Identification of new antigenic peptide presented by HLA-Cw7 and encoded by several MAGE genes using dendritic cells transduced with lentiviruses

Antigens encoded by MAGE genes are of particular interest for cancer immunotherapy because they are tumor specific and shared by tumors of different histological types. Several clinical trials are in progress with MAGE peptides, proteins, recombinant poxviruses, and dendritic cells (DC) pulsed with peptides or proteins. The use of gene-modified DC would offer the major advantage of a long-lasting expression of the transgene and a large array of antigenic peptides that fit into the different HLA molecules of the patient. In this study, we tested the ability of gene-modified DC to prime rare Ag-specific T cells, and we identified a new antigenic peptide of clinical interest. CD8(+) T lymphocytes from an individual without cancer were stimulated with monocyte-derived DC, which were infected with a second-generation lentiviral vector encoding MAGE-3. A CTL clone was isolated that recognized peptide EGDCAPEEK presented by HLA-Cw7 molecules, which are expressed by >40% of Caucasians. Interestingly, this new tumor-specific antigenic peptide corresponds to position 212-220 of MAGE-2, -3, -6, and -12. HLA-Cw7 tumor cell lines expressing one of these MAGE genes were lysed by the CTL, indicating that the peptide is efficiently processed in tumor cells and can therefore be used as target for antitumoral vaccination. The risk of tumor escape due to appearance of Ag-loss variants should be reduced by the fact that the peptide is encoded by several MAGE genes.     

Sequence and expression pattern of the human MAGE2 gene

We reported previously identification of the human MAGE1 gene, which encodes an antigen recognized on human melanoma MZ2-MEL by autologous cytolytic T lymphocytes. In addition to MAGE1, melanoma MZ2-MEL expresses several closely related genes, one of which has been named MAGE2. The complete MAGE2 sequence was obtained and it comprises 3 exons homologous to those of MAGE1 and an additional exon homologous to a region of the first MAGE1 intron. Like the open reading frame of MAGE1, that of MAGE2 is entirely encoded by the last exon. The MAGE1 and MAGE2 sequences of this exon show 82% identity and the putative proteins show 67% identity. The MAGE2 gene is expressed in a higher proportion of melanoma tumors than MAGE1. It is also expressed in many small-cell lung carcinomas and other lung tumors, laryngeal tumors, and sarcomas. No MAGE1 and MAGE2 gene expression was found in a large panel of healthy adult tissues, with the exception of testis.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number L18920.     

Efficient induction of antitumor cytoxic T lymphocytes from a healthy donor using HLA-A2-restricted MAGE-3 peptide in vitro

 The antigenic peptides encoded by tumor-rejection antigen genes, MAGE-1 and -3, have been identified, and various methods have been utilized for the in vitro induction of MAGE-specific, cytotoxic T lymphocytes (CTL) from peripheral blood mononuclear cells (PBMC) using synthetic peptides. However, all of these methods are technically demanding and thus have a relatively limited usefulness. We herein report a simple and efficient method for the in vitro induction of specific CTL by using the HLA-A2-restricted MAGE-3 peptide from the PBMC of a healthy donor. CTL responses could thus be efficiently induced from unseparated PBMC by stimulation with freshly isolated, peptide-pulsed PBMC as antigen-presenting cells and by using interleukin-7 and keyhole limpet hemocyanin for the primary culture. The induced CTL could thus recognize and lyse not only HLA-A2 target cells pulsed with the peptide but also HLA-A2 tumor cells expressing MAGE-3, in an HLA-class-I-restricted manner. This simple method may, therefore, become a useful tool for investigating the potential peptides for tumor antigens as well as for developing various immunotherapeutic approaches for human malignant tumors. Received: 15 October 1996 / Accepted: 6 December 1996     

Identification of five MAGE-A1 epitopes recognized by cytolytic T lymphocytes obtained by in vitro stimulation with dendritic cells transduced with MAGE-A1.

