WT1-tetramer⁺ CD8⁺ T cells from WT1-vaccinated donors provide attenuated antileukemia activity against FBL3, and require CD4⁺ T cells for potent activity. Donor vaccination scheme was the same as in Figure 5A. (A) Representative analysis of WT-1–specific CD8 T cells from vaccinated donors that were identified and sorted based on 2-color positive staining for 2 WT1-tetramers and for CD44 expression. CD44^hiWT1-tetramer⁺ CD8 T cells were injected with or without CD4 T cells into irradiated C57BL/6 recipients. (B-D) Survival after allogeneic (B) LP/J→C57BL/6 and (D) C3H.SW→C57BL/6 transplantation (n = 10 mice per group) with 6 × 10⁶ CD4 T cells from IFA alone vaccinated donors (□), 1 × 10⁵ WT1-tetramer⁺ CD8⁺ T cells from WT1 peptide and IFA vaccinated donors (●), combination of 1 × 10⁵ WT1-tetramer⁺ CD8 T cells and 6 × 10⁶ CD4 T cells both from WT1 peptide and IFA vaccinated donors (▴; ▴ vs ●, B, P < .001, D, P < .001) and combination of 1 × 10⁵ WT1-tetramer⁺ CD8 T cells from WT1 peptide and IFA vaccinated donors and 6 × 10⁶ CD4 T cells from IFA alone vaccinated donors (■; ■ vs ●, B, P < .001; D, P < .001). (C-E) Bioluminescence intensity(mean ± SE) of only surviving mice at each imaging time point in panels B and D (■ vs ●, C, P < .001; E, P < .001 at day 14). (F) PBLs from recipient C57BL/6 mice were isolated 28 days after cell transfer of 1 × 10⁵ WT1-tetramer⁺ CD8⁺ T cells from WT1 peptide and IFA vaccinated donors and 6 × 10⁶ CD4 T cells from IFA alone vaccinated donors (group ■ in panel D). Representative flow cytometry plots show percent of gated CD8⁺ T cells staining 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). (H) PBLs from 5 recipient C57BL/6 mice from pilot and validation experiments, were isolated 50, 75, and 100 days after cell transfer of 1 × 10⁵ WT1-tetramer⁺ CD8⁺ T cells from WT1 peptide and IFA vaccinated donors and 6 × 10⁶ CD4 T cells from IFA alone vaccinated donors (group ■ in Figure 6D), and assayed for percent WT1-tetramer⁺ CD8⁺ T cells (means denoted by bars at day 50, 75, and 100 = 3.5%, 1.5%, and 0.56%, respectively).