As shown in Fig. 1A, significantly more dead Hydroxychloroquine in vivo and apoptotic cells, as judged by staining with 7-amino-actinomycin D (7-AAD) and annexin V, respectively, were presented in anti-CD3+IL-2-activated WT CD8+ T cells (54 and 72%, respectively) than in similarly activated TNFR2−/− CD8+ T cells (13 and 17%, respectively). In contrast, essentially identical 7-AAD and annexin V staining data were obtained for both WT and TNFR2−/− CD8+ T cells when monoclonal anti-CD28 antibodies were included in the AICD assays (data not shown). These results indicate that AICD in either WT or TNFR2−/− CD8+ T cells is not regulated by CD28 costimulation. We have reported previously that TNFR2−/− CD8+ T cells

undergo suboptimal proliferation relative to WT CD8+ T cells when stimulated by anti-CD3 antibodies 6. This observation is consistent with the hypothesis that TNFR2 participates in the optimal activation of anti-CD3-stimulated CD8+ T cells. Here, we found that anti-TNFR2 antibodies also inhibited the proliferation of anti-CD3 stimulated WT CD8+ T cells (Fig. 1B). The specificity of the blocking anti-TNFR2 antibody was demonstrated by its lack of effect on the proliferation of anti-CD3-activated TNFR2−/− CD8+ T cells. This result indicates that in WT CD8+ T cells, optimal proliferation after anti-CD3

stimulation is dependent on TNFR2. We next determined whether antibody-mediated blocking of TNFR2 in WT CD8+ T cells recapitulates the effect of the TNFR2−/− mutation in AICD. We found that the blocking NVP-BKM120 order anti-TNFR2 antibody dramatically increased the resistance of activated WT CD8+ T cells to AICD (Fig. 1C). The specificity of the blocking anti-TNFR2 antibody was again demonstrated by its lack of effect on AICD of TNFR2−/− CD8+ T cells. These data indicate that TNFR2 is essential in

both the optimal proliferation of anti-CD3-activated CD8+ T cells and for the induction of AICD that terminates the proliferative response. To test the hypothesis that intracellular levels of TRAF2 regulate AICD, we determined MTMR9 the expression level of TRAF2 in TNF-α-stimulated WT and TNFR2−/− CD8+ T cells. WT and TNFR2−/− CD8+ T cells were stimulated for 48 h with anti-CD3+IL-2 followed by stimulation with TNF-α for various times. Immunoblotting showed that the amount of TRAF2 protein in WT cells decreased by 6 h after adding TNF-α (Fig. 2A). In contrast, the amount of TRAF2 protein in TNFR2−/− cells remained unchanged, even after 12 h of TNF-α stimulation. Furthermore, we found that TRAF2 protein levels were lower in WT CD8+ T cells than in TNFR2−/− cells at 72 h after anti-CD3+IL-2 stimulation (Fig. 2B). These data indicate that TNFR2 signaling promotes the degradation of TRAF2 at a time when AICD occurs and suggests that intracellular levels of TRAF2 play a critical role in regulating AICD. We next determined the effect of TNFR2 blocking on intracellular TRAF2 levels.