MAGE genes are expressed by many human tumors of different histological types but not by normal cells, except for male germline cells. The Ags encoded by MAGE genes and recognized by T cells are therefore strictly tumor-specific. Clinical trials involving therapeutic vaccination of cancer patients with MAGE antigenic peptides or proteins are in progress. To increase the range of patients eligible for therapy with peptides, it is important to identify additional MAGE epitopes recognized by CTL. Candidate peptides known to bind to a given HLA have been used to stimulate T lymphocytes in vitro. In some instances, CTL clones directed against these synthetic peptides have been obtained, but these clones often failed to recognize tumor cells expressing the relevant gene. Therefore, we designed a method to identify CTL epitopes that selects naturally processed peptides. Monocyte-derived dendritic cells infected with a recombinant canarypoxvirus (ALVAC) containing the entire MAGE-A1 gene were used to stimulate CD8+ T lymphocytes from the blood of individuals without cancer. Responder cell microcultures that specifically lysed autologous cells expressing MAGE-A1 were cloned using autologous stimulator cells either transduced with a retrovirus coding for MAGE-A1 or infected with recombinant Yersinia-MAGE-A1 bacteria. The CTL clones were tested for their ability to lyse autologous cells loaded with each of a set of overlapping MAGE-A1 peptides. This strategy led to the identification of five new MAGE-A1 epitopes recognized by CTL clones on HLA-A3, -A28, -B53, -Cw2, and -Cw3 molecules. All of these CTL clones recognized target cells expressing gene MAGE-A1.     

Generation of specific antitumor reactivity by the stimulation of spleen cells from gastric cancer patients with MAGE-3 synthetic peptide

The induction of cytotoxic T lymphocytes (CTL) from peripheral blood mononuclear cells (PBMC) using MAGE peptide has been investigated in order to use MAGE antigens immunotherapeutically. We therefore developed a simplified method for inducing peptide-specific CTL that kill tumor cells expressing MAGE from the PBMC of either healthy donors or even cancer patients. Since the spleen is a major lymphoid organ, we used a simple method to examine the capacity of spleen cells to generate MAGE-specific CTL by in vitro stimulation with MAGE peptide in gastric cancer patients. The CTL responses could thus be induced from unseparated spleen cells in HLA-A2 patients with gastric carcinoma expressing MAGE-3 by stimulating these cells with autologous spleen cells pulsed with HLA-A2-restricted MAGE-3 peptide as antigen-presenting cells and by using keyhole limpet hemocyanin and interleukin-7 for the primary culture. The induced CTL were thus able to lyse HLA-A2-positive carcinoma cells transfected with MAGE-3 and expressing MAGE-3, as well as the target cells pulsed with the peptide, in an HLA-class-I or -A2-restricted manner. Since MAGE-specific CTL could be induced from the spleen cells of gastric cancer patients, the spleen appears to play an important role in either clinical tumor vaccination or the treatment of cancer patients by adoptive immunotherapeutic approaches using the MAGE peptide. Received: 3 December 1998 / Accepted: 30 March 1999     

A new LAGE-1 peptide recognized by cytolytic T lymphocytes on HLA-A68 tumors

Antigens encoded by genes of the LAGE family, including LAGE-1 and NY-ESO-1, are of interest for cancer immunotherapy because they are tumor-specific and shared by tumors of different histological types. Several clinical trials are in progress with NY-ESO-1 peptides, protein, recombinant poxviruses, and dendritic cells pulsed with peptides. In this study, CD8 T lymphocytes from an individual without cancer were stimulated with dendritic cells infected with a recombinant avian poxvirus encoding a complete LAGE-1 protein. A CTL clone was isolated that recognized a new LAGE-1 peptide, ELVRRILSR, which corresponds to position 103–111 of the protein sequence. It is presented by HLA-A6801 molecules. When tumor cells expressing LAGE-1 were transfected with HLA-A68, they were lysed by the CTL clone, indicating that the peptide is processed in tumor cells. These results indicate that the LAGE-1.A68 peptide can be used for antitumoral vaccination. We observed also that specific T cells could be detected in a blood sample with a high sensitivity by using an A68/LAGE-1 fluorescent multimer.     

Study of HLA class I restriction and the directed antigens of cytotoxic T lymphocytes at the tumor sites of ovarian cancer

