INVOLVEMENT OF APOPTOSIS ANTIGEN FAS IN CLONAL DELETION OF HUMAN THYMOCYTES

Apoptosis appears to play a major role in the differentiation and sele ction of T and B lymphocytes, but the mechanisms of clonal deletion of T cells in thymus are not well understood. We have prepared an anti-h uman Fas IgM mAb with associated apoptosis-inducing activity in Fas an tigen-positive target cells including human T cells. We analyzed the e xpression of apoptosis antigen Fas on human thymocytes by cytofluorome try showing low, but significant amounts of Fas antigen on double-nega tive and double-positive undifferentiated thymocytes. On the contrary, most of the differentiated thymocytes (single-positive or CD3-brighte st) expressed undetectable levels of Fas antigen. About 1-2% of thymoc ytes expressed high amounts of Fas antigen, and these cells, which wer e CD3-bright, were CD4-bright and CD8-low at the stage of late double- positive lineage. Immunohistological analysis shows these Fas-bright c ells on the edge of the medulla, Stimulation through the TCR complex w as shown to induce the expression of Fas antigen on thymocytes at the late double-positive stage and prolonged stimulation through the TCR c omplex rendered the Fas-bright thymocytes sensitive to apoptosis-induc ing activity of anti-fas. To show the involvement of the Fas system in the negative selection/clonal deletion of thymocytes, we organ-cultur ed human thymus in the presence of the superantigen, staphylococcus en terotoxin B (SEE), and new antagonistic anti-fas mAb, which can inhibi t the apoptosis-inducing activity of the original anti-fas mAb. The SE E-reactive TCR complex on thymocytes was at first down-regulated by SE E, then the SEE-reactive clone was deleted by apoptosis, which was inh ibited by an antagonistic anti-fas mAb. Thus, Fas antigen is shown to be involved in the negative selection/ clonal deletion of superantigen -reactive thymocytes.