Figure 1.

TCR-independent and TCR-dependent mechanisms of DP thymocyte apoptosis. Antibodies against cell surface molecules that possess costimulatory/coactivating activity and antibodies against TNFR family members were assessed for their ability to induce apoptosis of rigorously purified DP thymocytes. Control EtBr percentages ranged between 18 and 30% in all experiments presented in this report. (a) Only TNF-α and fas induce death of DP thymocytes in the absence of TCR stimulation. DP thymocytes isolated from young adult female B6 mice were stimulated by platebound antibodies or by 100 ng/ml recombinant murine TNF-α. Cells were harvested after overnight incubation and percent cell death was quantitated by EtBr staining (see Materials and Methods). (b) Only CD28 cooperates with TCR to induce death of DP thymocytes. Cells were stimulated by platebound antibody combinations as indicated. Anti–TCR-β (H5-597) was plated at a concentration of 10 μg/ml. Even though anti-CD28 antibody was unique in its ability to induce cell death, every experimental antibody enhanced TCR-mediated upregulation of CD5 (data not shown). In a and b, each experimental antibody (other than anti-CD28) was plated at both 10 and 50 μg/ml concentrations. Results from the 10 μg/ml plating preparations are shown and were indistinguishable from results with 50 μg/ml concentrations. Cells were harvested and percent cell death was quantitated by EtBr staining (Materials and Methods).