Figure 6
A20 reduces CD95 expression by inhibition of NF-κB activation.
(a) Histograms display MFI of CD95 ex vivo on Lm OVA-specific CD8+ T cells at the indicated time points p.i. (b) Ex vivo kinetic of active caspase-3/7 in Lm OVA-specific CD8+ T cells at the indicated time points p.i. (c) CD8+ T cells were in vitro stimulated with anti-CD3/CD28 and flow cytometric analysis for CD95 was performed at the indicated time points. (d) Frequency of living cells was determined after in vitro stimulation with anti-CD3/CD28. Cell death was determined by flow cytometry, staining for annexin V and 7-AAD. (e) T cells were stimulated with anti-CD3/CD28, treated with IKK-inhibitor or were left untreated. mRNA was isolated and levels of CD95 were determined. (f) CD8+ T cells were stimulated in vitro with anti-CD3/CD28 and recombinant CD95L for the indicated time points. Active caspase 3/7 was measured using DEVD substrate. (g) Frequency of living cells was determined after CD8+ T cells were stimulated with CD95L for 2 h. To inhibit apoptosis, Z-VAD-FMK was added prior to the stimulation. (h) Active caspase-3/7 was determined before and after AICD. (a,b) Cells were gated on singlets and H2-Kb SIINFEKL+ CD8+ T cells. A representative of 2 independent experiments is shown, with 3 mice per group. Error bars indicate + SEM. (a–d,f,h) Student’s t-test. (e,g) Tukey’s multiple comparison test; *p < 0.05; **p < 0.01.
