Mapping of V beta 11+ helper T cell epitopes on mycobacterial antigen in mouse primed with Mycobacterium tuberculosis

Antigenic epitopes for Mycobacterium tuberculosis-reactive T cell immune responses have been mapped using the purified Mycobacterium protein antigen. Lymph node cells from C57BL/6 mice that had been immunized with heat-killed M. tuberculosis were cultured with various Mycobacterium protein antigens and their reactivity was monitored by proliferative response. Usage of the TCR beta chain repertoire was analyzed by flow cytometry. Stimulation of M. tuberculosis-primed lymph node cells with MPT59 (antigen 85B, alpha antigen) induced proliferative response, production of IL-2 and IFN-gamma, and the expansion of V beta 11+ CD4+ T cells in conjunction with antigen- presenting cells in an I-Ab-restricted manner. Lymph node cells from non-primed mice failed to proliferate in response to MPT59. Using peptides covering the complete mature 285 amino acids long MPT59 protein as 15-mer molecules overlapping by five amino acids, we identified the antigenic epitope for MPT59-specific V beta 11+ T cells. The 15-mer peptide, covering amino acid residues 240-254 of MPT59 [peptide-25 (amino acids 240-254)], contains the motif that is conserved for I-Ab and requires processing by antigen-presenting cells to trigger peptide-25-specific V beta 11+ CD4+ T cells. We conclude from these results that MPT59 and peptide-25 (amino acids 240-254) are not superantigens and require antigen processing in order to stimulate V beta 11+ Th1 cells. This experimental system will provide us with a useful tool for delineating the regulation of T cell development in a particular subset of M. tuberculosis infection and for developing antigenic peptides for Th1-dominant immune responses.      

Deficient antigen processing of a protein quaternary structure can be overcome by receptor-mediated uptake

Human chorionic gonadotropin (hCG) is a dimer of non-covalently associated alpha (hCG-α) and beta (hCG-β) subunits. This molecule was used to study whether receptor-mediated uptake influences the presentation of a protein quaternary structure. Unprimed splenocytes and a B cell lymphoma were capable of presenting only the free (hCG-α) but not the combined (hCG) α subunit to hCG-α T cell hybridomas, while hCG-α-primed lymph node cells (LNC) responded to both hCG-α and hCG. As antigen (Ag)-specific antigen-presenting cells (APC) present in the hCG-α-primed LNC population may be potentially effective for presenting hCG, we investigated the role of specific Ag capture, through mIg and FcγR, in the processing and presentation of hCG and hCG-a to HAG 5, a T cell hybridoma directed against the immunodominant region (amino acids 61-81) of hCG-α. Results showed that only B cells bearing membrane immunoglobulin capable of recognizing hCG-α and hCG, and present in hCG-α-primed mice, were extremely effective in presenting the free as well as the combined a subunit. The effect of FcR-mediated uptake was analyzed using a B cell line transfected with the FcγRII-B2 gene to present immune complexes of either hCG-α or hCG. We found that hCG-α and hCG were presented equally well, whatever the Ag-binding site of each antibody to hCG or its a subunit. Using HBG 6, an hCG-β Tcell hybridoma, we performed similar experiments with the FcγRII-B2 cell line and determined that the potentiation of hCG presentation to HBG 6 was similar to that observed with HAG 5. Then kinetic experiments were performed to examine the effect of Ag uptake through FcR on processing. Results demonstrated that the uptake pathway drastically influenced the expression of α T cell determinants in the αβ dimer. In addition, treatment with cycloheximide, a protein synthesis inhibitor, only impaired the ability of APC to present specifically captured Ag. Thus, the processing pathway for specifically captured Ag might be different from the pathway used to process nonspecifically captured Ag. This observation might explain why receptor-enhanced uptake bypasses the inefficient processing of the hCG quaternary structure and enables similar efficiency in the presentation of α and β T cell specificities. These findings provide new insight into the antigenicity of oligomeric molecules, which is modified whether antigen capture is specific or not.     

