Exclusive role of CD4⁺ T cells in mediating antitumor activity. (A) A group of mice were vaccinated with A20/veh, A20/αGC, or left untreated (Nil) on day −7. A20/αGC-vaccinated mice were injected intraperitoneally with depleting Abs of CD4, CD8, or NK cells, respectively, on days −3 and −1 (n=5-7 mice per group) before all mice were inoculated intravenously with live A20 on day 0. Survival was monitored daily. (B) BALB/c mice were vaccinated with A20/αGC. At 7 days later, CD4⁺ or CD8⁺ cells from lymphoid cells of A20/αGC-vaccinated mice were sorted and transferred intravenously into syngeneic naive mice (2-3 × 10⁷/transfer) on day 0 (n=4-5 mice per group). A20/αGC-vaccinated mice (day −7) were used as a positive control. All mice were inoculated intravenously with live A20 on day 0, and the survival was monitored daily. P values are calculated in comparison with the nonvaccinated (Nil) group. (C) BALB/c mice were vaccinated with A20/αGC. At 7 days later, CD1d tetramer⁻CD4⁺ cells from lymphoid cells of A20/αGC-vaccinated mice were sorted and transferred intravenously into syngeneic naive mice (2 × 10⁷/transfer) on day 0. Nontreated naive mice were used as a control (Nil). All mice were inoculated intravenously with live A20 cells on day 0, and the survival was monitored. P values are calculated in comparison with nontreated group. (D,E) CD1d tetramer⁻CD4⁺ T cells were sorted from vaccinated mice by using FACSAria (n=3 per group). Sorted CD4⁺ T cells (4 × 10⁵/well) were cultured in the presence or absence of irradiated A20 cells (1 × 10⁵/well) for 24 hours, and ELISPOT (D) or intracellular cytokine staining (E) was performed for analysis of IFN-γ and TNF-α. Values in panel D are means (±SEM). Values in panel E are percentages of the gated cells.