CD8α expression is up-regulated on CD4⁺ and CD8⁺ murine and human T9_IL-33 cells and increases their killing of leukemia cells. (A) Transcriptome analysis of CD8α expression in sorted WT T9_IL-33 versus ST2^−/− T9_IL-33 CD8 and CD4 cells (n = 3; see Fig. S2). (B) Representative plots of CD8α expression on CD4⁺ (top) and CD8β⁺ (bottom) T cells from in vitro differentiated murine T1, T9, WT T9_IL-33, and ST2^−/− T9_IL-33 cells, and bar graphs showing the frequency and mean florescence intensity (MFI) of CD4⁺CD8a⁺ and CD8α⁺CD8β⁺ T cells for each condition (n = 6, from three independent experiments, unpaired t test; data are shown as mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001). (C) Representative plots of Gzmb expression in CD8β⁺ T cells differentiated under T9_IL-33 conditions plus either anti-CD8α or isotype control, and bar graphs showing the frequency of Gzmb⁺CD8β⁺ cells (n = 3, from three independent experiments, unpaired t test; data are shown as mean ± SEM; *, P < 0.05). (D) Cytolytic assays of in vitro differentiated B6 T9_IL-33 incubated with BALB/c MLL-AF9 cells in the presence of anti-CD8α or isotype control for 6 h (n = 3, from three independent experiments, unpaired t test; data are shown as mean ± SEM; **, P < 0.01). (E) Cytolytic assays. B6 T9_IL-33 cells were differentiated in MLR conditions with anti-CD8α blocking antibody or isotype control. After 5 d, T9_IL-33 cells were incubated with BALB/c MLL-AF9 cells for 6 h (n = 3, from three independent experiments, unpaired t test; data are shown as mean ± SEM; *, P < 0.05; **, P < 0.01). (F) ImageStream cell images of syngeneic T9_IL-33 or allogeneic T9_IL-33 cells incubated with BALB/c eGFP–MLL-AF9 cells and anti-CD8α blocking antibody or isotype control for 3 h (n = 3, unpaired t test; data are shown as mean ± SEM; *, P < 0.05). (G) Cytolytic assays of in vitro differentiated B6 T1 or T9_IL-33 incubated with BALB/c MLL-AF9 cells in the presence of anti-CD8α or isotype control for 6 h (n = 3, mean ± SEM). (H) Cytolytic assay of in vitro differentiated B6 WT T9_IL-33 or CD8α^−/− T9_IL-33 cells in MLR conditions. After 5 d, both WT and ST2^−/− T9_IL-33 cells were incubated with BALB/c MLL-AF9 cells for 6 h (n = 3, unpaired t test; data are shown as mean ± SEM; *, P < 0.05; ***, P < 0.001). (I) Survival curves for BALB/c mice receiving 10⁴ cells of the syngeneic MLL-AF9 leukemic cell line with allogeneic WT (n = 7 mice per group, **, P < 0.01, log-rank test). Pie charts show relapses. (J) Representative plots of CD8α and GzmK expression from human in vitro differentiated T1, T9, and T9_IL-33 cells and a bar graph showing the frequency of GzmK⁺CD8α⁺ T cells from each condition (n = 3, from three independent experiments, unpaired t test; data are shown as mean ± SEM; *, P < 0.05). (K) Cytolytic assays of human T9_IL-33 cells differentiated with anti-CD8α blocking antibody or isotype control and incubated with MOLM14 cells for 6 h (n = 3, from three independent experiments, unpaired t test; data are shown as mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001).