Structure of the gene of tum- transplantation antigen P35B: presence of a point mutation in the antigenic allele.

Mutagen treatment of P815 tumour cells produces tum- variants that are rejected by syngeneic mice because they express new transplantation antigens. These 'tum-' antigens elicit a cytolytic T lymphocyte (CTL) response but no detectable antibody response. The DNA of tum- variant P35 was transfected into P815 cell line P1.HTR. Transfectants expressing tum- antigen P35B were identified on the basis of their ability to stimulate anti-P35B CTL. This was repeated with a cosmid library and a cosmid carrying the sequence encoding antigen P35B was recovered from a transfectant expressing the antigen. Gene P35B is 6 kb long and contains 11 exons. The sequence shows no homology with the previously identified tum- gene P91A nor with any gene presently recorded in the data banks. The antigenic allele of gene P35B differs from the normal allele by a point mutation located in exon 5. This mutation, which replaces a Ser by an Asn residue, was shown by site-directed mutagenesis to be responsible for the expression of the antigen. A synthetic decapeptide covering the sequence surrounding the tum- mutation rendered P815 cells sensitive to lysis by anti-P35B CTL. Surprisingly, the homologous peptide corresponding to the normal sequence of the gene had the same effect, indicating that this tum- mutation does not exert its effect by generating the aggretope or the epitope of the antigenic peptide. As observed previously with gene P91A, we found that fragments of gene P35B containing only exons 4 and 5, which were cloned in non-expression vectors, transferred efficiently the expression of the antigen.     

A new gene coding for an antigen recognized by autologous cytolytic T lymphocytes on a human renal carcinoma

Previous reports have described antigens that are recognized on human melanoma cells by autologous cytolytic T lymphocytes (CTL). The genes coding for a number of these antigens have been identified. Here we report the cloning of a gene that codes for an antigen recognized by autologous CTL on a human renal carcinoma cell line. This antigen is presented byHLA-B7 and is encoded by a new gene that we have namedRAGE1. No expression ofRAGE1 was found in normal tissues other than retina. RAGE1 expression was found in only one of 57 renal cell carcinoma samples, and also in some sarcomas, infiltrating bladder carcinomas, and melanomas. This represents the first identification of an antigen recognized by autologous CTL on a renal tumor.     

A MAGE-1 antigenic peptide recognized by human cytolytic T lymphocytes on HLA-A2 tumor cells

Cancer-germline genes such as those of the MAGE family are expressed in many tumors and in male germline cells, but are silent in normal tissues. They encode shared tumor-specific antigens that have been used in therapeutic vaccination trials of cancer patients. It was previously demonstrated that MAGE-1 peptide KVLEYVIKV was presented by HLA-A 0201 molecules on the surface of a human breast carcinoma cell line, but no human specific CTL had been isolated so far. Here, we have used HLA-A2/MAGE-1 fluorescent multimers to isolate from blood cells three human CTL clones that recognized the MAGE-1 peptide. These clones killed efficiently HLA-A2 tumor cells expressing MAGE-1, whether or not they were treated with IFN-, suggesting that the MAGE-1 antigen is processed efficiently by both the standard proteasome and the immunoproteasome. These results indicate that the MAGE-1.A2 peptide can be used for antitumoral vaccination.     

Transfection and expression of a gene coding for a human melanoma antigen recognized by autologous cytolytic T lymphocytes

Human melanoma line MZ2-MEL expresses several antigens recognized by autologous cytolytic T lymphocytes (CTL). As a first step towards the cloning of the gene coding for one of these antigens, we tried to obtain transfectants expressing the antigen. The DNA recipient cell was a variant of MZ2-MEL which had been selected with a CTL clone for the loss of antigen E. It was cotransfected with genomic DNA of the original melanoma line and with selective plasmid pSVtkneo. Geneticin-resistant transfectants were obtained at a frequency of 2 × 10^–4. These transfectants were then screened for their ability to stimulate the production of tumor necrosis factor by the anti-E CTL clone. One transfectant expressing antigen E was identified among 70 000 drug-resistant transfectants. Its sensitivity to lysis by the anti-E CTL was equal to that of the original melanoma cell line. When this transfectant was submitted to immunoselection with the anti-E CTL clone, the resulting antigen-loss variants were found to have lost several of the transfected pSVtkneo sequences. This indicated that the gene coding for the antigen had been integrated in the vicinity of pSVtkneo sequences, as expected for cotransfected DNA. Address correspondence and offprint requests to: T. Boon.     

