![IL-9/IL-9R signaling as well as TNF-α and IFN-γ expression in CD4+ T cells correlate with worse prognosis in patients with AML. (A) Colony forming assays with primary LSCs treated overnight in the presence or absence of rhTNF-α (100 pg/mL) or rhIFN-γ (100 pg/mL), before plating in methylcellulose in the primary plating. Secondary plating was without addition of rhTNF-α or rhIFN-γ (n = 6 patients with AML; each value indicates the average of 3 replicates of 1 individual patient sample). (B) Colony forming assays performed with primary LSCs treated overnight with or without supernatant of IL-9 activated AML CD4+ T cells in the presence or absence of neutralizing αTNF-α (1 μg/mL) or αIFN-γ antibodies (1 μg/mL), before plating in methylcellulose in the primary plating. No further treatment was added for secondary platings (n = 3 patients with AML; each value is average of 3 replicates of 1 individual patient sample). The results were normalized to the colony numbers of LSCs that have been treated with Veh (A) or left untreated (B; dotted line). (C) Kaplan-Meier plots of overall survival (OS) for pooled patients with AML from 3-independent AML cohorts (HOVON-SAKK data set, E-MTAB-3444; NL-Valk data set, GSE6891; Metzeler data set, GSE12417), according to the IL9 and IL9R gene expression (n = 1339 patients with AML). The cutoff for high or low gene expression was assessed by the X-Tile program. (D) Kaplan-Meier plots of OS according to the IL9 gene expression in LSCs or IL9R gene expressions in FACS-purified BM–infiltrating CD4+ T cells from patients with AML (n = 28 LSCs and n = 30 CD4+ T cells). (E) GSEA of 16 LSC-signature genes (IL9 high vs IL9 low in purified LSCs, as determined by the X-Tile program cutoff [D]). (F) Heat map of 16 LSC-signature genes. (G) Correlation between TNF and IFNG in CD4+ T cells and the geometric mean of 16 LSC-signature genes in purified LSCs. (H) Kaplan-Meier plots of OS according to the TNF and IFNG gene expressions in FACS-purified BM–infiltrating CD4+ T cells from patients with AML (n = 30). Statistics: 2-way ANOVA with multiple comparisons and Dunnett post hoc test (A-B) and log-rank test (C-D,H), Pearson correlation (G). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. ns, not significant.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/144/8/10.1182_blood.2024024000/1/blood_bld-2024-024000-gr7.jpeg?Expires=1761397510&Signature=Ogr14VZaF68FYrF3bJWhQghPLKNDOuv5Qd55oEtl~vw5GXQMqYgsKggjmKN7dNQuKvEo5kyfjCiVwtcwxR2CH8KUNOFQBk~r~GQzRvcgjaLOg1pGuFJpR3euNToHzyvu4zqQ2o-xkvJglkJtxuBq3Ofd~R7q94xHrHATHB~MkcB2CWQ3AJw-FfB~Ap3v-S8mO6oUMrChkTeBvSEeXHaPDn-pIUlRUksYg2fVkO5Kii6QzZM7ec-TOXEE~XlsFSHDK9NTqWu400WvPFct9dOVCr50eKccA6jeOgl4lA3a-uXKy8~RxNBTt7uBONx~R2TrC~eDNMH5aYwakBujkzcvGg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
IL-9/IL-9R signaling as well as TNF-α and IFN-γ expression in CD4+ T cells correlate with worse prognosis in patients with AML. (A) Colony forming assays with primary LSCs treated overnight in the presence or absence of rhTNF-α (100 pg/mL) or rhIFN-γ (100 pg/mL), before plating in methylcellulose in the primary plating. Secondary plating was without addition of rhTNF-α or rhIFN-γ (n = 6 patients with AML; each value indicates the average of 3 replicates of 1 individual patient sample). (B) Colony forming assays performed with primary LSCs treated overnight with or without supernatant of IL-9 activated AML CD4+ T cells in the presence or absence of neutralizing αTNF-α (1 μg/mL) or αIFN-γ antibodies (1 μg/mL), before plating in methylcellulose in the primary plating. No further treatment was added for secondary platings (n = 3 patients with AML; each value is average of 3 replicates of 1 individual patient sample). The results were normalized to the colony numbers of LSCs that have been treated with Veh (A) or left untreated (B; dotted line). (C) Kaplan-Meier plots of overall survival (OS) for pooled patients with AML from 3-independent AML cohorts (HOVON-SAKK data set, E-MTAB-3444; NL-Valk data set, GSE6891; Metzeler data set, GSE12417), according to the IL9 and IL9R gene expression (n = 1339 patients with AML). The cutoff for high or low gene expression was assessed by the X-Tile program. (D) Kaplan-Meier plots of OS according to the IL9 gene expression in LSCs or IL9R gene expressions in FACS-purified BM–infiltrating CD4+ T cells from patients with AML (n = 28 LSCs and n = 30 CD4+ T cells). (E) GSEA of 16 LSC-signature genes (IL9 high vs IL9 low in purified LSCs, as determined by the X-Tile program cutoff [D]). (F) Heat map of 16 LSC-signature genes. (G) Correlation between TNF and IFNG in CD4+ T cells and the geometric mean of 16 LSC-signature genes in purified LSCs. (H) Kaplan-Meier plots of OS according to the TNF and IFNG gene expressions in FACS-purified BM–infiltrating CD4+ T cells from patients with AML (n = 30). Statistics: 2-way ANOVA with multiple comparisons and Dunnett post hoc test (A-B) and log-rank test (C-D,H), Pearson correlation (G). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. ns, not significant.