The molecular basis of T-cell-mediated recognition of ovarian cancer cells remains to be fully addressed. In this study we investigated HLA class I restriction and directed antigens of cytotoxic T lymphocytes (CTL) at the sites of ovarian cancer. Three HLA-class-I-restricted CTL lines were established from the tumor sites of ovarian cancer by culturing tumor-infiltrating lymphocytes or tumor-associated ascitic lymphocytes with interleukin-2: (1) HLA-A2402-restricted and ovarian-adenocarcinoma-specific CTL, (2) HLA-A2-restricted CTL recognizing histologically different cancers, and (3) HLA-B52-restricted and ovarian-cancer-specific CTL. HLA-A0201, HLA-A0206 and HLA-A0207 tumor cells were lysed by the HLA-A2-restricted CTL. HLA-B52 restriction of the third CTL line was confirmed by the transfection of HLA-B5201 cDNA into the tumor cells. The HLA-A2-restricted CTL recognized the SART-1, but not the MAGE-1 or MAGE-3 antigen. These results may facilitate a better understanding of the molecular basis of tumor-specific immunity at the tumor site of ovarian cancer. Received: 30 December 1998 / Accepted: 2 March 1999     

Identification of peptide epitopes of MAGE-1, -2, -3 that demonstrate HLA-A3-specific binding

 The MAGE gene family of tumour antigens are expressed in a wide variety of human cancers. We have identified 43 nonamer peptide sequences, from MAGE-1, -2 and -3 proteins that contain binding motifs for HLA-A3 MHC class I molecules. The T2 cell line, transfected with the cDNA for the HLA-A3 gene, was used in a MHC class I stabilisation assay performed at 37°C and 26°C. At 37°C, 2 peptides were identified that stabilised HLA-A3 with high affinity (fluorescence ratio, FR >1.5), 4 peptides with low affinity (FR 1.11 – 1.49) and 31 peptides that did not stabilise this HLA haplotype (FR <1.1). At 26°C, 12 peptides were identified that stabilised HLA-A3 with high affinity, 8 peptides with low affinity and 17 peptides that did not stabilise this HLA haplotype. Two peptides stabilised HLA-A3 at both temperatures. Small changes in one to three amino acids at positions distinct from the anchor residues altered peptide affinity. Data were compared to a similar study in which a peptide competition assay was used to investigate MAGE-1 peptide binding to several HLA haplotypes. This study demonstrates that anchor residues do not accurately predict peptide binding to specific HLA haplotypes, changes in one to three amino acids at positions distinct from anchor residues influence peptide binding and alternative methods of determining peptide binding yield different results. We are currently investigating the ability of these peptides to induce antitumour cytotoxic T lymphocyte activity as they may be of potential therapeutic value. Received: 4 January 1996 / Accepted: 20 March 1996     

An HLA-A3-binding prostate acid phosphatase-derived peptide can induce CTLs restricted to HLA-A2 and -A24 alleles

We previously reported peptide vaccine candidates for HLA-A3 supertype (-A3, -A11, -A31, -A33)-positive cancer patients. In the present study, we examined whether those peptides can also induce cytotoxic T lymphocyte (CTL) activity restricted to HLA-A2, HLA-A24, and HLA-A26 alleles. Fourteen peptides were screened for their binding activity to HLA-A*0201, -A*0206, -A*0207, -A*2402, and -A*2601 molecules and then tested for their ability to induce CTL activity in peripheral blood mononuclear cells (PBMCs) from prostate cancer patients. Among these peptides, one from the prostate acid phosphatase protein exhibited binding activity to HLA-A*0201, -A*0206, and -A*2402 molecules. In addition, PBMCs stimulated with this peptide showed that HLA-A2 or HLA-A24 restricted CTL activity. Their cytotoxicity toward cancer cells was ascribed to peptide-specific and CD8⁺ T cells. These results suggest that this peptide could be widely applicable as a peptide vaccine for HLA-A3 supertype-, HLA-A2-, and -A24-positive cancer patients.     

Lack of tumor recognition by cytolytic T lymphocyte clones recognizing peptide 195–203 encoded by gene <Emphasis Type="Italic">MAGE-A3</Emphasis> and presented by HLA-A24 molecules

