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Peptides with dual binding specificity for classical HLA class I and non-classical HLA-E molecules have been identified in virus-encoded proteins, but not in cellular proteins from normal or neoplastic cells. Expression screening of a melanoma cDNA library with a CTL clone recognizing an HLA-A2-restricted tumor-specific epitope encoded by mutant peroxiredoxin 5 (Prdx5), a stress-inducible peroxidase, led to the identification of two alternatively spliced isoforms of the same gene. These isoforms, which lack the catalytic cysteine fundamental for enzymatic activity, showed widespread expression in neoplastic and normal tissues but were unstable at the protein level, being detectable, following transient transfection, only after lactacystin treatment to inhibit proteasomal degradation. Isoform-specific sequences which formed, respectively, as result of exon 1 splicing to either exon 3 or 4, encoded two distinct nonapeptides (AMAPIKTHL and AMAPIKVRL, not present in the full-length protein) with anchor residues for HLA-A2 and HLA-E molecules and able to stabilize HLA-A2 and HLA-E cell surface expression. HLA-E+ targets, loaded with these peptides, were not recognized by NK cells expressing CD94/NKG2A inhibitory or CD94/NKG2C activatory receptors. However, both peptides were recognized, although with low avidity, by HLA-E-restricted CD8+ CTL. The nonapeptide AMAPIKVRL was used to elicit HLA-A2-restricted CTL clones that killed peptide-pulsed lymphoblastoid cell lines and melanoma cells expressing the corresponding Prdx5 isoform. Our results suggest that alternatively spliced isoforms of Prdx5, through the generation of HLA-E- and HLA-A2-restricted peptides may be part of immune-mediated stress response contributing to the detection and elimination of damaged normal or neoplastic cells.

The molecular identification and characterization of antigenic epitopes recognized by T cells on human cancers has rapidly evolved since the cloning in 1991 of MAGEA1, the first gene reported to encode a CTL-defined human tumor antigen. In the expanding field of human tumor immunology, unique tumor antigens constitute a growing class of T cell-defined epitopes that exhibit strong immunogenicity. Some of these antigens, which often derive from mutation of genes that have relevant biological functions, are less susceptible to immunoselection and may be retained even in advanced tumors. Immunogenicity and constitutive expression of the unique tumor antigens provide a strong rationale for the design of novel, patient-tailored therapies that target such determinants. Here we discuss the immunologic relevance of unique tumor antigens in the light of the prospects for exploiting such epitopes as targets for patient-specific immune intervention strategies.

Human melanomas can express unique tumor antigens, resulting from mutated proteins, and shared epitopes encoded for by normal genes, but these two classes of antigens have not been previously compared for immunogenicity and retention in metastatic cells. Here, we identified a new unique antigen generated by a point mutation in the peroxiredoxin 5 (Prdx5) gene in an HLA-A*0201(+) human metastatic melanoma lacking the wild-type allele. An antioxidant assay, with recombinant Prdx5 proteins, and evaluation of peroxide accumulation in transiently transfected cells, indicated that the mutant protein retained its enzymatic activity. The mutation in the Prdx5 protein did not generate a new HLA agretope but yielded an HLA-A*0201-restricted T cell epitope (Prdx5(110-119)). By HLA-tetramer analysis, in a tumor-invaded lymph node, >50% of mutant Prdx5-specific CD8(+) T cells (frequency 0.37%/CD8(+)) showed a CCR7(+/-) CD45RA(-) "T(CM)" or "T(EM)" phenotype, as found in Melan-A/MART-1-specific T cells (frequency 0.68%/CD8(+)) in the same tissue. In agreement with their memory phenotype, the Prdx5-specific T cells readily expanded in vitro in mixed lymphocyte-tumor culture, as did the Melan-/MART-1-specific T cells. By immunohistochemistry of the invaded lymph node, the mutant Prdx5 protein was expressed in all neoplastic cells, in contrast with the heterogeneous expression of shared antigens as Melan-A/MART-1, gp100 and tyrosinase. Thus, a unique tumor antigen can be as immunogenic as the melanoma differentiation antigens but, in contrast to the latter, may be retained in all metastatic cells possibly as result of the relevant cellular function exerted by the mutated protein.

