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 malig nant melanomas as well as the same TIL grown in the presence of interl eukin 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, w hich was then amplified by PCR using 22 different 5' primers specifica lly recognizing the sequences of 20 Vbeta gene families and a 3' prime r annealing to the constant region of the beta chain. The TCR-alpha co nstant region (Calpha) gene was co-amplified as a standard for the cal culation of the percentage of each TCR-Vbeta gene expressed. The frequ ency of individual Vbeta regions expressed on TIL was computed from th e ratio of cpm Vbeta to cpm Calpha for each Vbeta region in relation t o the total of all 22 ratios. With fresh TIL obtained from 8 different melanomas, oligoclonal distribution of Vbeta genes expressed on TIL w as observed, in comparison with a broader and unrestricted distributio n seen with peripheral-blood T cells of 8 normal individuals. The olig oclonal patterns of Vbeta-gene expression in fresh melanoma TIL were d istinct in every tumor. Several of the Vbeta-genes usually expressed i n 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. Alt hough in 4/5 TIL cultures this selection involved the Vbeta7 gene, no relationship could be established between Vbeta gene expression in fre sh TIL and that in T-cell lines outgrowing in long-term cultures. Sele ction in culture of CD3+CD8+ T-cell lines with Vbeta-gene expression r estricted 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 re activity 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.