LSCs but not BM–infiltrating CD4⁺ T cells in AML produce IL-9 that expands LSCs in vitro. (A) Quantification of IL-9 protein in BM fluid of patients with AML (n = 5) and HDs (n = 12). BM fluid from HD was either collected from BM aspirates (n = 7) or from orthopedic patients who underwent vertebroplasty (n = 5). (B) Quantification of IL-9 protein in culture supernatants of FACS-purified LSCs/HSCs, BM–infiltrating CD4⁺ and CD8⁺ T cells (n = 6 patients with AML and 3 HDs). (C) Gene expression levels of IRF4 in HSCs/LSCs and CD4⁺ T cells (n = 28-30 patients with AML and 7 HDs). Fold differences represented as AML vs HD. (D) Gene expression levels of different isoforms of IL9R in HSCs/LSCs and CD4⁺ T cells (n = 9 patients with AML and 5 HDs). (E) IL-9R protein quantification in LSCs/HSCs and CD4⁺ T cells (n = 9 patients with AML and 5 HD; ΔMFI: MFI αIL-9R–MFI isotype). (F) Uniform manifold approximation and projection (UMAP) representation of primitive-, granulocyte-macrophage progenitors (GMPs)–, and differentiated-like cells as well as T, B, NK cells and monocytes in the BM of patients with AML (scRNA-seq; n = 20 patients with AML), or HSC, GMP, T, B, NK cells and monocytes in the BM of HD (scRNA-seq; n = 6 HDs). (G) The Reactome IL9 signaling signature, which includes 9 genes (IL9, IL9R, IL2RG, JAK1, JAK3, STAT1, STAT5A, STAT3, and STAT5B) in various cell subsets studied in scRNA-seq for both AML and HD. It was scored using AUCell in R, which calculates the area under the curve of gene set enrichment scores for each cell. (H) The Reactome IL9 signaling signature in scRNA-seq analysis in different subtypes of CD4⁺ T cells in the BM of patients with AML and HD (TBX21⁺ Th1, GATA3⁺ Th2, RORC⁺ Th17, FOXP3⁺ Treg, and SELL/LEF1⁺ naïve). (I) IL9 gene expression in FACS-purified BM–infiltrating LSC/HSC, LPC/HPC, CD4⁺ T cells, CD8⁺ T cells, CD19⁺ B cells, NK cells, or CD14⁺ monocytes assessed by quantitative polymerase chain reaction (qPCR; n = 4 patients with AML and 3 HDs). (J-K) Colony forming assays using FACS-purified AML LSCs (J) or HSCs from HD (K), cultured overnight in the presence or absence of αIL-9 (1μg/mL) before plating in methylcellulose in the primary plating. In secondary platings, rhIL-9 was not added (n = 6 patients with AML and 4 HDs; each value indicates the average of 3 replicates of 1 individual patient sample). Statistics: 2-sided Wilcoxon test (G-H), 2-tailed unpaired t test (J-K). ∗P < .05; ∗∗∗∗P < .0001.