Mapping of the genes encoding tum- transplantation antigens P91A,P35B,and P198

Tum ^- comprises a class of genes, mutation of which in P815 tumor cells has led to the acquisition of new cytotoxic T cell-recognized epitopes. The cells carrying the mutant alleles have impaired tumorigenicity compared with their progenitors due to in vivo induction of a cytotoxic T-cell response specific for tum ^- antigens. Two tum ^- genes, P91A and P35B, were found to be single copy loci mapping to chromosomes 11 and 15 respectively. A third, P198, was found to map to chromosome 7 and to be a member of a small gene family with other members on chromosomes 13, 14, and 15. Multiple P198-related sequences were found in other mammalian species suggesting the P198 related gene family is a general feature of mammalian genomes.     

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.     

Isolation of a new melanoma antigen, MART-2, containing a mutated epitope recognized by autologous tumor-infiltrating T lymphocytes

Using cDNA expression cloning, a cDNA encoding a novel human melanoma Ag, MART-2 (melanoma Ag recognized by T cells-2), recognized by HLA-A1-restricted CD8(+) T cells from tumor-infiltrating lymphocytes (TIL1362) was isolated from an autologous melanoma cell line, 1362 mel. Homologous sequences to the cDNA had been registered in the EST database. This gene encoded an uncharacterized protein expressed ubiquitously in most normal and cancer cells. A mutation (A to G transition) was found in the cDNA obtained from the1362 mel melanoma cell line in the sequences encoding the phosphate binding loop (P-loop) that resulted in loss of the ability to bind GTP. Transfection of NIH-3T3 with the mutated MART-2 did not result in the development of significant foci. By screening 36 various cancer cell lines using single-strand conformation polymorphism, a possible mutation in the P-loop of MART-2 was found in one squamous cell lung cancer cell line, EBC1. The T cell epitope for TIL1362, FLEGNEVGKTY, was identified to be encoded by the mutated sequence of the MART-2 Ag. The mutation substituted glycine in the normal peptide with glutamic acid at the third amino acid of the epitope, which is an important primary anchor amino acid for HLA-A1 peptide binding. The normal peptide, FLGGNEVGKTY, was not recognized by TIL1362, suggesting that this T cell response was specific for the autologous tumor. Although transforming activity was not detected in the NIH-3T3 assay, MART-2 with the mutation in the P-loop may be involved in the generation of melanoma through a loss of GTP binding activity.     

Characterization of a conserved T cell epitope in HIV-1 gp41 recognized by vaccine-induced human cytolytic T cells.

A human CTL epitope located in a region of the HIV-1 envelope protein gp41 that is highly conserved among various HIV-1 strains was identified. This epitope was recognized by CD4+ CTL clones that were induced in seronegative humans by immunization with recombinant gp160. Fusion proteins carrying portions of the HIV-1 env gene and synthetic peptides were used to localize this epitope to amino acids 584-595 of the HIV-1 BRU env sequence. Only two positions within this epitope showed variation among North American HIV-1 isolates, and the substitutions were conservative in nature. The Lys to Arg substitution at position 593 abolished recognition, probably by interfering with the peptide-MHC interactions. This epitope was recognized in association with at least one subtype of the widely distributed human class II MHC specificity DPw4, namely DPw4.2. The relatively high frequency of this allele (27.2% among Caucasians) makes it likely that a larger fraction of the population would generate a response directed at this epitope than would be the case for epitopes recognized in the context of gene products of most other class II and class I loci. Interestingly, the closely related DP beta-chain allele types 4.1 and 2.1, which differ from 4.2 by 3 and 1 amino acids, respectively, were unable to present this gp41 peptide to DPw4.2-restricted clones. Comparison of the structure of this epitope with that of other peptides recognized in the context of DPw4.2 led to the identification of a consensus sequence for DPw4.2 binding peptides. Because the gp41 CTL epitope 584-595 identified here is highly conserved and is recognized in the context of a common DP allele, it may represent an important target region for vaccine development. Our results indicate that vaccines containing this epitope may induce in a significant fraction of those immunized CTL active against at least half of all HIV-1 strains.     