Identification of an immunodominant T cell epitope on cholera toxin

Cholera toxin (CT), the enterotoxin of Vibrio cholerae, is a potent mucosal immunogen as well as a strong mucosal adjuvant to related and unrelated antigens. The mucosal immune response to CT is T cell dependent and MHC class II restricted. The epitopes on CT recognized by T cells have not been identified. The purpose of this study was to determine the fine specificity of T cell recognition of both the CT A subunit (CT-A) and the CT B subunit (CT-B) by using a range of synthetic peptides. After immunization with CT-B or CT-A in CFA subcutaneously, the peripheral lymph node T cells were stimulated with different synthetic peptides in vitro The peptide specificity of T cell recognition was identified by assaying T cell proliferation and interleukin-3 production. T cells from C57BL/6 (H-2^b) high responder mice recognized one immunodominant epitope (peptide 89–100) and one weak epitope (peptide 31–50) on CT-B and two epitopes (peptide 21–39 and 180–194) on CT-A. The immunization of C57BL/6 mice with synthetic immunodominant CT-B peptide 89–100 induced T cell immunity to the pentameric CT-B. Induction of tolerance to CTB peptide 89–100 by i.v. injection in high responder C57BL/6 mice induced unresponsiveness to mucosal immunization with CT, compatible with an immunodominant role for this T cell epitope.     

Identification of minimal CD8+ and CD4+ T cell epitopes in the Plasmodium yoelii hepatocyte erythrocyte protein 17kDa

Immunization of mice with subunit vaccines based on the Plasmodium yoelii 17kDa hepatocyte erythrocyte protein (PyHEP17), orthologue of Plasmodium falciparum exported protein 1 (PfExp1), induces antigen-specific immune responses and protects against sporozoite challenge. To aid in the characterization of candidate subunit vaccines based on this antigen, we have mapped the immunodominant and subdominant CD8+ and CD4+ T cell epitopes on PyHEP17. Using a panel of 29 15-mer synthetic peptides representing the complete sequence of PyHEP17 (amino acids 1-153), and overlapping each other by 10 residues, we identified an immunogenic region between amino acids 61-85. To define the minimal CD4+ and CD8+ T cell epitopes within this region, we synthesized 25 9-mer peptides overlapping each other by one residue. We screened the capacity of the 15-mer and 9-mer peptides to be recognized by splenocytes and lymph node cells from mice immunized with PyHEP17 plasmid DNA or peptides in Freund's adjuvant, as assessed by cytokine secretion, lymphoproliferation, and cytotoxicity. The profile of response to the T cell epitopes varied depending upon the immunization regimen. Antigen-specific T cell responses were detected to three 15-mer peptides (residues 61-75, 66-80 and 71-85) representing two 10-mer epitopes mapping to residues 66-75 (LTKNKKSLRK) and 71-80 (KSLRKINVAL). IFN-gamma responses after DNA immunization predominantly mapped to two overlapping 9-mer peptides (residues 73-81 and 74-82) sharing an eight amino acid overlap (residues 74-81, RKINVALA), whereas CTL responses predominantly mapped to four 9-mer peptides (residues 61-69, 70-78, 76-84, and 84-92). In addition, a subdominant 10-mer CD8+ T cell epitope recognized by peptide immunization but not DNA immunization mapped to residues 31-40 (GKYGSQNVIK). The identification of these epitopes will allow the evaluation of delivery systems for malaria vaccine candidates as well as the delineation of protective immune mechanisms.     

