was twenty times more efficient in activating a T-cell hybridoma than the free peptide as calculated on a molar basis. We show that such conjugates are able to prime in vivo the precursors of peptide-specific T cells and to induce proliferation of naive lymphocytes from transgenic mice expressing a peptide-specific T-cell receptor in both CD4 and CD8 T-cell subsets. Our results suggest that peptides enzymatically linked to the carbohydrate moieties of immunoglobulins, using galactose residues as peptide acceptor, can be used as a safe and efficient delivery system of protective epitopes for the prevention of infectious diseases. The enzymatic engineering of immunoglobulins may also allow the development of immunotherapeutic agents to deliver antagonist peptides to autoreactive T cells or to direct immunomodulatory agents such as interleukins or cytolytic drugs to tumor cells.     

Definition of natural T cell antigens with mimicry epitopes obtained from dedicated synthetic peptide libraries

Progress has recently been made in the use of synthetic peptide libraries for the identification of T cell-stimulating ligands. T cell epitopes identified from synthetic libraries are mimics of natural epitopes. Here we show how the mimicry epitopes obtained from synthetic peptide libraries enable unambiguous identification of natural T cell Ags. Synthetic peptide libraries were screened with Mycobacterium tuberculosis-reactive and -autoreactive T cell clones. In two cases, database homology searches with mimicry epitopes isolated from a dedicated synthetic peptide library allowed immediate identification of the natural antigenic protein. In two other cases, an amino acid pattern that reflected the epitope requirements of the T cell was determined by substitution and omission mixture analysis. Subsequently, the natural Ag was identified from databases using this refined pattern. This approach opens new perspectives for rapid and reliable Ag definition, representing a feasible alternative to the biochemical and genetic approaches described thus far.     

Epitope repertoire of human CD4+ T cells on tetanus toxin: identification of immunodominant sequence segments

Sequence regions of tetanus toxin-forming CD4+ cell epitopes in 8 HLA-disparate subjects were identified. Overlapping synthetic peptides corresponding to the complete tetanus toxin sequence were used to test, in a proliferation assay, unselected blood CD4+ cells or CD4+ cell lines propagated by stimulation with tetanus toxoid. The CD4+ cell lines recognized most peptides recognized by the blood CD4+ cells and they recognized additional peptides. Their responses were stronger than those of unselected blood CD4+ cells. Two peptides were recognized by all subjects: one largely overlapped a tetanus toxin sequence region previously identified as a "universal" T cell epitope. Thirteen other peptides elicited a CD4+ cell response in 6 or 7 of the 8 subjects, and another 10 elicited responses in 5 subjects.     

Comparison of primary sensitization of naive human T cells to varicella-zoster virus peptides by dendritic cells in vitro with responses elicited in vivo by varicella vaccination

Dendritic cells (DC) are potent APC during primary and secondary immune responses. The first objective of this study was to determine whether human DC mediate in vitro sensitization of naive CD4+ T cells to epitopes of the immediate early 62 (IE62) protein of varicella zoster virus (VZV). The induction of CD4+ T cell proliferative responses to eight synthetic peptides representing amino acid sequences of the VZV IE62 protein was assessed using T cells and DC from VZV-susceptible donors. The second objective was to compare in vitro responses of naive T cells with responses to VZV peptides induced in vivo after immunization with varicella vaccine. T cell proliferation was induced by three peptides, P1, P4, and P7, in 71-100% of the donors tested before and after vaccination using DC as APC. Monocytes were effective APC for VZV peptides only after immunization. Two peptides, P2 and P8, induced naive T cell proliferation less effectively and were also less immunogenic for T cells from vaccinated or naturally immune donors. T cell recognition of specific peptides was concordant between naive, DC-mediated responses, and postvaccine responses using monocytes as APC in 69% of comparisons (p = 0.05; chi2); the predictive value of a positive response to an IE62 peptide before immunization for T cell sensitization in vivo was 82%. These observations indicate that primary T cell responses detected in vitro using DC as APC may be useful to characterize the potential immunogenicity of viral protein epitopes in vivo.     