Delineation of Ia:nominal antigen complementary determinants recognized by T cells in studies of gene complementation in response to insulin

The immune response to beef insulin in mice is controlled by genes in the IA subregion. We have previously shown that B6.C-H-2bm12 (bm12) mice, an A beta gene mutation of B6, have a selective loss of responsiveness to beef insulin, whereas other IAb controlled responses such as (TG)AL and collagen are unchanged. F1 hybrid mice between two nonresponder genotypes Ik and Ibm12 were found to be good responders to beef insulin suggesting functional complementation. In this report, we define the cellular and molecular basis of this complementation by investigating the determinants on Ia molecules and nominal antigen that are recognized by (B10.A X bm12)F1 proliferating T cells. Genetic analyses demonstrated that the Ik region was the only nonresponder genotype that complemented Ibm12, thus restoring responsiveness to beef insulin. More precisely an IAk and not an IEk gene product was found to be responsible for this complementation. Antibody blocking studies furthermore showed that the A alpha b:A beta k hybrid Ia mediated the response to beef insulin in (B10.A X bm12)F1 mice. Clonal analyses of the response to beef insulin in these F1 mice confirmed these conclusions, because the insulin-specific response in all 21 F1-T cell clones studied thus far was found to be dependent upon presentation via the A alpha b:A beta k hybrid Ia molecule. Dissection of the antigenic specificity of the F1-T cell clones demonstrated recognition of at least two insulin determinants, one A-loop (A8-A10) associated and the other non-loop (A4 or B chain) associated. Therefore these studies identify the molecular and antigenic basis of the Ir gene complementation seen in the response to beef insulin of (B10.A X bm12)F1 hybrids.     

Contribution of the T cell receptor BJ gene to recognition of the P91A tumor antigen in DBA/2 mice

 To understand specific immune responses against a tumor, it is important to characterize T cells that recognize the tumor antigen. The mouse P91A antigen is one of the well-defined tumor antigens that is expressed on the P911 cell line, and T cells responding to the antigen in DBA/2 mice were reported to be restricted to BV8S2/S3 families in their T cell receptor (TCR) BV gene usage. We have further characterized the P91A-responding T cells in DBA/2 mice, focusing on TCR BJ gene usage and using the polymerase chain reaction/enzyme-linked immunosorbent assay and DNA sequencing studies of their third complementarity-determining (CDR3) regions. As a result, T cells with cytotoxic activity to the P91A antigen, induced from murine spleen cells both in vivo and in vitro, showed predominant use of the BJ2S1 gene segment in both BV8S2 and BV8S3 T cells compared to unmanipulated murine spleen cells. Sequencing studies of the CDR3 regions in the BV8S3 T cells revealed clonal expansion of T cells with the BV8S3-BJ2S1 combination in two of three DBA/2 mice tested. In the remaining mouse, clonal expansion was not detected despite predominant use of the BJ2S1 segment by these T cells. These data suggest that P91A-recognizing T cells would predominantly use the BV8S2/S3-BJ2S1 combination. Analysis of T cells with these TCR BV-BJ gene combinations may contribute to the evaluation, monitoring and development of a T-cell-mediated immunotherapeutic strategy. Received: 3 July 1997 / Accepted: 17 November 1997     

Epitopes of the Mycobacterium tuberculosis 65-kilodalton protein antigen as recognized by human T cells

A synthetic peptide approach has been used to identify the epitopes recognized by clonal and polyclonal human T cells reactive to the recombinant mycobacterial 65-kDa protein Ag. Three of the four epitopes identified were recognized as cross-reactive between Mycobacterium tuberculosis and Mycobacterium leprae, although their amino acid sequence in two of three cases was not identical. The peptide (231-245) defining an epitope recognized as specific to the M. tuberculosis complex contains two substitutions compared with the homologous M. leprae region of which one or both are critical to T cell recognition. The reactive T cell clones showed helper/inducer phenotype (CD4+, CD8-), and secrete IL-2, granulocyte-macrophage-CSF, and IFN-gamma upon Ag stimulation. The same clones display cytotoxicity against macrophages pulsed with the relevant peptides or mycobacteria.     

A peptide encoded by the human MAGE3 gene and presented by HLA-1344 induces cytolytic T lymphocytes that recognize tumor cells expressing MAGE3

The humanMAGE3 gene is expressed in a significant proportion of tumors of various histological types, but is silent in normal adult tissues other than testis and placenta. Antigens encoded byMAGE3 may therefore be useful targets for specific antitumor immunization. Two antigenic peptides encoded by theMAGE3 gene have been reported previously. One is presented to cytolytic T lymphocytes (CTL) by HLA-A1, the other by HLA-A2 molecules. Here we show that MAGE3 also codes for a peptide that is presented to CTL by HLA-1344.MAGE3 peptides containing the HLA-1344 peptide binding motif were synthesized. Peptide MEVDPIGHLY, which showed the strongest binding to HLA-1344, was used to stimulate blood T lymphocytes from normal HLA-1344 donors. CTL clones were obtained that recognized not only HLA-B44 cells sensitized with the peptide, but also HLA-B44 tumor cell lines expressingMAGE3. The proportion of metastatic melanomas expressing theMAGE3/HLA-1344 antigen should amount to approximately 17% in the Caucasian population, since 24% of individuals carry theHLA-B44 allele and 76% of these tumors express MAGE3.     

