Analysis of T cell receptor alpha beta variability in tumor-infiltrating lymphocytes in primary and metastatic melanoma.

The T cell receptor (TCR) alpha beta variable (V) gene family usage of tumor-infiltrating lymphocytes (TIL) in different primary human malignant melanomas and corresponding metastatic lesions were characterized using a recently developed method using the reverse transcription coupled polymerase chain reaction (RT-PCR). This semiquantitative RT-PCR method could be adapted to analysis of formalin-fixed, paraffin-embedded histopathological samples of primary tumor material and demonstrated to be reproducible and to be useful for the assessment of V alpha- and V beta-gene family usage in tumor samples. The TIL in primary tumors were observed to preferentially express certain TCR V alpha- and V beta-gene families: V alpha 4, and V beta 8 were highly expressed in several of the primary tumors analyzed using this method. With respect to V alpha 22 and V beta 8, the preferential expression of these V-gene families was demonstrated to be due in situ clonal expansion of T cells by means of cloning and sequencing of the CDR3 regions (V-J or V-D-J, respectively) corresponding to the RT-PCR products from one of the primary tumors. The observed preferential usage of certain TCR V alpha and V beta-genes strongly suggest the in situ clonal expansion of specific populations of T cells in accordance with recent results from others. These clonal T cell populations probably react with certain melanoma-associated peptides presented by specific HLA molecules. The preferential usage of certain V alpha- and V beta-gene families observed in several tumors further supports the involvement of a limited number of shared melanocyte or melanoma-associated peptides. Since the HLA status of the patients is obviously important to interpret these results, some of the patients were typed for HLA-A1 and -A2, the two most well-characterized restriction elements for melanoma-associated antigens, either serologically or by a newly developed RT-PCR method which similarly could by applied directly to the tumor material. In TIL in primary melanomas, a possible correlation was suggested between HLA-A2 and the preferential usage of the TCR V-gene families V alpha 4, V alpha 5, V alpha 22 and V beta 8, whereas the V beta 3-gene family appeared to be expressed together with HLA-A1. The V-gene families which were highly expressed in the primary tumors were generally not, or only very weakly, expressed in the corresponding metastases and vice versa, possibly reflecting a substantial change in the phenotype of the metastatic melanoma target cells. Continued studies of larger patient materials will be necessary to extend and validate these conclusions and of obvious interest for the further analysis of the T cell response in melanoma.