Vaccine-induced, pseudorabies virus-specific, extrathymic CD4+CD8+ memory T-helper cells in swine

Pseudorabies virus (PRV; suid herpesvirus 1) infection causes heavy economic losses in the pig industry. Therefore, vaccination with live attenuated viruses is practiced in many countries. This vaccination was demonstrated to induce extrathymic virus-specific memory CD4+CD8+ T lymphocytes. Due to their major histocompatibility complex (MHC) class II-restricted proliferation, it is generally believed that these T lymphocytes function as memory T-helper cells. To directly prove this hypothesis, 15-amino-acid, overlapping peptides of the viral glycoprotein gC were used for screening in proliferation assays with peripheral blood mononuclear cells of vaccinated d/d haplotype inbred pigs. In these experiments, two naturally processed T-cell epitopes (T1 and T2) which are MHC class II restricted were identified. It was shown that extrathymic CD4+CD8+ T cells are the T-lymphocyte subpopulation that responds to epitope T2. In addition, we were able to show that cytokine secretion can be induced in these T cells through recall with inactivated PRV and demonstrated that activated PRV-primed CD4+CD8+ T cells are able to induce PRV-specific immunoglobulin synthesis by PRV-primed, resting B cells. Taken together, these results demonstrate that the glycoprotein gC takes part in the priming of humoral anti-PRV memory responses. The experiments identified the first T-cell epitopes so far known to induce the generation of virus-specific CD4+CD8+ memory T lymphocytes and showed that CD4+CD8+ T cells are memory T-helper cells. Therefore, this study describes the generation of virus-specific CD4+CD8+ T cells, which is observed during vaccination, as a part of the potent humoral anti-PRV memory response induced by the vaccine.     

Effect of taurine on angiotensin II-induced hypertrophy of neonatal rat cardiac cells

T Lymphocytes (T cells) recognize short antigenic peptides bound to either MHC I or II molecules, in contrast to antibodies which can bind to native antigen. The mechanism by which antigens are processed into peptides, and the nature of the interactions of antigenic peptides with MHC molecules and with the T cell receptor have now been defined in some detail. Of significance to glycobiologists is the recent appreciation that the carbohydrate of glycoprotein antigens can contribute to the T cell recognition of epitopes presented by MHC molecules. Experiments using model T cell epitopes have demonstrated that carbohydrate can modulate T cell responses in a variety of ways; for example, there are a number of cases where glycopeptide-specific T cell responses have been identified. Many of these glycopeptide-specific T cell responses involve a peptide bearing a single glycosyl residue, thus it appears very likely that both glycan and peptide make contact with the T cell receptor binding site. Significantly, glycopeptide-specific T cell responses have also been detected to native glycoproteins. The ability of carbohydrate to influence T cell recognition of antigen has important consequences for a wide range of immune responses as well as the current strategies for mapping T cell determinants.     

Dendritic cells, interleukin 12, and CD4+ lymphocytes in the initiation of class I-restricted reactivity to a tumor/self peptide

Cell-mediated immunity involving CD8+ lymphocytes is effective in mediating rejection of murine mastocytoma cells bearing P815AB, a tumor-associated and self antigen showing similarity to tumor-specific shared antigens in humans. Although this antigen may act as an efficient target for class I-restricted responses in immunized mice, neither P815AB expressed on tumor cells nor a related synthetic nonapeptide will activate unprimed CD8+ cells for in vivo reactivity, measured by skin test assay. We review evidence showing that the failure of P815AB to initiate CD8+ cell reactivity may be due to defective recruitment of accessory and Th1-like cells to the afferent phase of the response initiated by transfer of mice with dendritic cells pulsed in vitro with the P815AB peptide. Although the copresence of a T helper peptide in dendritic cell priming in vitro with P815AB may compensate for the poor generation of accessory and Th1 cells in the adoptively transferred mice, recombinant IL-12 can replace the helper peptide in both effects. Effective priming to P815AB in vivo is achieved by either exposing dendritic cells to IL-12 prior to P815AB priming or administering the recombinant cytokine in vivo. Different approaches suggest that IL-12 may act both on accessory cells to improve presentation of previously undescribed class II-restricted epitopes of P815AB and on CD4+ cells to improve recognition of such epitopes. In particular, at the CD4+ cell level, IL-12 apparently acts as an adjuvant and an inhibitor of anergy induction. These data offer useful information for developing vaccination strategies using dendritic cells and class I-restricted tumor peptides in humans.     