We report here the identification of a new shared human melanoma antigen recognized by a human leukocyte antigen (HLA)-A*68011-restricted cytotoxic T lymphocyte clone (CTL 128). The cDNA encoding this antigen is composed of a partially spliced form of the melanocyte differentiation antigen tyrosinase-related protein (TRP)-2, containing exons 1-4 with retention of intron 2 and part of intron 4 (TRP-2-INT2). The sequence coding for the antigenic epitope is located at the 5' end of intron 2 and is available for translation in the same open reading frame of the fully spliced TRP-2 mRNA. This peptide is also recognized by CTL 128 when presented by the HLA-A*3301, a member of the HLA-A3-like supertype that includes the HLA-A*68011. Quantitative reverse transcription PCR analysis carried out on total and/or cytoplasmic mRNA demonstrated that, in contrast to the fully spliced TRP-2 mRNA expressed in melanomas, normal skin melanocytes, and retina, the TRP-2-INT2 mRNA could be detected at significant levels in melanomas but not in normal cells of the melanocytic lineage. Instead, in these normal samples, both the spliced and the unspliced transcript of gp100 were expressed at high levels. Absence of endogenous TRP-2-INT2 expression in melanocytes was also confirmed by lack of recognition of HLA-A*68011-transduced, TRP-2(+) melanocyte lines by CTL 128. These results indicate that a partially spliced form of a differentiation antigen mRNA, present in the cytoplasmic compartment of neoplastic but not normal cells of the melanocytic lineage, can be the source of a melanoma-restricted T cell epitope.

T cells infiltrating pre- and postvaccine metastases obtained from melanoma patients vaccinated with either dinitrophenyl (DNP)-modified autologous tumor or with the MAGE-3.A1 peptide display selective T cell receptor (TCR) beta chain variable region (BV) repertoire changes at the tumor site as a consequence of vaccination. Restricted sets of BV families expand in all postvaccine lesions when compared with prevaccine specimens and often contain dominant clones. A protocol devised to obtain T cell lines highly enriched for expression of a given BV region through the use of anti-BV monoclonal antibodies was used to understand whether responses to specific antigen(s) accounted for these clonal expansions. In one of the patients vaccinated with DNP-modified tumor cells, BV-driven selection of the T lymphocytes expanded in two infiltrated postvaccine metastases resulted in T cell lines able to exert HLA class I-restricted lysis of the autologous tumor. These results indicate that TCR repertoire analysis at the tumor site facilitates the detection of T cell responses elicited by a vaccine and potentially cytotoxic for the autologous tumor.

The peptide-binding and presentation characteristics of seven naturally occurring HLA-A2 subtypes were studied using M3(271), a peptide derived from the tumor-specific Ag encoded by gene MAGE-3, which has been shown to be processed and presented by A*0201+ melanoma lines. Three independent M3(271)-specific CTL clones were obtained from two unrelated A*0201+ donors. B lymphoblastoid cell lines (BLCLs) expressing A*0201, A*0207, or A*0209 could be sensitized to lysis by all three clones upon incubation with the relevant peptide. Furthermore, the same BLCLs were able to present endogenous M3(271) in IFN-gamma release assays. These findings demonstrate, for the first time, the existence of a functional overlap between A*0207 and other A*02 subtypes. One of the CTL clones also lysed M3(271)-pulsed BLCLs expressing A*0204 and A*0206, while the other two clones recognized M3(271) only in the context of either of these two subtypes. Peptide-pulsed BLCLs expressing A*0202 or A*0205 were not lysed, although A*0205 and, with lower affinity, A*0202 molecules were shown to bind peptide M3(271). These findings have implications for the selection of cancer patients for specific immunotherapy with peptide M3(271).

In this study we determined TCR alpha and beta chain nucleotide sequences of HLA-Cw* 1601-restricted cytoxic T lymphocyte (CTL) clones obtained from the peripheral blood lymphocytes (PBL) of a melanoma patient. These clones were previously shown to be involved in the recognition of melanoma-associated antigenic epitopes SAYGEPRKL and AARAVFLAL encoded by gene MAGE-1 and BAGE respectively. All (3/3) anti-MAGE-1 CTL clones displayed TCRBV5 usage and one clonotype was found twice, > 1 year apart, in patient's PBL. Two out of three anti-BAGE CTL clones showed the same TCRAV/AJ and TCRBV/BJ combinations and differed in the alpha chain CDR3 for two residues and in the beta chain CDR3 for a single nucleotide which, however, did not change translation. These results suggest a pattern of TCR conservation in CTL selected for recognition of MAGE-1 or BAGE peptides on the autoiogous melanoma.

