Pharmacodynamics of decitabine and ipilimumab. (A) Number of differentially expressed genes across cell types between before decitabine treatment (Screening) and after one cycle of decitabine (Decitabine, Lead-in) from scRNA-seq dataset shows predominantly myeloid-specific effect of decitabine (pink) (Log₂FC > 0.25, −log₁₀FDR > 10). (B) Gene set enrichment analysis of differentially expressed genes in myeloid cells. (C) Soluble IL8 in peripheral blood plasma quantified using Olink assay throughout treatment. Statistical testing using Wilcoxon rank-sum test. NPX – Normalized protein expression. (D) Mean gene expression of CXCL8 (encoding IL8) across cell subsets shows preferential expression in CD14⁺ monocytes and other myeloid cells, while expression is absent in T and NK cells. (E) Number of differentially expressed genes across cell types after 1 cycle of decitabine (Decitabine) and after combined treatment of decitabine and ipilimumab (Ipilimumab) shows ipilimumab-specific effect on CD4⁺ T cells (blue box) (log₂FC > 0.25, −log₁₀FDR > 10). (F) Gene set enrichment analysis of differentially expressed genes in CD4⁺ T cells. (G) Soluble CXCL13 in peripheral blood plasma throughout treatment quantified using Olink assay. Statistical testing using Wilcoxon rank-sum test. (H-I) Percentage of Tregs in bone marrow aspirates measured using single cell sequencing (H) and multiplexed immunofluorescence (I) shows ipilimumab-induced increase of Tregs. Data from ETCTN 9204 were obtained before (Pre-Ipi) and after (Post-Ipi) ipilimumab monotherapy. Tissue biopsies from ETCTN 9204 were obtained from bone marrow (n = 17) and extramedullary AML sites (skin n = 3; breast n = 2; soft tissue n = 1), while tissue biopsies from ETCTN 10026 were exclusively bone marrow (n = 36). Statistical testing using Wilcoxon rank-sum test. (J) Validation of ipilimumab-induced increase in Tregs (CD3⁺ FOXP3⁺) in tissue biopsies from ETCTN/CTEP 10026 study using multiplexed immunofluorescence staining at screening, after 1 cycle of decitabine monotherapy (Decitabine, Lead-in) and after combination treatment (Ipilimumab). NPX, normalized protein expression.