A human NK and K cell subset shares with cytotoxic T cells expression of the antigen recognized by antibody OKT8

The antigen recognized by monoclonal antibody OKT8 is expressed on the cell membrane of 30 to 50% of human NK/K cells. The reactivity of OKT8 with NK/K cells was determined by indirect methods (treatment of the effector cells with OKT8 antibody and complement (C) and separation of OKT8(+) and (-) effector cell populations by fluorescence-activated cell sorting or by rosetting techniques) and, at single cell level, by C-dependent lysis of effector NK cells that bind and kill K562 targets. Analysis by indirect immunofluorescence (flow cytofluorometry) of lymphocyte subpopulations mediating NK/K cytotoxic activity and deprived of OKT8(+) T cells reveals that the NK/K cell subset bears OKT8 antigen at a density lower than that present on cytotoxic T cells. The OKT8 antigen on NK/K cells is trypsin- and pronase-sensitive, but it is resynthesized by the same effector cells during 24 hr of culture at 37 degrees C. OKT8 antibody does not inhibit NK killing, and, on a per cell basis, OKT8(+) cells within the NK/K subset mediate the same level of cytotoxic activity as OKT8(-) NK/K cells. Analogous results were obtained by using anti-Leu-2a, an antibody with the same specificity as OKT8 on cytotoxic/suppressor T cells, but not when OKT5 was used, which might identify a distinct epitope on the same antigenic molecule. The possible significance of these findings in understanding the cell lineage of NK/K cells is discussed.     

N protein is the predominant antigen recognized by vesicular stomatitis virus-specific cytotoxic T cells.

The specificity of anti-vesicular stomatitis virus (VSV)-specific cytotoxic T cells was explored with cell lines expressing VSV genes introduced by electroporation. Low levels of nucleocapsid (N) protein were detected on the surface of VSV-infected cells, but N protein could not be detected on the plasma membrane of transfected EL4 cells. Intracellular N protein was detectable by enzyme-linked immunosorbent assay or immunoprecipitation in some of the transfected cell lines but not in others, unless the transfected genes were induced by sodium butyrate. However, all of the stably transfected EL4 cell lines expressing the VSV-Indiana N protein were efficiently lysed by serotype-specific and cross-reactive anti-VSV cytotoxic T cells (CTLs). Primary cross-reactive anti-VSV CTLs appeared to be specific solely for N protein, based on cold-target competition assays using infected and transfected target cells. Cell lines expressing 100- to 1,000-fold less N protein than did VSV-infected cells were efficiently lysed by both primary and secondary anti-VSV CTLs. Cell lines expressing 100-fold less G protein than did VSV-infected cells were not lysed by either population of effectors. Significantly, cold-target competition studies with secondary CTLs demonstrated that N protein-expressing cell lines were more efficient competitors than were VSV-infected cells even though the latter expressed 100- to 1,000-fold more N protein. This was not an artifact of viral infection since infection of the transfected cell lines did not affect their ability to compete. The possibility that cell lines constitutively expressing internal virus proteins present antigen more effectively than infected cells do is discussed.     

Unrestricted recognition of a nonpeptide antigen by CD8+ cytolytic T lymphocytes

CD8+ murine CTL that are specific for an unusual nonpeptide Ag, the heme moiety of hemoglobin, have been derived by in vitro stimulation of spleen cells with hemin. Such CTL demonstrate a requirement for the expression of class I Ag on target cells, yet appear to be unrestricted to the extent that both syngeneic and allogeneic targets precoated with hemin are sensitive to lysis. A series of CTL clones with specificity for hemin was derived from C57BL/6 mice. They exhibited the same type of promiscuous recognition that was observed in CTL populations from a number of different strains. The possibility that hemin acts as a nonspecific mediator of lysis by CTL was ruled out by the fact that a variety of CTL populations and clones specific for different Ag did not exhibit hemin-specific lysis. Some explanations offered to explain these results include 1) the possibility that hemin is recognized by binding to a site on the MHC other than the Ag-binding groove, and 2) the possibility that TCR recognition of a rigid molecule, such as hemin, may be less sensitive to polymorphic variation in the MHC than is recognition of a conventional peptide Ag whose conformation may differ significantly when bound to MHC molecules whose sequences differ within the Ag-binding groove.     

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.