Tumor-infiltrating lymphocytes (TILs), through displaying a T-cell receptor (TCR) repertoire as heterogeneous as that of normal peripheral blood T cells, show overexpression of TCR variable-gene segments that include clonally expanded TCR sequences. Here, Marialuisa Sensi and Giorgio Parmiani analyze the available information on TCR usage by T cells present in the infiltrate of histologically different tumors and suggest that the analysis of TCR sequences represents a powerful new tool to assess the in vivo immune response to growing tumors. Ultimately, this strategy may lead to the identification and manipulation of T-cell populations endowed with antitumor reactivity.

To determine whether T-cell-receptor (TCR) usage by T cells recognizing a defined human tumor antigen in the context of the same HLA molecule is conserved, we analyzed the TCR diversity of autologous HLA-A2-restricted cytotoxic T-lymphocyte (CTL) clones derived from five patients with metastatic melanoma and specific for the common melanoma antigen Melan-A/MART-1. These clones were first identified among HLA-A2-restricted anti-melanoma CTL clones by their ability to specifically release tumor necrosis factor in response to HLA-A2.1+ COS-7 cells expressing this tumor antigen. A PCR with variable (V)-region gene subfamily-specific primers was performed on cDNA from each clone followed by DNA sequencing. TCRAV2S1 was the predominant alpha-chain V region, being transcribed in 6 out of 9 Melan-A/MART-1-specific CTL clones obtained from the five patients. beta-chain V-region usage was also restricted, with either TCRBV14 or TCRBV7 expressed by all but one clone. In addition, a conserved TCRAV2S1/TCRBV14 combination was expressed in four CTL clones from three patients. None of these V-region genes was found in a group of four HLA-A2-restricted CTL clones recognizing different antigens (e.g., tyrosinase) on the autologous tumor. TCR joining regions were heterogeneous, although conserved structural features were observed in the complementarity-determining region 3 sequences. These results indicate that a selective repertoire of TCR genes is used in anti-melanoma responses when the response is narrowed to major histocompatibility complex-restricted antigen-specific interactions.

Several tumor antigens are recognized by autologous cytolytic T lymphocytes (CTL) on human melanoma MZ2-MEL. Some of them are encoded by genes MAGE-1 and MAGE-3, which are not expressed in normal tissues except in testis. Here, we report the identification of a new gene that codes for another of these antigens. This gene, named BAGE, codes for a putative protein of 43 aa and seems to belong to a family of several genes. The antigen recognized by the autologous CTL consists of BAGE-encoded peptide AARAVFLAL bound to an HLA-Cw 1601 molecule. Gene BAGE is expressed in 22% of melanomas, 30% of infiltrating bladder carcinomas, 10% of mammary carcinomas, 8% of head and neck squamous cell carcinomas, and 6% of non-small cell lung carcinomas. Like the MAGE genes, it is silent in normal tissues with the exception of testis. Because of its tumor-specific expression, the BAGE-encoded antigen may prove useful for cancer immunotherapy.

Human melanoma cell line MZ2-MEL expresses several antigens recognized by autologous cytolytic T lymphocyte (CTL) clones. We reported previously the identification of a gene, named MAGE-1, which codes for antigen MZ2-E which is presented by HLA-A1. Gene MAGE-1 is expressed in many tumors of several types but not in normal tissues except for testis. We show here that gene MAGE-1 directs the expression of another antigen recognized by CTL on the MZ2-MEL cells. This antigen, which was named MZ2-Bb, consists of MAGE-1-encoded peptide SAYGEPRKL bound to major histocompatibility molecule HLA-Cw*1601. The HLA-Cw*1601 allele was found to be expressed by 7 out of 99 individuals from a Caucasian population. Our results extend the range of tumor patients who could be eligible for immunization against MAGE antigens.

