(A, B) S4KO, EKO, ES4KO (gray), and control mice (black) were activated in vitro and stimulated with cytokines. Graphs show cytotoxic T lymphocytes (CTLs) analyzed 96 hr after T cell receptor (TcR) stimulation. Bars are CTLs stimulated with rIL-2 alone (filled bars), rIL-2 plus SB431542 (no fill), and rIL-2 plus transforming growth factor β (TGFβ) (hatched). Data are means ± standard deviation (SD) (3 mice per group). p values were calculated using Student’s t tests. Two independent experiments produced similar results. (A) Percentages of CD8 T cells that expressed CD103. (B) Percentages of CD8 T cells that expressed CD62L. (C) S4KO, EKO, and ES4KO mice were infected with X31-OVA and anti-viral CTLs were analyzed with MHCI tetramers at 33 dpi. Anti-CD8 antibodies were injected 5 min before sacrifice. Contour plots show OVA-specific CTLs analyzed for CD103 and CD62L expression. (D, E) S4KO and S4Ctrl cells were activated in vitro (48 hr) and transduced with a retroviral vector encoding EOMES (Eomes-IRES-GFP) or the empty vector control (EV-IRES-GFP). (D) GFP+ cells were analyzed for CD103 and CD62L expression after 6 days in culture. (E) S4KO and S4Ctrl cells were transferred to B6 mice that were previously infected (24 hr) with X31-OVA. GFP+ cells in the lungs were analyzed for CD62L and CD103 expression at 35 dpi. Data are means ± SD (3 mice per group). Two independent experiments produced similar results.