Clonal analysis of CD4-CD8- human thymocytes expressing a T cell receptor γ/δ chain. Direct evidence for the de novo expression of CD8 surface antigen and of cytolytic activity against tumor targets

CD4^-CD8^- human thymocytes were obtained by treating total thymocyte suspensions with anti-CD4 and anti-CD8 monoclonal antibodies (mAb) and complement. The resulting cell populations contained virtually no CD4⁺, CD8⁺ or WT31⁺ cells and 17-65% CD3⁺ cells. In addition, analysis of cell reactivity with delta-TCS-1 mAb (specific for the C(γ)2-encoded, nondisulfide-linked form of TcR γ/δ), revealed the presence of a variable proportion of δ-TCS-1⁺ cells (the % of δ-TCS-1⁺ cells were lower than the percentage of CD3⁺ cells). Upon culture in recombinant interleukin 2 (IL 2, in the presence of irradiated mononuclear cells), CD4^-CD8^- thymocytes underwent extensive proliferation. In addition, a progressive increase of CD8⁺ cells (but not of CD4⁺ or WT31⁺ cells) could be detected. Cells also progressively acquired cytolytic activity against K-562 or fresh melanoma cells. Fresh CD4^-CD8^- thymocytes were cloned under limiting dilution conditions. The cloning efficiencies were relatively high (1/3 cells); in addition, virtually all the clonal progenies obtained displayed cytolytic activity and expressed the CD3⁺WT31^-δ-TCS-1⁺ surface phenotype. About half of the clones analyzed were CD8⁺, whereas none expressed CD4 antigens. We conclude that (a) only δ-TCS-1-reactive, TcR γ/δ⁺ cells can be isolated from CD4^-CD8^- thymocytes cultured in IL 2, and (b) the expression of CD8 antigen and of cytolytic activity reflects a true in vitro phenotypic change of CD8^-, noncytolytic precursors (and not the preferential growth of few contaminating cells).