To see whether different antigens expressed by the same tumor are recognized by distinct T-cell receptors (TCR), we used cytotoxic T-lymphocyte (CTL) lines known to lyse in vitro the syngeneic BALB/c adenocarcinoma C-26. Four of these CD3+ CD8+ lines showed 4 different patterns of lysis on a panel of MHC-class-I-compatible targets. The activity was H-2d-restricted and could be blocked by anti-CD3 and anti-TCR-alpha/beta monoclonal antibodies (MAbs). The CTL lines were also effective, although to a different extent, in adoptive immunotherapy of mice bearing lung metastases. Phenotypic analysis revealed in all the lines a high frequency of cells positive for CD45, asialo GM1 (ASGM1), lymphocyte-function-associated antigen-1 (LFA-1), intercellular adhesion molecule-1 (ICAM-1) and CD44, but negligible expression of L-selectin (LAM-1) and very late antigen-4 (VLA-4); 2 lines expressed the vitronectin-receptor (VN-R). Analysis of TCR V beta-chains used by the 4 lines showed selective presence of V beta 6, V beta 8.2, V beta 8.3 and of V beta 13 chains. MAbs directed to these V beta chains blocked their lytic activity in vitro. V alpha-chain transcripts of the lines were identified by polymerase chain reaction (PCR) as V alpha ITT11 and V alpha 52 in 2 lines, while one could not be identified. Analysis of V beta s in mixed lymphocyte-tumor-cell cultures (MLTC) of T cells deriving from tumor-infiltrating lymphocytes (TIL) or from spleen of C-26 tumor-bearing or immune animals indicated that the TCR of the CTL lines were representatives of the TCR repertoire recognizing C-26 tumor, since their V beta s were shown to be selectively expanded in MLTC.

The objective of our study was to determine whether and how frequently MHC class I gene alterations occur in fibrosarcomas induced in BALB/c mice by the carcinogen 3-methylcholanthrene (3-MCA). Southern blot was performed to analyze the genomic organization of class I genes in a panel of twenty tumors of different immunogenic strength used at early in vivo passages. Our results rule out the presence of gross rearrangements or deletions in the H-2 class I genes of the tumors tested and indicate that the frequency of detectable class I alterations giving rise to restriction fragments length polymorphism (RFLP) should be very low.

The possibility of obtaining a syngeneic antitumor effect by immunization with normal allogeneic cells was investigated by tests of the lytic activity of peritoneal exudate cells (PEC) of BALB/c mice immunized with lymphoid cells of either a single strain or a pool of six different allogeneic strains on the syngeneic Moloney virus-induced lymphoma YC8 target and on one of its clones designated YC8-D1. Significant cytotoxicity on both targets but not on two other BALB/c lymphomas was obtained with PEC of BALB/c mice singly immunized to the non-H-2-incompatible but H-2-compatible DBA/2 or B10.D2 lymphoid cells. The lack of lysis of YC8 cells by PEC of BALB/c mice immune to B10.A (H-2k,d) suggests that the in vitro killing was restricted by Kd-IEd region products of the major histocompatibility complex. Pool immunization was effective in generating antitumor cytotoxic lymphocytes only when DBA/2 lymphoid cells were included in the pool. The pattern of reactivity of effectors elicited in (BALB/c x DBA/2)F1 and in (BALB/c x B10.D2)F1 mice by immunization with DBA/2 and B10.D2 cells showed that at least two sets of antigens are recognized on YC8 targets, one shared by DBA/2 and B10.D2 tissues and the other expressed by DBA/2 cells only. Cold target blocking experiments indicated that the same effectors recognized non-H-2 antigens of DBA/2 and the cross-reacting YC8 determinants. The antitumor effect was mediated by T-cells, since it was abrogated by treatment of effectors with anti-Thy 1.2 serum plus complement. These data indicate that determinants defined by cytotoxic T-lymphocytes are expressed on the BALB/c lymphoma YC8 and cross-react with non-H-2 antigens of DBA/2 and B10.D2 strains.

The presence of H-2Ld antigens was evaluated in methylcholanthrene-induced BALB/c fibrosarcomas by a variety of approaches. Transplantation experiments showed that BALB/c-H-2dm2 mice, a mutant strain whose cells do not express H-2Ld antigens, after immunization with BALB/c normal tissues developed a resistance to the growth of two tumours (C-3 and GI-17), but not to a third neoplasm, C-1, which is known to have H-2d- as well as H-2k-like alien antigens. In vitro experiments with cytotoxic T lymphocytes generated against Ld antigens confirmed a loss of Ld antigens on C-1 but not on C-3 tumour cells. Serological experiments with an anti-Ld serum again revealed the presence of H-2Ld determinants on C-3 but not on C-1 cells. Biochemical analysis in SDS-PAGE of immunoprecipitates obtained by specific anti-H-2 sera with NP40 lysates of the tumours studied could detect H-2Kd, H-2Dd and H-2Ld antigens in C-3 fibrosarcoma cells whereas Kd and Dd were the only H-2d molecules found in C-1 lysate along with the H-2k-like specificities. The possible genetic mechanisms which may explain this apparent gain and loss modification of the H-2 profile of C-1 are discussed.