T cell reactivity of defined peptides from a major Plasmodium falciparum vaccine candidate: the Pf155/RESA antigen

Several immunodominant B-cell epitopes of the P. falciparum antigen blood stage Pf155/RESA, a major vaccine candidate antigen, are located in the molecular regions containing amino acid repeats. We started to map Pf155/RESA for T cell reactive epitopes. For this purpose, short synthetic peptides corresponding to the 3'- and 5' repeat regions of the molecule as well as to non-repeated sequences outside these regions were prepared. T cells from P. falciparum primed donors from two highly endemic areas of Africa were tested for their responsiveness to the peptides by thymidine incorporation and/or interferon gamma (IFN-gamma) release. There was a considerable variation in the response to the different peptides. However, the strongest and most frequent responses were seen with a few peptides from the 3'- and 5'-repeat regions. Thus, the immunodominant B cell epitope regions of Pf155/RESA, contain several T cell epitopes. Since the repeat regions are known to be conserved in different P. falciparum strains, the T cell epitopes reported here may be suitable constituents of a P. falciparum subunit vaccine.     

Inhibition of T-cell responses by feeding peptides containing major and cryptic epitopes: studies with the Der p I allergen.

H-2b mice respond to the 222 residue allergen Der p I by producing T cells sensitized to the dominant epitopes encompassed in peptides 21-49, 78-100, 110-131 and 197-212. Immunization with the synthetic peptides 120-143 and 144-169, however, revealed cryptic epitopes which could sensitize T cells for responses to the respective peptides and, providing splenic adherent cells were added to lymph node cultures, to the whole allergen. It is shown that feeding recombinant fusion peptides can markedly inhibit the ability of the whole antigen to immunize mice, as measured by the in vitro interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-3 release on stimulation with protein or peptides, although inhibition measured by IL-2 release was more marked. The inhibition extended to epitopes other than those in the fusion peptides used for feeding. Thus feeding peptide 101-154 inhibited responses to 110-131 and 78-100. Fusion peptides 1-14 and 188-222 did not inhibit responses, although 188-222 did contain an epitope. Inhibition was also obtained when mice were fed a fusion containing the cryptic epitope 144-169. The ability of peptides containing the cryptic epitopes to inhibit responses has significant implications for peptide-based immunotherapy.     

Tolerance and autoimmunity to a gastritogenic peptide in TCR transgenic mice

The catalytic alpha and glycoprotein beta subunits of the gastric H/K ATPase are major molecular targets in human and mouse autoimmune gastritis. We have previously shown that the H/K ATPase beta subunit is required for the initiation of mouse gastritis and identified a gastritogenic H/K ATPase beta subunit peptide (H/Kbeta253-277). Here we report the generation of MHC class II-restricted TCR transgenic mice using V(alpha)9 and V(beta)8.3 TCR chains with specificity for the gastritogenic H/Kbeta253-277 peptide. We found an 8-fold reduction in CD4(+) T cells in the thymus of the transgenic mice. Despite the reduction in intrathymic CD4(+) T cells, V(beta)8. 3-expressing T cells comprised the majority (>90%) of peripheral spleen and lymph node T cells. These peripheral T cells retained their capacity to proliferate in vitro to the H/Kbeta253-277 peptide. Using the responsive T cells, we have restricted the gastritogenic T cell epitope to H/Kbeta261-274. Despite the capacity of the peripheral T cells to proliferate in vitro to the peptide, the majority ( approximately 80%, 13 of 16) of transgenic mice remained free of gastritis while a minority (20%, three of 16) spontaneously developed an invasive and destructive gastritis. Our results confirm that H/Kbeta261-274 is a gastritogenic peptide. The data also suggest that CD4 T cell tolerance to the gastritogenic peptide in the transgenic mice is maintained by a combination of intrathymic and peripheral tolerance mechanisms.     

The autoimmune response of different mouse strains to T-cell epitopes of the human acetylcholine receptor alpha subunit.

