Figure 5. Mcl-1 is up-regulated and dephosphrylated at Ser159/Thr163 by IL-7 in tumor-exposed T cells.

(A) T cells or tumor-exposed T cells harvested immediately (0h) and 12h after co-incubation with Tu167 tumor cells were harvested. Tumor-exposed T cells + IL-7 were harvested 0.5h, 2h or 12h after co-incubation with tumor. Tumor-exposed T cells + IL-7 and LY294002, AKT inhibitor IV, or GSK3β inhibitor X for 12h were harvested. Total Mcl-1, pSer159/Thr163-Mcl-1 and β-actin (loading control) in all samples were analyzed by Western blot. (B) T cells were coincubated with Tu167 tumor cells, washed and cultured in media with or without a single dose of IL-2 (100 U/ml), IL-7 (20ng/ml), or IL-12 (20ng/ml). The expression of Mcl-1 was assessed by flow cytometry on day 2, 4, and 7; the mean fluorescence intensity (MFI) of Mcl-1 is shown. (C) Control and Mcl-1-siRNA transfected T cells were cultured for 36h, then co-incubated with Tu167 for 6h at a ratio of 1:1 and then cultured with or without IL-7. Mcl-1 expression was examined by Western blotting (lower panel). On day 5, we evaluated loss of CD27 and CD28 by flow cytometry. Representative data are shown in the upper panel. (D) TW37 but not ABT737 abrogates IL-7 protection. Tumor-exposed T cells +/− IL-7 with and without TW37 or ABT737 were cultured for 7 days before staining for loss of CD27 and CD28 expression. Data from 3 independent experiments are shown in (D), with each bar representing the mean of triplicate values ±SD.