CCR5 activation induces a caspase-dependent death of CD4 T cells. (a) CD4 T cells were incubated with 293T cells transfected with empty vector or 293T cells expressing the HIV_JRFL-env (R5env), either untreated or treated with soluble CD4 at different concentrations (sCD4). 293T cells were lysed and blotted by duo, tropic anti-gp160 to verify envelope expression (left panel). CD4 T cells were left untreated, pretreated with Z-IETD (a caspase 8 inhibitor) or Z-LHED (a caspase 9 inhibitor) before incubation with 293T cells expressing empty vector or R5env (right panel). (b) CD4 T cells treated or not treated with Z-VAD, SDF1α, or MIP1β were incubated with 293T cells expressing R5env or empty vector pretreated or not pretreated with sCD4, respectively. (c) CD4 T cells were treated with IgG-matched isotype control, anti-CD4 Leu-3a (αCD4 Ab), or anti-CCR5 MAB183 (αCCR5 Ab), followed by their cross-linking with goat anti-mouse Ab’s. Each point was assessed for caspase-dependent death (Z-VAD) as well as for Fas susceptibility (αFas). (d) CD4 T cells from a healthy donor with the CCR5 Δ32 mutation or from a control donor lacking the Δ32 CCR5 deletion were pretreated or not pretreated with Z-VAD and coincubated with 293T cells expressing R5env or empty vector, pretreated or not pretreated with sCD4.