IL-9 secreted by AML LSCs activated CD4+ T cells and induced a Th1-phenotype through JAK-STAT signaling and histone H3 methylation. (A) Circus plot illustrating the top 10-upregulated genes involved in several of the dysregulated pathways based on the GO analysis (Figure 5E; CD4+ T cells + rhIL-9 vs untreated CD4+ T cells). (B) GSEA of H3-K4me and H3-K27me gene signatures in CD4+ T cells treated with hIL9 vs untreated and in CD4+ T cells coincubated with LSCs vs CD4+ T cells cocultured with LSCs in the presence of αIL-9. (C) Quantification of 21-histone H3 modifications (15-different lysine methylation, 4-different lysine acetylation, and 2-different serine phosphorylation) in extracted histones from FACS purified in the same experimental groups (n = 3 biological replicates per condition). The dotted square lines represent significant changes. (D) In silico prediction of IL9 and KMT2A or KMT2C gene interactions (GeneMANIA database). (E) Expression levels of crucial genes for lysine methyltransferases, JAK-STAT, phospho ERK, and canonical NF-κB signaling, as well as IFNG and TNF genes assessed by qPCR (n = 3 AML and 3 HD per condition). Fold differences in gene expression, as depicted in bar charts, were calculated in comparison to unstimulated, untreated CD4+ T cells. Corresponding heat maps, illustrating the relative expression levels of the analyzed genes in individual samples. (F) Heat map demonstrating fold differences in gene expression for lysine methyltransferases, JAK-STAT, important genes of canonical NF-κB signaling, as well as TBX21, IFNG, and TNF in CD4+ T cells from patients with AML after gene silencing using siKMT2A, siKMT2C, or siKMT2E in the presence or absence of rhIL-9. The fold changes in gene expression were calculated according to siCtrl treated CD4+ T cells without addition of rhIL-9 as the control condition (n = 4 patients with AML per condition). (G) Heat map demonstrating fold differences in gene expression in CD4+ T cells from patients with AML after in vitro treatment with rhIL-9 plus NF-κB inhibitor (BAY 11-7082) or JAK1/JAK2 inhibitor (ruxolitinib). The fold changes in gene expression were calculated using vehicle (Veh) treated cells as the control condition (n = 4 patients with AML per condition). (H-I) Proliferation of CD4+ T cells from BM of patients with AML treated in vitro with or without rhIL-9 plus siKMT2A, siKMT2C, siKMT2E, NF-κB (BAY 11-7082) or JAK1/JAK2 (ruxolitinib) inhibitors. Following the CFSE staining, the proliferation and replication indexes were determined using FlowJo proliferation modeling module. The proliferation and replication indices were compared to control CD4+ T cells treated with siCtrl or veh without addition of rhIL-9 (n = 3-4 patients with AML per condition). (J) H3K4me3 occupancy at the promoters of NFKB1, RELA, TBX21, IFNG, and TNF by chromatin immunoprecipitation (ChIP)-qRT-PCR in FACS–purified AML CD4+ T cells cultured 48 hours in the presence or absence of rhIL-9. H3K4me3 occupancy at the GAPDH promoter was used as a positive control (n = 4 AML samples). Statistics: 2-way analysis of variance (ANOVA) with multiple comparisons and Dunnett post hoc test (E), 2-tailed unpaired t test (H-J). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. GOBP, gene ontology biological process; pos, positive; reg, regulation; siCtrl, siRNA control; sig, signaling.

IL-9 secreted by AML LSCs activated CD4+ T cells and induced a Th1-phenotype through JAK-STAT signaling and histone H3 methylation. (A) Circus plot illustrating the top 10-upregulated genes involved in several of the dysregulated pathways based on the GO analysis (Figure 5E; CD4+ T cells + rhIL-9 vs untreated CD4+ T cells). (B) GSEA of H3-K4me and H3-K27me gene signatures in CD4+ T cells treated with hIL9 vs untreated and in CD4+ T cells coincubated with LSCs vs CD4+ T cells cocultured with LSCs in the presence of αIL-9. (C) Quantification of 21-histone H3 modifications (15-different lysine methylation, 4-different lysine acetylation, and 2-different serine phosphorylation) in extracted histones from FACS purified in the same experimental groups (n = 3 biological replicates per condition). The dotted square lines represent significant changes. (D) In silico prediction of IL9 and KMT2A or KMT2C gene interactions (GeneMANIA database). (E) Expression levels of crucial genes for lysine methyltransferases, JAK-STAT, phospho ERK, and canonical NF-κB signaling, as well as IFNG and TNF genes assessed by qPCR (n = 3 AML and 3 HD per condition). Fold differences in gene expression, as depicted in bar charts, were calculated in comparison to unstimulated, untreated CD4+ T cells. Corresponding heat maps, illustrating the relative expression levels of the analyzed genes in individual samples. (F) Heat map demonstrating fold differences in gene expression for lysine methyltransferases, JAK-STAT, important genes of canonical NF-κB signaling, as well as TBX21, IFNG, and TNF in CD4+ T cells from patients with AML after gene silencing using siKMT2A, siKMT2C, or siKMT2E in the presence or absence of rhIL-9. The fold changes in gene expression were calculated according to siCtrl treated CD4+ T cells without addition of rhIL-9 as the control condition (n = 4 patients with AML per condition). (G) Heat map demonstrating fold differences in gene expression in CD4+ T cells from patients with AML after in vitro treatment with rhIL-9 plus NF-κB inhibitor (BAY 11-7082) or JAK1/JAK2 inhibitor (ruxolitinib). The fold changes in gene expression were calculated using vehicle (Veh) treated cells as the control condition (n = 4 patients with AML per condition). (H-I) Proliferation of CD4+ T cells from BM of patients with AML treated in vitro with or without rhIL-9 plus siKMT2A, siKMT2C, siKMT2E, NF-κB (BAY 11-7082) or JAK1/JAK2 (ruxolitinib) inhibitors. Following the CFSE staining, the proliferation and replication indexes were determined using FlowJo proliferation modeling module. The proliferation and replication indices were compared to control CD4+ T cells treated with siCtrl or veh without addition of rhIL-9 (n = 3-4 patients with AML per condition). (J) H3K4me3 occupancy at the promoters of NFKB1, RELA, TBX21, IFNG, and TNF by chromatin immunoprecipitation (ChIP)-qRT-PCR in FACS–purified AML CD4+ T cells cultured 48 hours in the presence or absence of rhIL-9. H3K4me3 occupancy at the GAPDH promoter was used as a positive control (n = 4 AML samples). Statistics: 2-way analysis of variance (ANOVA) with multiple comparisons and Dunnett post hoc test (E), 2-tailed unpaired t test (H-J). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. GOBP, gene ontology biological process; pos, positive; reg, regulation; siCtrl, siRNA control; sig, signaling.

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