Figure 6. Host CD4⁺ T cells are required for the development of a tumor-specific memory T-cell response, but not for tumor protection mediated by chNKG2D-expressing T cells. (A) C57BL/6 and MHC Class II-deficient mice bearing 5-week ID8 tumors were treated with wtNKG2D-expressing T cells or chNKG2D-expressing T cells. Eight weeks after tumor cell injection, the number of solid tumors on the peritoneal wall and number of tumor cells in the peritoneal wash was assessed. Cumulative data of two independent experiments are shown. The average of each group and SD (n = 8) are shown (**p < 0.01; ***p < 0.001 as compared with C57BL/6 mice receiving wtNKG2D-expressing T cells; ^†p < 0.05 as compared with C57BL/6 mice receiving chNKG2D-expressing T cells). (B) MHC Class II-deficient mice bearing ID8 tumors were treated with wtNKG2D-expressing, chNKG2D-expressing or purified CD8⁺ chNKG2D-expressing T cells 5 weeks after tumor inoculation. The number of solid tumors on the peritoneal wall and number of tumor cells in the peritoneal wash was assessed. Cumulative data of two independent experiments are shown. The average of each group and SD (n = 8) are shown. (***p < 0.001 as compared with mice receiving wtNKG2D-expressing T cells; ^†p < 0.05 as compared with mice receiving chNKG2D-expressing T cells). (C) C57BL/6 or MHC Class II-deficient mice bearing ID8 tumors were treated with chNKG2D-expressing T cells and control C57BL/6 mice were treated with Hank’s balanced salt solution (HBSS) 1, 2 and 3 weeks after tumor-cell inoculation. The survival of the mice was measured (n = 8–10 mice) (*p < 0.01 as compared with HBSS-treated mice). (D) Two-hundred days after tumor-cell injection, spleen cells from surviving or naïve mice were cultured with ID8 cells, RMA tumor cells or medium alone. Cell-free supernatants were analyzed for interferon γ (IFNγ) production (***p < 0.001 as compared with spleen cells from naïve mice; ^†p < 0.01 vs. as compared with spleen cells from MHC Class II-deficient mice). (E) Spleen cells from tumor-surviving mice (day 200) or from naïve mice were stimulated with ID8 tumor cells and IFNγ production by CD8b⁺ T cells was assessed by intracellular flow cytometry (*p < 0.05 as compared with spleen cells from naïve mice).