Naturally acquired immune responses against human cancers often include CD8 ؉ T cells specific for the cancer testis antigen NY-ESO-1. Here, we studied T cell receptor (TCR) primary structure and function of 605 HLA-A*0201/NY-ESO-1 157-165-specific CD8 T cell clones derived from five melanoma patients. We show that an important proportion of tumor-reactive T cells preferentially use TCR AV3S1/BV8S2 chains, with remarkably conserved CDR3 amino acid motifs and lengths in both chains. All remaining T cell clones belong to two additional sets expressing BV1 or BV13 TCRs, associated with ␣-chains with highly diverse VJ usage, CDR3 amino acid sequence, and length. Yet, all T cell clonotypes recognize tumor antigen with similar functional avidity. Two residues, Met-160 and Trp-161, located in the middle region of the NY-ESO-1 157-165 peptide, are critical for recognition by most of the T cell clonotypes. Collectively, our data show that a large number of ␣␤ TCRs, belonging to three distinct sets (AVx/BV1, AV3/BV8, AVx/BV13) bind pMHC with equal antigen sensitivity and recognize the same peptide motif. Finally, this in-depth study of recognition of a self-antigen suggests that in part similar biophysical mechanisms shape TCR repertoires toward foreign and self-antigens.antigen recognition ͉ cytolytic T lymphocytes ͉ melanoma ͉ T cell receptors ͉ tumor immunity T he specificity of CD8 ϩ cytolytic T lymphocyte (CTL) responses relies on the interaction of clonotypically distributed antigen receptors (TCR) on the surface of the effector cell with small immunogenic peptide fragments displayed at the surface of a target cell by self-MHC class I molecules (pMHC) (1). Binding of the heterodimeric ␣␤-chains of the TCR to the pMHC complex is a key step leading to T cell activation and cell killing. The ␣␤ TCRs bind agonist pMHCs with relatively low affinity (K d Х1-100 M) through complementary determining regions (CDR) present on their variable domains (2). The mature TCR repertoire is shaped by both positive and negative intrathymic selection, leading to an estimated 2.5 ϫ 10 7 different TCR clonotypes in the human peripheral T cell pool (3). A relatively small number of CTL precursor cells is normally selected in response to the antigenic stimulus and comprises cells bearing several TCRs (Ϸ10 to Ϸ50 clonotypes) differing from each other yet having the ability to recognize the same pMHC complex. The recent development and availability of multimerized MHC-peptide complexes combined to TCR spectratyping with high-throughput DNA sequencing allowed the study of the development of antigen-driven CD8 ϩ T lymphocyte response to chronic antigenic exposure, e.g., in viral infection (CMV, EBV, HIV) or in cancer patients. The impact of TCR diversity on recognition of single antigenic pMHC complexes has been extensively investigated, and the relative contributions of each TCR ␤-chain have been addressed in several models. In a few systems, strong biases in the TCR repertoire selection of antigenspecific T cells, resulting in the preferentia...

T cell responses to viral epitopes are often composed of a small number of codominant clonotypes. In this study, we show that tumor Ag-specific T cells can behave similarly. In a melanoma patient with a long lasting HLA-A2/NY-ESO-1-specific T cell response, reaching 10% of circulating CD8 T cells, we identified nine codominant clonotypes characterized by individual TCRs. These clonotypes made up almost the entire pool of highly differentiated effector cells, but only a fraction of the small pool of less differentiated “memory” cells, suggesting that the latter serve to maintain effector cells. The different clonotypes displayed full effector function and expressed TCRs with similar functional avidity. Nevertheless, some clonotypes increased, whereas others declined in numbers over the observation period of 6 years. One clonotype disappeared from circulating blood, but without preceding critical telomere shortening. In turn, clonotypes with increasing frequency had accelerated telomere shortening, correlating with strong in vivo proliferation. Interestingly, the final prevalence of the different T cell clonotypes in circulation was anticipated in a metastatic lymph node withdrawn 2 years earlier, suggesting in vivo clonotype selection driven by metastases. Together, these data provide novel insight in long term in vivo persistence of T cell clonotypes associated with continued cell turnover but not replicative senescence or functional alteration.

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We have identified a novel complementary DNA (cDNA) corresponding to a gene overexpressed in the rat ventral prostate after castration. This cDNA displays 89.4% identity with 453 bp of a mouse EST and 81.5% identity with 157 bp of a human EST and was named PARM-1 for prostatic androgen-repressed message-1. The complete cDNA is 1187 bp long and codes for a protein of 298 amino acids that contains four potential glycosylation sites and three half cystinyl residues. The PARM-1 gene was found to be expressed at quite low levels in most rat tissues including those of the urogenital tract. The kinetic of induction of PARM-1 gene in the prostate was highly correlated to the development of apoptosis in the whole organ. Supplementation of castrated animals with androgens reversed both the process of apoptosis and the overexpression of PARM-1 gene. Supplementation with estrogens did not result in an increase in the PARM-1 messenger RNA levels when compared with the castration alone. However, the treatment resulted in a more rapid return to intact levels in the castrated plus estrogen group. When apoptosis of testis and prostate was induced in vivo by hypophysectomy, it was found that PARM-1 was only overexpressed in the prostate. Therefore, PARM-1 seems to be regulated by androgens only in the prostate. Using in situ hybridization and immunohistological techniques, we have shown that PARM-1 gene product is found exclusively in the epithelial cells of involuting prostate. Analysis by flow cytometry of MAT LyLu epithelial cells transiently expressing PARM-1 protein did not allow us to demonstrate a direct effect of PARM-1 gene overexpression on the programmed death of the transfected cells. Treatment of MAT LyLu cells by transforming growth factor-beta induced apoptosis but had no effect on PARM-1 production. However PARM-1 protein has been detected by Western blotting in various cell lines such as MAT LyLu, MAT Lu, and PIF, which are androgen independent. This would suggest that PARM-1 gene product would be a marker for acquired androgen-independence of these tumor cells.