Gene MAGE-A3 encodes tumor-specific antigenic peptides recognized by T cells on many tumors. MAGE-A3 peptides presented by HLA class I molecules have been identified using CD8 lymphocytes stimulated with cells that either expressed gene MAGE-A3 or were pulsed with candidate peptides. One antigen identified with the latter method is peptide MAGE-A3(195-203) IMPKAGLLI, presented by HLA-A24 molecules. It has been used to vaccinate advanced cancer patients. Here, we have used HLA/peptide tetramers to detect T cells recognizing this peptide. Their frequency was estimated to be 2 x 10(-8) of the blood CD8 cells in non-cancerous HLA-A24(+) individuals, which is tenfold lower than the reported frequencies of T cells against other MAGE peptides. In the blood of a patient vaccinated with MAGE-A3, the estimated frequency was 5 x 10(-7). Anti-MAGE-3.A24 cytolytic T cell clones were derived, that lysed peptide-pulsed cells with half-maximal effect at the low concentration of 500 pM. However, these CTL did not recognize a panel of HLA-A24(+) tumor cells that expressed MAGE-A3 at levels similar to those found in HLA-A1(+) tumor cells recognized by anti-MAGE-3.A1 CTLs. Furthermore, 293-EBNA cells transfected with MAGE-A3 and HLA-A24 constructs were hardly recognized by the anti-MAGE-3.A24 CTL clones. These results suggest that peptide MAGE-A3(195-203) is poorly processed and is not an appropriate target for cancer immunotherapy.     

A geometric study of the amino acid sequence of class I HLA molecules

 HLA class I alleles are studied by representing them in a metric space where each dimension corresponds to each one of the amino acid positions. Their similarity in reference to their ability to present peptides to T cells is then evaluated by calculating the correlation matrix between the amino-acid-composition tables (or binding affinity tables) for the sets of peptides presented by each allele. This correlation matrix is considered an empirical similarity matrix between HLA alleles, and is modeled in terms of possible structures defined in the metric space of HLA class I amino acid sequences. These geometric structures are adequate models of the peptide-binding data currently available. The following clusters of HLA class I molecules are identified in reference to their ability to present peptides: Cluster I) HLA-A3/ HLA-A11/ HLA-A31/ HLA-A33/ HLA-A68; Cluster II) HLA-B35/ HLA-B51/ HLA-B53/ HLA-B54/ HLA-B7; and Cluster III) HLA-A29/ HLA-B61/HLA-B44; the last cluster showing possible similarities between alleles from different loci. In modeling these natural clusters, the geometric structures with more predictive power confirm the importance of those positions in the peptide-binding groove, particularly those in the B pocket. In addition, other positions (46, 79, 113, 131, 144, and 177) appeared to bear some relevance in determining which peptides can be presented by which HLA alleles. Received: 20 January 1998 / Revised: 30 March 1998     

Alloreactive cytotoxic T-lymphocyte-defined HLA-B7 subtypes differ in peptide antigen presentation

We investigated T-cell-defined HLA-B7 subtypes using cDNA sequencing, analysis of bound peptides, and reactivity with a panel of alloreactive cytotoxic T-lymphocyte (CTL) clones. Three subtypes (HLA-B*0702, HLA-B*0703, and HLA-B*0705) differ in nucleotide and predicted amino acid sequence. CTL reactivity and pooled peptide sequencing show that these three HLA-B7 subtypes bind distinct but overlapping sets of peptides. In particular B*0702 expresses D pocket residue Asp 114 and binds peptides with P3 Arg, whereas B*0705 expresses D pocket residue Asn 114 and binds peptides with P3 Ala, Leu, and Met. Consistent with different peptide-binding specificities, three alloreactive CTL differentiate between cells expressing B*0702, B*0703, and B*0705 by detecting specific peptide/HLA-B7 complexes. In contrast, three other T-cell-defined HLA-B7 subtypes are identical to HLA-B*0702. The B*0702-expressing cell lines are differentiated by two of ten CTL clones. One CTL clone differentiates B*0702-expressing cells by their ability to present peptide antigen. Thus differences in peptide presentation can explain differential CTL recognition of cell lines expressing structurally identical and variant HLA-B7.     

Use of HLA-B27 tetramers to identify low-frequency antigen-specific T cells in <Emphasis Type="Italic">Chlamydia</Emphasis>-triggered reactive arthritis

