Pharmacological blockade of Tpl2 significantly impairs human CTL cytokine secretion and lytic activity.

<p>CD8⁺CCR7^lo T_EM CTLs were sorted from healthy human PBMCs. (A) Sorted cells were stimulated with plate-bound anti-CD3 and rhIL-2 (200 U/mL)±indicated kinase inhibitors. Supernatants were collected at 24 h post stimulation. IFN-γ (top panel) and TNF-α (bottom panel) in the supernatant were measured using ELISA and mean±SEM is plotted for a representative of five similar experiments. (B) Sorted CD8⁺CCR7^lo T_EM CTLs were used in redirected lysis assay at the indicated Effector:Target ratios using anti-CD3 coated THP-1 target cells±Tpl2 inhibitor at the indicated concentrations. Killing of target cells is plotted as percent specific lysis, mean±SD plotted. Data shown are representative of two similar experiments from separate healthy adult donors. (C) Purified human CD8⁺ T cells were expanded for 5 days on anti-CD3/anti-CD28-coated plates in the presence of rhIL-12. Cells were rested for 24 h then restimulated with biotin-anti-CD8 + biotin-anti-CD3 with crosslinking streptavidin for 10 min in the absence or presence of the Tpl2 inhibitor at the indicated concentrations. Cell lysates were prepared and assessed for p-ERK and ERK by western blotting. Data are representative of 3 separate experiments.