As shown in Fig. 1A, significantly more dead Acalabrutinib concentration 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 5-Fluoracil solubility dmso 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 Phenylethanolamine N-methyltransferase 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.