3A), and their increased resistance to AICD (Fig. 1C). To directly test whether AICD in activated CD8+ T cells depends on the level TRAF2, we determined whether increasing TRAF2 levels in WT CD8+ T cells by expressing an exogenous TRAF2 protein would increase the resistance of these cells to AICD. We

used a retroviral expression method to overexpress the TRAF2-EGFP fusion protein in activated WT CD8+ T cells as described in the Materials and methods. FACS analysis indicated that the infection efficiency of the control EGFP and TRAF2-EGFP vectors was similar (data not shown). The EGFP+ and TRAF-EGFP+ cells were purified and stimulated with R428 ic50 anti-CD3+IL-2 and the percentages of live/dead/apoptotic cells analyzed at the indicated time points. Our data showed that the overexpression Rapamycin of TRAF2-EGFP increased the percentage of live cells from 11.1% (in cells transfected with the control EGFP vector) to 40.2% (in cells transfected with the TRAF2-EGFP vector) and reduced the number of dead cells from 64 to 48.1% after 24 h of restimulation with anti-CD3+IL-2 (Fig. 3B). Similar

results were observed after 48 h of restimulation with anti-CD3+IL-2 (Fig. 3B). However, there was no significant difference in the percent of apoptotic cells at either 24 or 48 h of restimulation with anti-CD3+IL-2 (Fig. 3B). Similar results were also observed after 6 or 12 h of restimulation of the transfected cells (data not shown). These data indicate that the TRAF2

overexpression promotes the survival of activated WT CD8+ T cells in the AICD assay. Our data support the hypothesis that the TNFR2-induced decrease in TRAF2 levels is required for TNFR2-induced cell death and AICD. Thus, decreasing the expression of TRAF2 in the TNFR2−/− CD8+ T cells would mimic the TNF-induced decrease in TRAF2 seen in the WT cells Myosin and should result in enhanced cell death. To provide support for this hypothesis we used small interfering RNA (siRNA) to knock down endogenous TRAF2 expression in activated TNFR2−/− CD8+ cells and determined its effect on AICD in these cells. Two TRAF2-specific siRNA oligonucleotides (si523 and si537) were used to decrease TRAF2 protein level in both activated WT and TNFR2−/− CD8+ T cells as described in the Materials and methods. The TRAF2-specific oligonucleotides (si523 or si537) were very efficient in abrogating the expression of TRAF2 (Fig. 4A). Furthermore, the specificity of TRAF2 knock down was indicated by the lack of effect on TRAF2 expression following the expression of TRAF1-specific oligonucleotides (si807 or si828) under the same conditions (Fig. 4A). We found that TRAF2 knockdown rendered anti-CD3+IL-2-activated TNFR2−/− CD8+ T cells as sensitive to AICD as similarly activated WT CD8+ T cells since similar percentages of dead and apoptotic cells were observed in both groups in the AICD assay (Fig. 4B).