Reports of the use of HLA-B27/peptide tetrameric complexes to study peptide-specific CD8⁺ T cells in HLA-B27⁺-related diseases are rare. To establish HLA-B27 tetramers we first compared the function of HLA-B27 tetramers with HLA-A2 tetramers by using viral epitopes. HLA-B27 and HLA-A2 tetramers loaded with immunodominant peptides from Epstein–Barr virus were generated with comparable yields and both molecules detected antigen-specific CD8⁺ T cells. The application of HLA-B27 tetramers in HLA-B27-related diseases was performed with nine recently described Chlamydia-derived peptides in synovial fluid and peripheral blood, to examine the CD8⁺ T cell response against Chlamydia trachomatis antigens in nine patients with Chlamydia-triggered reactive arthritis (Ct-ReA). Four of six HLA-B27⁺ Ct-ReA patients had specific synovial T cell binding to at least one HLA-B27/Chlamydia peptide tetramer. The HLA-B27/Chlamydia peptide 195 tetramer bound to synovial T cells from three of six patients and HLA-B27/Chlamydia peptide 133 tetramer to synovial T cells from two patients. However, the frequency of these cells was low (0.02–0.09%). Moreover, we demonstrate two methods to generate HLA-B27-restricted T cell lines. First, HLA-B27 tetramers and magnetic beads were used to sort antigen-specific CD8⁺ T cells. Second, Chlamydia-infected dendritic cells were used to stimulate CD8⁺ T cells ex vivo. Highly pure CD8 T cell lines could be generated ex vivo by magnetic sorting by using HLA-B27 tetramers loaded with an EBV peptide. The frequency of Chlamydia-specific, HLA-B27 tetramer-binding CD8⁺ T cells could be increased by stimulating CD8⁺ T cells ex vivo with Chlamydia-infected dendritic cells. We conclude that HLA-B27 tetramers are a useful tool for the detection and expansion of HLA-B27-restricted CD8⁺ T cells. T cells specific for one or more of three Chlamydia-derived peptides were found at low frequency in synovial fluid from HLA-B27⁺ patients with Ct-ReA. These cells can be expanded ex vivo, suggesting that they are immunologically functional.     

Cytolytic T lymphocytes recognize an antigen encoded by MAGE-A10 on a human melanoma.

From melanoma patient LB1751, cytolytic T lymphocytes (CTL) were generated that lysed specifically autologous tumor cells. To establish whether these CTL recognized one of the Ags that had previously been defined, a CTL clone was stimulated with cells expressing various MAGE genes. It produced TNF upon stimulation with target cells expressing MAGE-A10. The Ag was found to be nonapeptide GLYDGMEHL (codons 254-262), which is presented by HLA-A2.1. This is the first report on the generation of anti-MAGE CTL by autologous mixed lymphocyte-tumor cell culture (MLTC) from a melanoma patient other than patient MZ2, from whom the first MAGE gene was identified. MAGE genes are expressed in many tumors but not by normal tissues except male germline cells and placenta, which do not express HLA molecules. Therefore, the identification of an antigenic peptide derived from MAGE-A10 adds to the repertoire of tumor-specific shared Ags available for anti-tumoral vaccination trials.     

Specificity of peptide binding by the HLA-A2.1 molecule

The HLA-A2 molecule contains a putative peptide binding site that is bounded by two alpha-helices and a beta-pleated sheet floor. Previous studies have demonstrated that the influenza virus matrix peptide M1 55-73 can sensitize target cells for lysis by HLA-A2.1-restricted virus-immune CTL and can induce CTL that can lyse virus-infected target cells. To assess the specificity of peptide binding by the HLA-A2.1 molecule, we examined the ability of seven variant M1 peptides to be recognized by a panel of M1 55-73 peptide-specific HLA-A2.1-restricted CTL lines. The results demonstrate that five out of the seven variant M1 55-73 peptides could be recognized by A2.1-restricted M1 55-73 peptide-specific CTL lines. The two variant peptides that were not recognized by any CTL could bind to HLA-A2.1 as indicated by their ability to compete for presentation of the M1 55-73 peptide. In addition, 5 of a panel of 24 unrelated peptides tested could also compete for M1 55-73 presentation by HLA-A2.1. One peptide derived from the sequence of a rotavirus protein could sensitize HLA-A2.1+ targets for lysis by M1 55-73 peptide-specific CTL. We conclude from these studies that: 1) the HLA-A2.1 molecule can bind a broad spectrum of peptides; 2) T cells selected for the ability to recognize one peptide plus a class I molecule can actually recognize an unrelated peptide presented by that same class I molecule; and 3) a stretch of three adjacent hydrophobic amino acids may be an important common feature of peptides that can bind to HLA-A2.1.     

