Fig 1: The reverse signaling of tmTNF-a enhances the sensitivity of T cells to TNF-a-induced AICD through upregulating TNFR(A, B, C, D) Jurkat cells were stimulated with PHA-P (5 µg/ml) in the absence or presence of TNF-a pAb (1:100) for 24 h. PHA preactivated T cells were reactivated by aCD3 (10 µg/ml) with or without TNF-a pAb (1:100) for 24 h. The expression of tmTNF-a (A, B) and two types of TNFR (C, D) was detected by flow cytometry. (E, F) Jurkat cells activated for 3 h by PHA-P (5 µg/ml) with or without TNF-a pAb were incubated for 4 h with sTNF-a (50 U/ml) or tmTNF-a stably transfected and fixed NIH3T3 cells at an effector/target ratio of 10:1. The apoptosis was detected by Annexin V/PI. (G, H, I) Jurkat cells transfected with 100 nM siRNA for TNFR1 or TNFR2 for 48 h were activated by PHA-P (5 µg/ml) with TNF-a pAb for 3 h, followed by incubation for 4 h with sTNF-a (50 U/ml) or tmTNF-a stably transfected and fixed NIH3T3 cells at an effector/target ratio of 10:1. The expression of TNFR1 and TNFR2 was analyzed by flow cytometry (G) and the apoptosis was detected by Annexin V/PI (H, I). For neutralization of tmTNF-a, the effector cells were treated with TNF-a pAb for 30 min prior to the addition to the target cells. All the quantitative data represent means ± S.D. of at least three independent experiments. *p<0.05, ** p<0.01, *** p<0.001.