The specific recognition of the acetylcholine receptor and its alpha-subunit by T cells derived from patients with myasthenia gravis or mice with experimental autoimmune myasthenia gravis raises the question of the role of autoreactive T cells in the myasthenic process. Sequences of the acetylcholine receptor alpha-subunit previously shown to be immunogenic in myasthenic patients were tested for their immunogenicity in various inbred mouse strains. High, intermediate and low T-cell proliferative responses could be observed to peptides representing sequences 195-212 and 259-271 of the human acetylcholine receptor alpha-subunit. Following immunization with the Torpedo acetylcholine receptor, lymphocytes of SJL mice proliferated efficiently to p 195-212 but not to p259-271. On the other hand, lymph node cells of BALB/c mice responded well to p259-271 but not to p195-212. Thus, the influence of the genetic make-up of the examined mice on the immune response to the two peptides could be clearly demonstrated by the existence of strain-dependent immunodominant and cryptic regions on the autoantigen. The differences between the strains were less pronounced when antibody responses were measured to these two T-cell epitopes, although a partial correlation with the proliferative responses could be observed. It can be concluded that epitopes specifically recognized by T lymphocytes of patients with myasthenia gravis also represent specific T-cell epitopes in the autoreactivity to the acetylcholine receptor in mice and that immune responsiveness to these peptides is influenced by the genetic make-up of the responding mouse strains.     

The immune response to a synthetic peptide analogous to the 109-145 beta hCG carboxyl-terminus is directed against two major and two minor regions

The immune response to a 37-amino acid synthetic peptide analogous to the carboxyl-terminal part (109-145) of the human chorionic gonadotropin beta subunit (beta hCG) was studied with monoclonal antibodies selected from 31 cell fusion experiments. Analysis of the immunogenic determinants borne on the synthetic peptide (CTP) showed a prevailing response to two immunodominant regions. The first was located on the 110-116 amino acid sequence of the CTP which is also the most hydrophilic region: 50% of anti-CTP antibodies selected for their high binding to 125I beta hCG were directed to this sequence. A second immunodominant portion was recognized by four antibodies, and comprised amino acids 134 to 139, representing a highly O-glycosylated region on the native protein. Moreover, a unique antibody designated FB13 bound to a region located on the last seven amino acids (139-145) of beta hCG. Finally, a hypothetical conformational determinant was recognized by antibody FB02 within the 121-145 region. Thus, the immune response to CTP was directed against two major and two minor regions. These antigenic determinants were demonstrated to be accessible for antibody binding on both the hCG molecule and its beta subunit. Localization of these epitopes suggests a relationship between the hydrophilicity and the immunological potency of different CTP regions.     

Recognition of peptide epitopes of the 16,000 MW antigen of Mycobacterium tuberculosis by murine T cells.

The T-cell repertoire to a prominent immunogen of Mycobacterium tuberculosis has been investigated on the assumption that differences in epitope specificity could influence the protective and pathogenic host reactions. Proliferative responses of lymph node and spleen cells to overlapping peptides, spanning the entire sequence of the 16,000 MW protein antigen were analysed in C57BL/10 and B10.BR mice. Following footpad priming and in vitro challenge with homologous peptide, 12 out of the 14 peptides tested were found to be immunogenic. However, only two peptides of residues 31-40 and 71-91 stimulated strong proliferative responses of T cells from mice which had been presensitized with either killed or live M. tuberculosis organisms; another three peptides were only weakly stimulatory. These epitopes have been immunodominant in both H-2b and H-2k mouse strains, indicating the genetically permissive nature of their recognition. Furthermore, both major immunodominant epitopes were found to be species specific for the M. tuberculosis complex and therefore potentially suitable for the early diagnosis of tuberculous infection.     

人绒毛膜促性腺激素β亚基的T细胞表位

本文根据人绒毛膜促性腺激素 β亚基 (β hCG )序列用多针同步固相多肽合成技术合成了一系列短肽 ,用T细胞增殖法进行T细胞表位的筛选。结果表明 ,合成的肽纯度高 ,结构正确。完全满足细胞表位筛选的要求。β hCG (19 30 )、 (37 48)、 (85 96 )、 (136 145 )段肽与对照相比有显著性差异 ,为免疫活性较高的区域。     

Characterization of a helper T cell epitope recognized by mice of a low responder major histocompatibility type.

