CROSS-LINKING THE TCR COMPLEX INDUCES APOPTOSIS IN CD4(-A()8(+) THYMOCYTES IN THE PRESENCE OF CYCLOSPORINE)

Although it is generally agreed that TCR ligation is a minimal require ment fur negative selection in the CD4(+)8(+) double-positive (DP) thy mocyte subset, the costimulatory requirements and specific signaling e vents necessary to induce apoptosis are not well defined. We have expl ored the consequences of cross-linking CD3/TCR complexes on thymocytes from H-Y TCR transgenic (Tg) mice. In agreement with previous reports , we demonstrate that culturing DP thymocytes with plate-bound anti-TC R antibody induces downregulation of CD4 and CD8 and upregulation of C D69 expression. Nevertheless, the activated cells did not undergo apop tosis, as determined by viable cell recoveries and by quantitation of DNA fragmentation using the TUNEL assay. However, specific depletion o f the DP subset occurred within 24 hr when thymocytes were incubated i n the presence of both anti-TCR and the immunosuppressant cyclosporin A (CsA). CsA also induced depletion of anti-CD3 stimulated normal DP t hymocytes. Using mice homozygous for the lpr or gld mutation, we also have shown that Fas/Fas ligand interactions are not involved in the Cs A-induced death of TCR-stimulated DP thymocytes. These data verify tha t TCR cross-linking alone is insufficient to induce apoptosis of DP th ymocytes and further suggest that TCR stimulation activates a CsA-sens itive protective pathway that interferes with signaling events leading to apoptosis in DP thymocytes.