FREQUENCY OF CYTOTOXIC T-LYMPHOCYTE PRECURSORS (CTLP) INTERACTING WITH AUTOLOGOUS TUMOR VIA THE T-CELL RECEPTOR - LIMITING DILUTION ANALYSIS OF SPECIFIC CTLP IN PERIPHERAL-BLOOD AND TUMOR-INVADED LYMPH-NODES OF MELANOMA PATIENTS

The frequencies of cytotoxic T-lymphocyte precursors (CTLp) that lyse autologous tumor by a T-cell receptor (TCR)-dependent mechanism (speci fic CTLp) were evaluated by limiting dilution analysis (LDA) using lym phocytes from peripheral blood (PBL) and from surgically resected, tum or-invaded lymph nodes (LNL) in 9 melanoma patients. The frequency of specific CTLp was determined in PBLs and/or LNIs of all patients by a modified LDA assay, enabling us to measure lytic activity on the autol ogous tumor that could be significantly inhibited by an anti-CD3 monoc lonal antibody (MAb). This assay allowed us to detect frequencies of s pecific CTLp ranging from 1/720 to 1/32,037 in peripheral blood and fr om 1/328 to 1/22,061 in tumor-invaded lymph nodes. These frequencies i ndicated that lymphoid populations from PBLs or LNLs of melanoma patie nts may contain as low as 30 to as much as 3,000 specific CTLp/10(6) l ymphocytes. In addition, comparison of wells containing specific CTLp with those showing no inhibition by anti-CD3 MAb indicated that specif ic CTLp represent between 3 and 88% of all precursors with lytic activ ity on the tumor. In 6 of 9 patients, no marked differences between PB Ls and LNIs in specific CTLp frequencies were found. A 10-fold increas e of specific CTLp, in comparison to PBL and LNL, was found only in ly mphocytes isolated from a subcutaneous metastasis of one patient. Our results indicate that CTLp interacting with autologous tumor by a TCR- dependent mechanism exist in PBL and LNL of most melanoma patients, al though a wide variation in their absolute number is evident among diff erent patients. (C) 1994 Wiley-Liss, Inc.