E. Weidmann et al., USAGE OF T-CELL RECEPTOR V-BETA CHAIN GENES IN FRESH AND CULTURED TUMOR-INFILTRATING LYMPHOCYTES FROM HUMAN-MELANOMA, International journal of cancer, 54(3), 1993, pp. 383-390
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.