Most known helper T cell (Th) epitopes studied have naturally been immunodominant epitopes recognized by T cells from animals of high responder major histocompatibility complex (MHC) haplotype. We have previously found that most such immunodominant Th epitopes tend to be amphipathic alpha helices, that is, helices with hydrophobic residues on one side and hydrophilic residues on the other, and the corresponding peptide can usually elicit a response to the native protein. However, very few epitopes seen by MHC low responder T cells have been identified. Within the CNBr fragment of residues 1-55 of sperm whale myoglobin (SwMb), a Th epitope is known to exist that stimulates T cells from low responder H-2k mice, but it has not yet been localized to a length of 8-12 residues, the usual length of a Th epitope. To determine whether this low responder epitope would have similar properties, we located it using 10 evenly overlapping 15-residue peptides that span the region. Analysis of this region by the computer program predicted the site covered by two peptides (residues 26-40 and 31-45 which overlap by 10 residues) to be the most likely site for a Th epitope. Of the 10 peptides tested experimentally, only one peptide (residues 26-40) was able to stimulate two low responder Th clones that are specific for the 1-55 region. The peptide was able to prime T cells of low responder B10.BR mice in vivo for in vitro response to the native SwMb as well as to the peptide fragment of residues 1-55. Immunization of low responder mice with SwMb showed that, of the 10 overlapping peptides, the major site of response within the 1-55 region is to the identified peptide. Finally, an extended peptide of residues 24-42 was made to increase the amphipathic score. This extended peptide induced greater proliferation of the clones. Thus, this low responder epitope has properties similar to those of immunodominant epitopes recognized by high responders.     

Differential thymus dependence of rat CD8 isoform expression.

Expression of the rat CD8 molecule was studied using five novel monoclonal antibodies (mAb), four of which are specific for the V-like domain of CD8 alpha, whereas one reacts either with the beta chain or with a determinant only expressed on the CD8 alpha/beta heterodimer. mAb to both chains effectively blocked purified lymph node CD8 T cells in mixed lymphocyte reaction and in cell-mediated cytotoxicity. Flow cytometric analysis showed that CD8 T cells from lymph nodes or spleen of normal rats almost exclusively express the alpha/beta isoform, regardless of the T cell receptor isotype (alpha/beta or gamma/delta). In contrast, natural killer (NK) cells carry only CD8 alpha chains. This CD8 alpha + beta - phenotype was also prominent among CD8 T cells from athymic rats and from intestinal epithelium of normal rats. CD8 alpha homodimers can also be expressed as a result of activation, as shown by analysis of CD4 CD8 double-positive T cells obtained from highly purified lymph node CD4 T cells by in vitrok stimulation. Such CD4+CD8 alpha + beta - cells also represent a major subset among adult intestinal intraepithelial lymphocytes (IEL), suggesting local activation. Taken together, the difference in CD8 isoform expression among T cells from athymic rats, NK cells, and gut IEL versus CD8 T cells from peripheral lymphatic organs of euthymic animals suggests that like in mice, expression of the CD8 heterodimer is more dependent on intrathymic maturation than that of the homodimer. Since the more stringent thymus dependence of CD8 alpha + beta + T cells may be due to a requirement for thymic selection on self major histocompatibility complex class I antigens, the virtually exclusive CD8 alpha + beta + phenotype of peripheral rat gamma/delta T cells could mean that antigen recognition by this subset is also restricted by MHC class I molecules.     

