IL-9 secreted by leukemia stem cells induces Th1-skewed CD4⁺ T cells, which promote their expansion

• IL-9 secreted by AML LSCs epigenetically activates CD4⁺ T cells and induces Th1 skewing.

• IFN-γ and TNF-α produced by activated CD4⁺ T cells expand LSCs.

In acute myeloid leukemia (AML), leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) interact with various cell types in the bone marrow (BM) microenvironment, regulating their expansion and differentiation. To study the interaction of CD4⁺ and CD8⁺ T cells in the BM with LSCs and LPCs, we analyzed their transcriptome and predicted cell-cell interactions by unbiased high-throughput correlation network analysis. We found that CD4⁺ T cells in the BM of patients with AML were activated and skewed toward T-helper (Th)1 polarization, whereas interleukin-9 (IL-9)–producing (Th9) CD4⁺ T cells were absent. In contrast to normal hematopoietic stem cells, LSCs produced IL-9, and the correlation modeling predicted IL9 in LSCs as a main hub gene that activates CD4⁺ T cells in AML. Functional validation revealed that IL-9 receptor signaling in CD4⁺ T cells leads to activation of the JAK-STAT pathway that induces the upregulation of KMT2A and KMT2C genes, resulting in methylation on histone H3 at lysine 4 to promote genome accessibility and transcriptional activation. This induced Th1-skewing, proliferation, and effector cytokine secretion, including interferon gamma (IFN-γ) and tumor necrosis factor α (TNF-α). IFN-γ and, to a lesser extent, TNF-α produced by activated CD4⁺ T cells induced the expansion of LSCs. In accordance with our findings, high IL9 expression in LSCs and high IL9R, TNF, and IFNG expression in BM–infiltrating CD4⁺ T cells correlated with worse overall survival in AML. Thus, IL-9 secreted by AML LSCs shapes a Th1-skewed immune environment that promotes their expansion by secreting IFN-γ and TNF-α.