Substitution analog peptide derived from HER-2 can efficiently induce HER-2-specific, HLA-A24 restricted CTLs

In order to broaden the possibility for anti-HER-2/neu (HER-2) immune targeting, it is important to identify HLA-A24 restricted peptide epitopes derived from HER-2, since HLA-A24 is one of the most common alleles in Japanese and Asian people. In the present study, we have screened HER-2-derived, HLA-A24 binding peptides for cytotoxic T lymphocyte (CTL) epitopes. A panel of HER-2-derived peptides with HLA-A24 binding motifs and the corresponding analogs designed to enhance HLA-A24 binding affinity were selected. Identification of HER-2-reactive and HLA-A24 restricted CTL epitopes were performed by a reverse immunology approach. To induce HER-2-reactive and HLA-A24 restricted CTLs, PBMCs from healthy donors were repeatedly stimulated with monocytes-derived, mature DCs pulsed with HER-2 peptide. Subsequent peptide-induced T cells were tested for the specificity by enzyme linked immunospot, cytotoxicity and tetramer assays. CTL clones were then obtained from the CTL lines by limiting dilution. Of the peptides containing HLA-A24 binding motifs, 16 peptides (nine mers) including wild type peptides (IC₅₀<1,000 nM) and substituted analog peptides (IC₅₀<50 nM) were selected for the present study. Our studies show that an analog peptide, HER-2(905AA), derived from HER-2(905) could efficiently induce HER-2-reactive and HLA-A24 restricted CTLs. The reactivity of the HER-2(905AA)-induced CTL (CTL905AA) was confirmed by different CTL assays. The CTL905AA clones also were able to lyse HER-2(+), HLA-A24(+) tumor cells and cytotoxicity could be significantly reduced in cold target inhibition assays using cold targets pulsed with the HER-2(905) wild type peptide as well as the inducing HER-2(905AA) analog peptide. A newly identified HER-2(905) peptide epitope is naturally processed and presented as a CTL epitope on HER-2 overexpressing tumor cells, and an MHC anchor-substituted analog, HER-2(905AA), can efficiently induce HER-2-specific, HLA-A24 restricted CTLs.     

HLA-B57-restricted cytotoxic T-lymphocyte activity in a single infected subject toward two optimal epitopes, one of which is entirely contained within the other

Viral peptides are recognized by cytotoxic T lymphocytes (CTL) as a complex with major histocompatibility complex (MHC) class I molecules, but the extent to which a single HLA allele can accommodate epitope peptides of different length and sequence is not well characterized. Here we report the identification of clonal CTL responses from the same donor that independently recognize one of two HLA-B57-restricted epitopes, KAFSPEVIPMF (KF11; p24(Gag) residues 30 to 40) and KAFSPEVI (KF8; p24(Gag) residues 30 to 37). Although lysis studies indicated that the KF11 peptide stabilized the HLA-B57-peptide complex more efficiently than the KI8 peptide, strong clonal responses were directed at each epitope. In samples from a second donor, the same phenomenon was observed, in which distinct CTL clones recognized peptide epitopes presented by the same HLA class I allele (in this case, HLA-A3) which were entirely overlapping. These data are relevant to the accurate characterization of CTL responses, which is fundamental to a detailed understanding of MHC class I-restricted immunity. In addition, these studies demonstrate marked differences in the length of peptides presented by HLA-B57, an allele which is associated with nonprogressive human immunodeficiency virus infection.     

Induction of human cytotoxic T lymphocytes that preferentially recognise tumour cells bearing a conformational p53 mutant

