Again, neutralizing TNF-α did not cause a decrease in TRAF2 expression levels in activated WT cells, presumably because the TNFR2-mediated degradation of TRAF2 opposes this effect of TNFR1 (Fig. 5D). These data indicate that signaling through TNFR1 is required for maintaining high TRAF2 levels in TNFR2−/− CD8+ T cells. They also provide further support for the hypothesis that in TNFR2−/− CD8+ T cells, TNFR1 functions as a survival receptor by Angiogenesis inhibitor regulating TRAF2 levels in these cells. NF-kB is a key transcription factor that regulates many pro-survival

genes in activated T cells 18, 19. To provide further evidence that TNFR1 functions as a pro-survival receptor in TNFR2−/− CD8+ T cells, we measured the level of NF-κB activation in these cells by quantifying the level of phosphorylated IκBα in these cells. We found that the AICD-resistant TNFR2−/− CD8+ T cells expressed higher levels

of phosphorylated IκBα compared with similarly activated WT CD8+ T cells (Fig. 6A). Consistent with the idea that TNFR2 signaling opposes NF-κB activation, we found that blocking TNFR2 in WT cells also led to increased levels of phosphorylated IκBα (Fig. 6A). As expected, the anti-TNFR2 antibody had no effect on phosphorylated IκBα levels in TNFR2−/− CD8+ T cells. We also determined that the effect of neutralizing endogenously produced TNF-α on the levels of phosphorylated IκBα in activated WT and TNFR2−/− CD8+ cells. In TNFR1+/+ TNFR2−/− CD8+ T cells, blocking TNF-α signaling Bcl-w led to selleck a decrease in the levels of phosphorylated

IκBα (Fig. 6B). Independent evidence for increased NF-κB activation in anti-CD3+IL-2-activated TNFR2−/− CD8+ T cells was obtained with the TransAM p65 Transcription Factor Assay. In this assay, an oligonucleotide containing an NF-κB consensus-binding site is immobilized to a 96-well plate. Activated NF-κB homodimers and heterodimers contained in nuclear extracts specifically bind to this consensus oligonucleotide. Binding of the p65 (RelA) subunit is detected by specific antibodies and the amount of binding is quantified by ELISA. We found that the nuclear extracts of activated TNFR2−/− CD8+ T cells possessed significantly more p65 binding activity relative to similarly activated WT CD8+ T cells (Fig. 6C). The specificity of the p65 binding to the NF-κB consensus site is indicated by complete abrogation of p65 binding with a WT oligonucleotide but not a mutated form of the oligonucleotide (Fig. 6C). Furthermore, blocking activated WT CD8+ T cells with anti-TNFR2 antibodies increased p65 binding to that observed in activated TNFR2−/− CD8+ T cells and neutralizing TNF-α decreased p65 binding in activated TNFR2−/− CD8+ T cells to WT levels (Fig. 6C).