Fig. 5.

Increased basal level of T-bet in SHIP-deleted T cells due to altered cytokine signaling. (A) Proliferation of naive OT-II CD4cre SHIP^fl/fl T cells (filled diamonds) vs. OT-II SHIP^fl/fl controls (empty diamonds) in the presence of irradiated splenocytes and the indicated concentration of OVA peptide. (B) Proliferation of naive 5CC7 CD4cre SHIP^fl/fl T cells (filled squares) vs. 5CC7 SHIP^fl/fl controls (empty triangles) in the presence of irradiated P13.9 fibroblasts as described in Methods. (C and D) SHIP deletion does not alter T-bet or GATA-3 induction on antigen stimulation of T cells. Naive CD4⁺ CD62L⁺ cells from 5CC7 CD4cre SHIP^fl/fl mice (black bars) and 5CC7 SHIP^fl/fl control mice (gray bars) were stimulated with mitomycin C-treated P13.9 fibroblast cells that had been preloaded with 0.001–1 μM pPCC. Cells were harvested after 24 h of incubation, and relative mRNA levels (compared with L32 gene) for T-bet (C) and GATA-3 (D) were measured by real-time PCR. The experiment was performed twice with the same result. (E) T-bet and GATA-3 mRNA expression in naive freshly isolated CD4⁺ CD62L⁺ T cells from CD4cre SHIP^fl/fl (black bars) and SHIP^fl/fl mice (gray bars). (F) SHIP mediates TGFβ1 inhibition of IFNγ-induced T-bet up-regulation. Purified CD4⁺ CD62L⁺ T cells were incubated with IFNγ with or without TGFβ for 24 h, and the relative level of mRNA T-bet was assessed by real-time PCR. (G) Increased STAT phosphorylation in freshly purified T cells from CD4cre SHIP^fl/fl vs. control SHIP^fl/fl mice. (H) Increased STAT phosphorylation in IFNγ-activated T cells from CD4cre SHIP^fl/fl (Right) vs. control SHIP^fl/fl mice (Left). Th1-skewed T cells were incubated for 24 h with IL12 to up-regulate the IFNγ receptor level, rested for 4 h, and incubated with IFNγ for 15 min. The proportion of cells expressing pSTAT1 was calculated as the difference between anti-pSTAT intracellular flow cytometry (solid line) minus unstimulated control (gray blot).