 The tumour-suppressor gene p53 is pivotal in the regulation of apoptosis, and point mutations within p53 are the commonest genetic alterations in human cancers. Cytotoxic T lymphocytes (CTL) recognise peptide-MHC complexes on the surface of tumour cells and bring about lysis. Therefore, p53-derived peptides are potential candidates for immunisation strategies designed to induce antitumour CTL in patients. Conformational changes in the p53 protein, generated as a result of point mutations, frequently expose the 240 epitope, RHSVV (amino acids 212–217), which may be processed differently from the wild-type protein resulting in an altered MHC-associated peptide repertoire recognised by tumour-specific CTL. In this study 42 peptides (37 overlapping nonameric peptides, from amino acids 193–237 and peptides 186–194, 187–197, 188–197, 263–272, 264–272, possessing binding motifs for HLA-A2) derived from the wild-type p53 protein sequence were assayed for their ability to stabilise HLA-A2 molecules in MHC class I stabilisation assays. Of the peptides tested, 24 stabilised HLA-A2 molecules with high affinity (fluorescence ratio>1.5) at 26 °C, and five (187–197, 193–200, 217–224, 263–272 and 264–272) also stabilised the complexes at 37 °C. Peptides 188–197, 196–203 and 217–225 have not previously been identified as binders of HLA-A2 molecules and, of these, peptide 217–225 stabilised HLA-A2 molecules with the highest fluorescence ratio. Peptide 217–225 was chosen to generate HLA-A2-restricted CTL in vitro; peptide 264–272 was used as a positive control. The two primary CTL thus generated (CTL-217 using peptide 217–225; and CTL-264 using peptide 264–272) were capable of specifically killing peptide-pulsed T2 or JY cells. In order to determine whether these peptides were endogenously processed and to test the hypothesis that mutants expressing different protein conformations would generate an alternative peptide repertoire at the cell surface, a panel of target cells was generated. HLA-A2⁺ SaOs-2 cells were transfected with p53 cDNA containing point mutations at either position 175 (R → H) or 273 (R → H) (SaOs-2/175 and SaOs-2/273). Two HLA-A2-negative cell lines, A431 and SKBr3, naturally expressing p53 mutations at positions 273 and 175 respectively, were transfected with a cDNA encoding HLA-A2. The results showed that primary CTL generated in response to both peptides were capable of killing SaOs-2/175 and SKBr3-A2 cells, which possess the same mutation, but not SaOs-2/273, A431-A2 or SKBr3 cells transfected with control vector. This suggests that these peptides are presented on the surface of SaOs-2/175 and SKBr3-A2 cells in a conformation-dependent manner and represent potentially useful target peptides for immunotherapy. Received: 23 March 2000 / Accepted: 22 June 2000     

Analysis of a rare melanoma patient with a spontaneous CTL response to a MAGE-A3 peptide presented by HLA-A1

We describe an HLA-A1 melanoma patient who has mounted a spontaneous cytolytic T cell (CTL) response against an antigenic peptide encoded by gene MAGE-A3 and presented by HLA-A1. The frequency of anti-MAGE-3.A1 CTLp was 5×10⁻⁷ of the blood CD8 cells, with a dominant clonotype which was present in six out of seven independent anti-MAGE-3.A1 CTL clones. After vaccination with a recombinant poxvirus coding for the MAGE-3.A1 antigen, the blood frequency of anti-MAGE-3.A1 CTLp increased tenfold. Twenty-two independent CTL clones were derived. Surprisingly, only one of them corresponded to the dominant clonotype present before vaccination. Two new clonotypes were repeated 12 and 7 times, respectively. Our interpretation of these results is that the spontaneous anti-MAGE-3.A1 CTL response pre-existing to vaccination was polyclonal, and that the vaccine restimulated only some of these clones. To estimate the incidence of spontaneous anti-MAGE-3.A1 CTL responses in melanoma patients with a tumor expressing gene MAGE-A3, we measured the blood frequency of anti-MAGE-3.A1 T cells in 45 patients, and found only two clear responses.     

Responses against antigens encoded by theH-3 histocompatibility locus: Antigens stimulating class I MHC- and class II MHC-restricted T cells are encoded by separate genes

The purpose of this work was to study the genetic basis of histocompatibility antigens encoded by the mouse minor histocompatibility (H) locusH-3. Both class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) and class II MHC-restricted helper T cells (TH) specific for antigens encoded by genes within theH-3 locus were isolated and analyzed. Typing a number of mouse strains for expression of antigens recognized by these TH and CTL suggested that there was a different strain distribution pattern of expression of the antigens recognized by TH compared with those recognized by CTL. Separation of the genes whose products stimulate TH from those whose products stimulate CTL was suggested by: (1) analysis of the strain B10.FS(92NX)/Grf that has undergone recombination within theH-3 region; (2) genetic segregation studies of (B10.UW-H-3 ^b/Sn×C57BL/10Sn)F₂ mice; and (3) F₁ complementation studies in which CTL specific for products of the TH-defined gene(s) could not be detected, even in the absence of immune responses to products of the CTL-defined genes. Taken together, these data suggest that in addition to two genes (B2m andCd-1) within theH-3 region whose products typically stimulate class I MHC-restricted CTL, there is at least one additional gene whose product selectively stimulates class II MHC-restricted TH. This new gene is located telomeric from the CTL-defined genes and between the lociwe andun on chromosome 2. These data demonstrate a novel degree of complexity of theH-3 “locus” and suggest selective presentation of minor H gene products in the context of class I or class II MHC proteins.