Enhanced antileukemia activity induced by WT1 peptide vaccination requires CD8⁺ T cells from WT1 peptide vaccinated donors combined with CD4⁺ T cells. (A) Vaccination and bone marrow transplant scheme was the same as in Figures 3A and 4A. (B and D) Survival after allogeneic LP/J→C57BL/6 (B) or C3H.SW→C57BL/6 (D) transplantation (n = 10 mice per group) with 5 × 10⁶ T-cell depleted bone marrow cells (○) from IFA vaccinated donors or with T-cell depleted bone marrow from IFA alone vaccinated donors and combination of 6 × 10⁶ CD4 T cells and 2 × 10⁶ CD8 T cells from IFA alone vaccinated donors (□), 6 × 10⁶ CD4 T cells from WT1 peptide and IFA vaccinated donors (■), 2 × 10⁶ CD8 T cells from WT1 peptide and IFA vaccinated donors (●), or combination of 6 × 10⁶ CD4 T cells and 2 × 10⁶ CD8 T cells from WT1 peptide and IFA vaccinated donors (▴; ● vs ▴, B, P < .001; D, P = .002). (C-E) Bioluminescence intensity (mean ± SE) of only surviving mice at each imaging time point in B and D (● vs ▴, C, P < .001; E, P < .001 at day 28). (F) Representative flow cytometry plots show percent of gated CD8⁺ T cells stained positively for CD44 and intracellular IFN-γ (box) after 24-hour coculture with irradiated FBL3 tumor cells or H11 tumor cells (negative control). (G) Means are of 5 mice per group (*P < .001). PBLs from recipient C57BL/6 mice in F and G were isolated 28 days after cell transfer of 6 × 10⁶ CD4 T cells and 2 × 10⁶ CD8 T cells from WT1 peptide and IFA-vaccinated donors (group ▴ in Figure 5D).