ANALYSIS OF T-CELL RECEPTOR BETA- AND GAMMA-GENES FROM PERIPHERAL-BLOOD, REGIONAL LYMPH-NODE AND TUMOR-INFILTRATING LYMPHOCYTE CLONES FROM MELANOMA PATIENTS

A total of 199 T cell clones from two melanoma patients were derived from progenitor T cells from recurrent melanoma, regional lymph nodes (either involved or uninvolved with malignancy) and peripheral blood by inoculating single cells directly into the wells of microtiter plates before in vitro expansion. The surface marker phenotype of most clones was CD4+CD8-, although some were CD4-CD8+. Genomic DNA prepared from all clones was analyzed by Southern blot hybridization using T cell receptor (TCR) beta and gamma gene probes, seeking clones with identical TCR gene rearrangement patterns as direct evidence for in vivo progenitor T cell clonal amplification. Probing HindIII-digested DNA with TCR-beta and TCR-gamma probes revealed several clones with identical TCR gene rearrangement patterns. These clones had subsequent probing of BamHI-digested DNA with TCR-beta and TCR-gamma probes, which showed all but 2 clones to have distinct rearrangement patterns. These analyses provide clear molecular evidence for in vivo polyclonal CD4+ T cell populations in each of several separate immune compartments in these patients.