Analysis of Type II Collagen Reactive T Cells in the Mouse

The specificity of the recognition of type II collagen (CII) by T cells in the DBA/l mouse was analysed using fragments of chick and rat CII obtained by cyanogen bromide (CB) cleavage. Firstly, DBA/l mice were immunized with chick CB fragments 5, 8, 9, 10, 11 and 12. Ten days later the draining lymph node cells were cultured with rat and mouse CII and the proliferative response was determined by incorporation of [3H]thymidine. All peptides were capable of triggering T cells recognizing rat CII but only CB9 immunized mice responded well to mouse CII. Secondly, lymph node cells from DBA/l mice immunized with rat and mouse CII were cultured with the CB fragments, including rat CB10 and CB11, and the proliferative response was determined. After immunization with rat CII, the response was strongly dominated by T cells recognizing CB11 with equal responses against chick and rat CB11. After immunization with mouse CII only rat CB10 gave a strong response. It is concluded that several epitopes on the CII molecule can be recognized by T cells in the DBA/l mouse and that most of these epitopes are shared by rat and chick CII but not mouse CII. These epitopes exhibit strong immunodominance. In mice immunized with intact heterologous CII, the immunodominant response is directed against one or more epitopes on the CB11 fragment present on several heterologous CII but apparently not on mouse CII. In mice immunized with autologous CII the immunodominant response is directed against one or more epitopes on the CB10 fragment, present on rat and mouse CII. They are either absent in chick CII or located in the carboxyterminal end of the CB10 fragment where a cyanogen bromide cleavage site is present in chick CII but not in rat CII. These results suggest that the proposed importance of CB11 in collagen-induced arthritis is due to activation of T cells reactive with heterologous CII only. These cells may be important for the induction of the strong auto-antibody-response after immunization with heterologous CII. Structures of importance for direct T cell involvement in the arthritic process and recognized by autoreactive T cells are suggested to be found on CB10.     

Characterization of T-cell responses to the house dust mite allergen Der p II in mice. Evidence for major and cryptic epitopes.

Major histocompatibility complex (MHC) congenic strains can be defined as high and low responders to the major house dust mite allergen Der p II on the basis of the ability to sensitize T cells for in vitro lymphokine release. Mice of the H-2b haplotype were high responders, H-2k were intermediate and H-2d low responders. Like responses to other proteins, only a limited number of epitopes could be located by the response of T cells from mice immunized with allergen to a series of overlapping peptides. The epitopes for H-2b mice were 11-35, 78-104 and 105-129, 36-50 and 78-104 for H-2k mice and 36-60 for H-2d. Immunization with the peptides however revealed that spleen-adherent cells were required for lymph node cells to recall responses to the whole protein and in addition that mice could be sensitized by cryptic epitopes defined by peptides 22-50 and 1-20 for H-2b mice. Peptides containing these cryptic epitopes did not normally induce responses in mice primed with the allergen, but when they were used for immunizing they could prime mice for responses to the peptide and the whole allergen. The results both help to define a model for studying the presentation of allergens and have significant implications for peptide-based immunotherapy.     

Expression and regulation of beta 7(beta p) integrins on mouse lymphocytes: relevance to the mucosal immune system

A mouse lymphocyte surface molecule which is selectively expressed by mucosal T cells and detected with the monoclonal antibody (mAb) M290 has provisionally been identified as a beta 7 integrin. Identification was based on close homology of its beta subunit at the N terminus with the recently reported, highly distinctive, human beta 7 sequence. mAb were prepared against the beta and alpha subunits of the mouse molecule, termed beta 7 and alpha M290, respectively, and used to study surface expression of the two components. beta 7 was present on most lymph node lymphocytes in association with alpha 4 rather than alpha M290. This integrin, beta 7 alpha 4, was shown to be identical to LPAM-1 (beta p alpha 4) the Peyer's patch homing receptor. Stimulation in vitro of mouse lymph node T cells with anti-CD3 in the presence of transforming growth factor (TGF)-beta increased beta 7 expression in about 40% of cells and changed the associated alpha chain from alpha 4 to the novel alpha M290 subunit, which, in most cells, was expressed de novo. Immunoprecipitation of beta 7 both from these cells and from intraepithelial lymphocytes gave closely similar results and showed predominance of the beta 7 alpha M290 integrin. It is suggested that in vivo this change in alpha-chain usage occurs in mucosal T cells in response to TGF-beta acting in the mucosal microenvironment and that the new integrin confers particular adhesive properties, possibly homing specificity, on the cells.     

