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*

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Abstract
CD4−CD8− human thymocytes were obtained by treating total thymocyte suspensions with antiCD4 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 Cγ2-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 (IL2, 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-l-reactive, TcRγ/δ+ cells can be isolated from CD4−CD8− thymocytes cultured in IL2, and (b) the expression of CD8 antigen and of cytolytic activity reflects a true in vitro phenotypic change of CDK, noncytolytic precursors (and not the preferential growth of few contaminating cells).

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