In situ demonstration of renal-cell-carcinoma-specific T-cell clones

Using mixed lymphocyte tumor-cell culture (MLTC) in a selected renal-cell carcinoma, we derived a tumor-specific T-cell line in which Vbeta14+ and Vbeta19+ T cells represented 70% of the whole T-cell population. Selected Vbeta19+ T cells were CD8+ and exhibited a HLA-restricted specific cytotoxicity against tumor cells. Independently, 2 CTL clones were obtained by direct cloning of tumor-infiltrating lymphocytes, VIIIC2 CTL expressing a Vbeta19 and VIIB10 CTL a Vbeta13 T-cell-receptor transcript. VIIB10 lysed autologous tumor cells, normal kidney cells and EBV-transformed B cells. In contrast, VIIIC2 lysed tumor cells exclusively, demonstrating that the antigen structure recognized is tumor-specific. In addition, we used a PCR-based method to search for the presence of these CTL in situ. TCR beta chain of VIIIC2 and VIIB10 CTL were sequenced and primers complementary to their N regions were synthesized. VIIIC2 CTL constituted up to 60% of Vbeta19 transcripts in MLTC T-cell lines derived from tumor-infiltrating lymphocytes, 23% in tumor and 26% in a tumor-draining lymph node, while VIIB10 was not detected. Thus, VIIIC2 CTL was successfully derived from lymphocytes infiltrating a renal-cell carcinoma by direct cloning as well as by MLTC, probably because it was highly expanded in vivo within the tumor composing almost 2% of the TIL.