Specific T helper cell requirement for optimal induction of cytotoxic T lymphocytes against major histocompatibility complex class II negative tumors

This study shows that induction of tumor-specific CD4+ T cells by vaccination with a specific viral T helper epitope, contained within a synthetic peptide, results in protective immunity against major histocompatibility complex (MHC) class II negative, virus-induced tumor cells. Protection was also induced against sarcoma induction by acutely transforming retrovirus. In contrast, no protective immunity was induced by vaccination with an unrelated T helper epitope. By cytokine pattern analysis, the induced CD4+ T cells were of the T helper cell 1 type. The peptide-specific CD4+ T cells did not directly recognize the tumor cells, indicating involvement of cross-priming by tumor-associated antigen-presenting cells. The main effector cells responsible for tumor eradication were identified as CD8+ cytotoxic T cells that were found to recognize a recently described immunodominant viral gag-encoded cytotoxic T lymphocyte (CTL) epitope, which is unrelated to the viral env-encoded T helper peptide sequence. Simultaneous vaccination with the tumor-specific T helper and CTL epitopes resulted in strong synergistic protection. These results indicate the crucial role of T helper cells for optimal induction of protective immunity against MHC class II negative tumor cells. Protection is dependent on tumor-specific CTLs in this model system and requires cross-priming of tumor antigens by specialized antigen-presenting cells. Thus, tumor-specific T helper epitopes have to be included in the design of epitope-based vaccines.     

Differential antigen recognition by T cells from the spleen and central nervous system of coronavirus-infected mice

CD8+ cytotoxic T lymphocytes (CTLs) isolated from the central nervous system (CNS) of C57BI/6 mice acutely infected with mouse hepatitis virus, strain JHM (MHV-JHM), and analyzed in a direct ex vivo cytotoxicity assay recognize two epitopes (H-2Db- and H-2Kb-restricted encompassing amino acids 510-518 and 598-605, respectively) within the surface (S) glycoprotein. In contrast, CD8+ T cells isolated from the spleens of mice inoculated intraperitoneally with MHV-JHM and restimulated in vitro only respond to the H-2Db-restricted epitope. In this report, the preferential recognition of the H-2Db-restricted epitope is confirmed using splenocytes stimulated in vitro with either MHV-JHM-infected MC57 cells or with a cell line expressing the S protein and analyzed in secondary CTL assays. To determine whether these results represent a difference in epitope recognition between the spleen and CNS, secondary CTL assays were performed using spleen cells coated with peptides encompassing the CTL epitopes as stimulators. Under these conditions, both epitopes sensitized cells for lysis by spleen-derived CTLs, suggesting that both epitopes were recognized by splenic CD8+ T cells after infection in vivo. Furthermore, limiting dilution analysis indicated that the precursor frequency of splenic CD8+ T cells specific for both the H-2Kb- and H-2Db-restricted epitopes were not significantly different. Thus, the results suggest that in vitro stimulation of splenocytes specific for the H-2Kb-restricted epitope is inefficient after endogenous processing but that this inefficiency can be corrected if peptide is provided exogenously at sufficiently high concentrations. As a consequence, the results also show that cells responsive to both of the previously identified CNS-derived CD8+ T cell epitopes are present in the infected spleen at nearly the same frequency.     