The proteasome regulator PA28alpha/beta can enhance antigen presentation without affecting 20S proteasome subunit composition

PA28alpha/beta is a regulatory complex of the 20S proteasome which consists of two IFN-gamma inducible subunits. Both subunits, alpha and beta, contribute equally to the formation of hexa- or heptameric rings which can associate with the 20S proteasome. Previously, we have shown that overexpression of the PA28alpha subunit enhanced the MHC class I-restricted presentation of two viral epitopes and that purified PA28alpha/beta accelerated T cell epitope generation by the 20S proteasome in vitro, indicating a role for PA28alpha/beta in antigen presentation. This conclusion was recently confirmed in PA28beta gene targeted mice which were severely deficient in MHC class I-restricted antigen presentation. These mice displayed a defect in the assembly of immunoproteasomes, suggesting that a lack of the proteasome subunits LMP2, LMP7, and MECL-1 may account for the deficiency in antigen presentation. In this study we investigated whether the effect of PA28alpha/beta on antigen presentation is dependent on a change of proteasome subunit composition. We have analyzed the assembly and subunit composition of proteasomes in fibroblast transfectants overexpressing both, alpha and beta subunits of PA28. In these transfectants we found a marked enhancement in the presentation of the immunodominant H-2Ld-restricted pp89 epitope of murine cytomegalovirus, although the 20S proteasome composition was the same as in recipient cells. We, therefore, conclude that PA28alpha/beta can enhance antigen processing independently of changes in 20S proteasome subunit composition or assembly.     

Increasing the frequency of T-cell precursors specific for a cryptic epitope of hen-egg lysozyme converts it to an immunodominant epitope

Efforts to understand the mechanisms that govern how immunodominant T-cell epitopes are selected from protein antigens have focused mostly on differences in the efficiency of processing and presentation of peptide/major histocompatibility complex (MHC) complexes by antigen-presenting cells, while little attention has been directed at the role of the T-cell repertoire. In this report, the influence of the T-cell repertoire on immunodominance was investigated using transgenic mice that express the beta chain from a T-cell receptor specific for a cryptic Ek restricted epitope of hen-egg lysozyme, HEL85-96. In these mice, the frequency of HEL85-96-specific T-cell precursors is increased 10-20-fold over non-transgenic mice. Transgenic mice respond as well as non-transgenic controls to intact HEL, even though they respond poorly or not at all to a variety of other antigens, including the dominant H-2k restricted epitopes of HEL. Following immunization with native HEL, the only HEL peptide that could recall a response in vitro in the transgenic mice was HEL85-96. Therefore, this normally cryptic epitope is the sole immunodominant epitope in the transgenic mice, and this alteration in immune response is due solely to an increase in the frequency of specific T-cell precursors. An analysis of four additional H-2k restricted cryptic epitopes of HEL suggests that three are similarly limited by T-cell frequency, and that only one is consistent with a defect in efficient antigen presentation. This indicates that there are at least two different types of cryptic epitopes, one in which crypticity is caused by inefficient processing or presentation, and another in which the frequency of specific T-cell progenitors is limiting.     

Distinct but overlapping T helper epitopes in the 37-58 region of SSX-2

Because of their specific expression in tumors of different histological types, the products of the SSX genes are important candidate targets for development of cancer vaccines. We have previously identified two immunodominant SSX-2-derived T cell epitopes recognized by HLA-A2-restricted CD8+ T cells (SSX-2 41-49) and HLA-DR11-restricted CD4+ T cells (SSX-2 45-59), respectively. In this study, we report the identification of an HLA-DR3-restricted epitope mapping to the 37-51 region of SSX-2, overlapping both previously identified epitopes. As about one fifth of individuals from several major ethnic groups express HLA-DR3, the identification of this epitope significantly increases the percent of patients that are expected to mount specific CD4+ T cell responses following vaccination with peptides in this region of SSX-2. Retrieval of multiple overlapping epitopes in a defined region of SSX-2 protein suggests the presence of a "hot spot" for T cell recognition that may prove sufficient for the induction of immune responses.