IL-9 secreted by LSCs activates BM–infiltrating CD4⁺ T cells and induces Th1 skewing. (A) PCA based on the transcriptomic profile of CD4⁺ T cells upon stimulation with rhIL-9 or untreated CD4⁺ T cells as controls as well as CD4⁺ T cells cocultured with paired AML LSCs and CD4⁺ T cell cocultured with AML LSCs together with αIL-9 (n = 3 biological replicates per condition). (B) Heat map of differentially expressed genes in CD4⁺ T cells + rhIL-9 vs untreated CD4⁺ T cells. (C) GSEA of T-cell activation, proliferation and differentiation-related gene signatures in CD4⁺ T cells + rhIL-9 vs untreated CD4⁺ T cells. (D) GSEA of the IL-9 stimulated_up signature (1073 genes, Figure 5C) in CD4⁺ T cells coincubated with LSCs vs untreated or CD4⁺ T cells coculture with LSCs in the presence of αIL-9. (E) Heat map illustrating the expression profile of key genes for CD4⁺ Th-cell polarization. (F) GSEA of AML CD4 vs CTRL_DE gene signature (386 genes, Figure 1B) in CD4⁺ T cells treated with rhIL-9 or coincubated with LSCs vs untreated CD4⁺ T cells. (G) Protein quantification of IFN-γ and TNF-α in supernatants of FACS–purified AML CD4⁺ T cells in the presence or absence of rhIL-9 and cocultured with LSCs in the presence or absence of αIL-9 (n = 3 patients with AML). (H) Protein quantification of IFN-γ and TNF-α in BM fluid of patients with AML (n = 5) and HDs (n = 12). Statistics: 2-tailed unpaired t test. ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001.