Upon CD95L and anti-CD95 treatment we observed significantly more viable thymocytes from vavFLIPR mice compared with the number of WT thymocytes (Fig. 3A and B). In contrast, dexamethasone (Dex)-induced cell death, which proceeds via the glucocorticoid receptor in a death receptor-independent pathway, was not affected by the c-FLIPR transgene (Fig. 3A and B). To have a closer look into the time-course of apoptosis, thymocytes from WT and vavFLIPR animals were stimulated with CD95L for

up to 8 h. After 4 h of CD95L-stimulation, more early apoptotic (AnnexinV+ 7AAD−) WT cells than vavFLIPR cells were identified (Fig. 3C and D). After 8 h of stimulation, higher frequencies of both late apoptotic (AnnexinV+ 7AAD+) and early apoptotic WT cells were observed in comparison to vavFLIPR MK-1775 thymocytes (Fig. 3C and D). Taken together, WT thymocytes were rapidly

undergoing apoptosis, whereas vavFLIPR thymocytes were more resistant to CD95-induced apoptosis. Next, we examined the apoptosis sensitivity of peripheral T and B CH5424802 cells. Sorted CD4+ and CD8+ T cells as well as CD19+ B cells were stimulated with CD95L and Dex. Significantly, more viable (AnnexinV− 7AAD−) vavFLIPR CD4+ and CD8+ cells were identified upon CD95L stimulation compared to WT cells, while the Dex-treated controls were comparable between WT and vavFLIPR cells (Fig. 4A and B). Furthermore, sorted CD19+ B cells were activated with lipopolysaccharide (LPS) for 2 days to induce expression of the CD95 receptor before CD95L- and Dex-stimulation. Although activated B cells were fairly insensitive toward both

CD95L- and Dex-induced apoptosis, we detected significantly lower specific apoptosis of vavFLIPR B cells than of WT B cells (Fig. 4C). Again, the specific apoptosis of Dex-treated B cells was comparable between WT and vavFLIPR samples (Fig. 4D). Reactivation Evodiamine of the T-cell receptor leads to subsequent apoptosis via the death receptor pathway and the CD95 receptor has been shown to be involved in activation-induced cell death (AICD) [20-23]. To assay AICD, peripheral lymph node cells from WT and vavFLIPR mice were isolated and T cells were activated for 2 days with plate-bound anti-CD3 and anti-CD28 in presence of IL-2. Activated T cells were further expanded for 3 days in medium containing IL-2. Subsequently, AICD was assessed by restimulating T cells with plate-bound anti-CD3 to induce cell death on day 5. Also in this assay, cells from vavFLIPR mice showed significantly less specific apoptosis compared to WT cells (Fig. 4E). Thus, the c-FLIPR transgene is functional and protects primary immune cells against CD95-induced apoptosis and AICD. Next, we analyzed lymphocyte populations in vavFLIPR mice, since inhibition of CD95-induced apoptosis is often associated with alterations in lymphocyte numbers. However, total cellularity in spleen, peripheral lymph nodes, and thymus, was overall comparable between WT and vavFLIPR mice (Table 1).