Proliferative and cytokine responses to class II HER-2/neu-associated peptides in breast cancer patients

Previous studies have characterized the reactivity of CD8+ CTLs with ovarian and breast cancer. There is little information about the antigens and epitopes recognized by CD4+ T cells in these patients. In this study, we analyzed the ability of T cells from peripheral blood mononuclear cells of breast cancer patients to recognize HER-2/neu (HER-2) peptides. We found that 13 of 18 patients responded by proliferation to at least one of the HER-2 peptides tested. Of these peptides, one designated G89 (HER-2: 777-789) was recognized by T cells from 10 patients. Seven of nine responding patients were HLA-DR4+, suggesting that this peptide is recognized preferentially in association with HLA-DR4. Analysis of the specificity and restriction of the cytokine responses to G89 by G89-stimulated T cells revealed that these cells secreted significantly higher levels of IFN-gamma than interleukin 4 and interleukin 10, suggesting priming for a Th0-T helper 1 response. The same pattern of cytokine responses was observed to the intracellular domain of HER-2 protein, suggesting that G89-stimulated T cells recognized epitopes of the HER-2 protein in association with HLA-DR4. Because HLA-DR4 is present in 25% of humans, characterization of MHC class II-restricted epitopes inducing Th0-T helper 1 responses may provide a basis for the development of multivalent HER-2-based vaccines against breast and ovarian cancer.     

Major histocompatibility complex class I-restricted T cells are required for all but the end stages of diabetes development in nonobese diabetic mice and use a prevalent T cell receptor alpha chain gene rearrangement

Nonobese diabetic (NOD) mice develop insulin-dependent diabetes mellitus due to autoimmune T lymphocyte-mediated destruction of pancreatic beta cells. Although both major histocompatibility complex class I-restricted CD8(+) and class II-restricted CD4(+) T cell subsets are required, the specific role each subset plays in the pathogenic process is still unclear. Here we show that class I-dependent T cells are required for all but the terminal stages of autoimmune diabetes development. To characterize the diabetogenic CD8(+) T cells responsible, we isolated and propagated in vitro CD8(+) T cells from the earliest insulitic lesions of NOD mice. They were cytotoxic to NOD islet cells, restricted to H-2Kd, and showed a diverse T cell receptor beta chain repertoire. In contrast, their alpha chain repertoire was more restricted, with a recurrent amino acid sequence motif in the complementarity-determining region 3 loop and a prevalence of Valpha17 family members frequently joined to the Jalpha42 gene segment. These results suggest that a number of the CD8(+) T cells participating in the initial phase of autoimmune beta cell destruction recognize a common structural component of Kd/peptide complexes on pancreatic beta cells, possibly a single peptide.     

Selective activation of CD8 T cell effector functions by epitope variants of lymphocytic choriomeningitis virus glycoprotein

We provide evidence for selective activation of different effector functions of CD8+ T lymphocytes by altered peptide ligands. A T cell epitope from the glycoprotein of lymphocytic choriomeningitis virus (p33-41) and single amino acid variants thereof were used for primary in vitro induction of CTL clones. When the CTL were analyzed for cytotoxicity, proliferation, IFN-gamma production, and Ca2+ mobilization, we found that some of the clones showed activation of only their cytotoxic effector function when stimulated with variants of their inducing peptides. For one clone, cytotoxic reactivity was readily detected to the inducing peptide and three of four variants, but only the former was also able to trigger proliferation, IFN-gamma production, and Ca2+ mobilization. Another clone also revealed this dichotomy, but in this case some of the altered peptide ligands in addition to the inducing peptide were able to stimulate the full spectrum of effector functions, whereas others only stimulated cytotoxicity. A third clone revealed inefficient triggering of some effector functions by the peptide variants. Our data suggest that, as described for CD4 T cells, altered peptide ligands may lead to partial activation of effector functions of CD8 T cells. In addition, ligands with glycine substitutions in potential TCR contact positions induced CTL, which were able to recognize peptides with a variety of amino acids in the former glycine position.     

Antigen processing by proteasomes: insights into the molecular basis of crypticity

Eight to eleven amino acid residues are the sizes of predominant peptides found to be associated with MHC class I molecules. Proteasomes have been implicated in antigen processing and generation of such peptides. Advanced methodologies in peptide elution together with sequence determination have led to the characterisation of MHC class I binding motifs. More recently, screening of random peptide phage display libraries and synthetic combinatorial peptide libraries have also been successfully used. This has led to the development and use of predictive algorithms to screen antigens for potential CTL epitopes. Not all predicted epitopes will be generated in vivo and the emerging picture suggests differential presentation of predicted CTL epitopes ranging from cryptic to immunodominant. The scope of this review is to discuss antigen processing by proteasomes, and to put forward a hypothesis that the molecular basis of immunogenicity can be a function of proteasomal processing. This may explain how pathogens and tumours are able to escape immunosurveillance by altering sequences required by proteasomes for epitope generation.     

