USAGE OF T-CELL RECEPTOR V-BETA CHAIN GENES IN FRESH AND CULTURED TUMOR-INFILTRATING LYMPHOCYTES FROM HUMAN-MELANOMA

Tumor-infiltrating lymphocytes (TIL) freshly obtained from human malignant melanomas as well as the same TIL grown in the presence of interleukin 2 (IL2) were studied for gene expression of the T-cell receptor (TCR) variable beta regions (Vbeta). To perform the TCR-Vbeta analysis, total RNA was isolated from TIL and reverse-transcribed into cDNA, which was then amplified by PCR using 22 different 5' primers specifically recognizing the sequences of 20 Vbeta gene families and a 3' primer annealing to the constant region of the beta chain. The TCR-alpha constant region (Calpha) gene was co-amplified as a standard for the calculation of the percentage of each TCR-Vbeta gene expressed. The frequency of individual Vbeta regions expressed on TIL was computed from the ratio of cpm Vbeta to cpm Calpha for each Vbeta region in relation to the total of all 22 ratios. With fresh TIL obtained from 8 different melanomas, oligoclonal distribution of Vbeta genes expressed on TIL was observed, in comparison with a broader and unrestricted distribution seen with peripheral-blood T cells of 8 normal individuals. The oligoclonal patterns of Vbeta-gene expression in fresh melanoma TIL were distinct in every tumor. Several of the Vbeta-genes usually expressed in normal PBL were not expressed in fresh TIL. In melanoma TIL cultured in the presence of IL2 and IL4 and in the absence of autologous tumor (AuTu) or antigen-presenting cells for 23 to 65 days, selection of T-cell lines expressing a restricted number of Vbeta genes occurred. Although in 4/5 TIL cultures this selection involved the Vbeta7 gene, no relationship could be established between Vbeta gene expression in fresh TIL and that in T-cell lines outgrowing in long-term cultures. Selection in culture of CD3+CD8+ T-cell lines with Vbeta-gene expression restricted to 1 or 2 Vbeta families did not correlate with the presence or level of AuTu cytotoxicity mediated by these T cells. The results indicate that in TIL cultures random selection of T-cell lines with reactivity not relevant to AuTu may account for poor expression or loss of AuTu cytotoxicity by most TIL cultured long-term in the presence of cytokines and in the absence of specific antigenic stimulation.