Figure 2. DGKα silencing or inhibition restores RICD in SAP-silenced T cells.

(A) Activated normal donor T cells were transfected with control or SAP siRNA and restimulated 4 days later with OKT3 Ab. After 24 hours, % cell loss was evaluated by PI staining. Data are mean ± SEM of 3 experiments performed in triplicate.

(B) SAP expression in siRNA-transfected T cells from (A) was measured by qRT-PCR (upper panel, mean ± SEM of 4 experiments) or by Western blotting, with actin as a loading control (lower panel).

(C–D) siRNA-transfected cells (A) were restimulated with OKT3 Ab following pretreatment with DMSO, DGK inhibitor R59949 or R59022 (5–10 μM). After 24 hours, the % cell loss was evaluated by PI staining. Data are mean ± SEM of 5 experiments (C), or 5 (control) and 8 (SAP siRNA) independent experiments (D) performed in triplicate.

(E) siRNA-transfected cells as in (A) were pretreated with DMSO or R59022 (10 μM) and restimulated with OKT3 (10 ng/ml). After 12 hours, the % apoptotic cells was evaluated by AnnexinV staining. Representative histograms are shown; marker numbers denote % AnnexinV⁺ cells. The net increase in AnnexinV⁺ cells at 12 hours is shown at right. Data are mean ± SD of 4 experiments.

(F) siRNA-transfected cells (A) were treated with C8-DAG (50 μM) and restimulated with OKT3 Ab. After 24 hours, % cell loss was evaluated by PI staining. Data are mean ± SEM of 5 experiments performed in triplicate. Asterisks denote statistical significance by two-way ANOVA with Sidak correction (A,C,D–F) or paired t-test (B,E).

(G) Schematic cartoon: pro-apoptotic TCR signaling is governed by DGKα inhibition in activated T cells.