Spontaneous autoimmune diabetes in monoclonal T cell nonobese diabetic mice

It has been established that insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice results from a CD4+ and CD8+ T cell-dependent autoimmune process directed against the pancreatic beta cells. The precise roles that beta cell-reactive CD8+ and CD4+ T cells play in the disease process, however, remain ill defined. Here we have investigated whether naive beta cell-specific CD8+ and CD4+ T cells can spontaneously accumulate in pancreatic islets, differentiate into effector cells, and destroy beta cells in the absence of other T cell specificities. This was done by introducing Kd- or I-Ag7-restricted beta cell-specific T cell receptor (TCR) transgenes that are highly diabetogenic in NOD mice (8.3- and 4.1-TCR, respectively), into recombination-activating gene (RAG)-2-deficient NOD mice, which cannot rearrange endogenous TCR genes and thus bear monoclonal TCR repertoires. We show that while RAG-2(-/-) 4.1-NOD mice, which only bear beta cell-specific CD4+ T cells, develop diabetes as early and as frequently as RAG-2+ 4.1-NOD mice, RAG-2(-/-) 8.3-NOD mice, which only bear beta cell-specific CD8+ T cells, develop diabetes less frequently and significantly later than RAG-2(+) 8.3-NOD mice. The monoclonal CD8+ T cells of RAG-2(-/-) 8.3-NOD mice mature properly, proliferate vigorously in response to antigenic stimulation in vitro, and can differentiate into beta cell-cytotoxic T cells in vivo, but do not efficiently accumulate in islets in the absence of a CD4+ T cell-derived signal, which can be provided by splenic CD4+ T cells from nontransgenic NOD mice. These results demonstrate that naive beta cell- specific CD8+ and CD4+ T cells can trigger diabetes in the absence of other T or B cell specificities, but suggest that efficient recruitment of naive diabetogenic beta cell-reactive CD8+ T cells to islets requires the assistance of beta cell-reactive CD4+ T cells.     

Identification of new melanoma epitopes on melanosomal proteins recognized by tumor infiltrating T lymphocytes restricted by HLA-A1, -A2, and -A3 alleles

To isolate melanoma Ags recognized by T cells, cDNA libraries made from melanoma cell lines were screened with four CTLs derived from tumor infiltrating lymphocytes (TIL) that were able to recognize melanoma cells in a HLA-A1, -A2, or -A3 restricted manner. Although cDNAs encoding the previously identified melanoma Ags, tyrosinase and gp100, were isolated, these TIL were found to recognize previously unidentified peptides. An HLA-A1-restricted CTL, TIL1388, was found to recognize a tyrosinase peptide (SSDYVIPIGTY), and an HLA-A3-restricted CTL, TIL1351, recognized a gp100 peptide (LIYRRRLMK). CTL clones isolated from the HLA-A2-restricted TIL1383 recognized a gp100 peptide (RLMKQDFSV). HLA-A2-restricted CTL, TIL1200, recognized a gp100 peptide (RLPRIFCSC). Replacement of either cysteine residue with alpha-amino butyric acid in the gp100 peptide, RLPRIFCSC, enhanced CTL recognition, suggesting that the peptide epitope naturally presented on the tumor cell surface may contain reduced cysteine residues. Oxidation of these cysteines might have occurred during the course of the synthesis or pulsing of the peptide in culture. These modifications may have important implications for the development of efficient peptide-based vaccines. These newly identified peptide epitopes can extend the ability to perform immunotherapy using synthetic peptides to a broader population of patients, especially those expressing HLA-A1 or HLA-A3 for whom only a few melanoma epitopes have previously been identified.