Longitudinal evaluation of local and systemic immune responses after IPI plus DEC therapy. (A) Kaplan-Meier overall survival curves in the post-HSCT (left panel) and the transplant-naïve (right panel) arms, separated by those with irAEs requiring systemic steroids (orange) and those without any irAE (purple). Two post-HSCT patients who required only topical steroids were not included in this analysis. (B) Multiplex immunofluorescence of bone marrow biopsies obtained serially from patients before and after combination DEC + IPI therapy. Immunohistochemical staining staining density was semi-quantified by Inform software (Akoya Biosciences, Marlborough, MA). The left panel shows serial multiplex immunofluorescence images with CD3 (purple), CD8 (white), and GZMB (green) immunohistochemical staining from patient 1002 (who achieved CR). Arrows indicate clusters of CD3⁺ CD8⁺ GZMB⁺ cells observed after 4 cycles of IPI + DEC treatment. The right panel shows dynamic changes in CD3⁺ T-cell subsets among 16 available paired samples before and after IPI + DEC treatment. Statistical testing was performed using the Wilcoxon signed-rank test for paired samples and the Wilcoxon rank-sum test for unpaired samples. (C) Serial flow cytometry-based immune phenotyping was performed using 10 paired blood samples collected at screening, after DEC lead-in, and after IPI + DEC combination therapy at the RP2D (IPI 10 mg/kg). The left panel shows a uniform manifold approximation and projection (UMAP) plot with cells colored according to 8 peripheral blood mononuclear cell populations obtained from FlowSOM (Bioconductor). Generated UMAPs were stratified by each timepoint (T0, T1, and T2). Based on unsupervised cluster analysis of immune cell types and checkpoint expression, accumulations of ICOS⁺ CD4⁺ T cells were observed (dashed line). The right panel shows the proportion of ICOS-positive cells in CD4⁺ T cells. (D) Comparison of the proportion of CD4⁺ Treg cells as a subset of total CD3⁺ T cells was performed. Box plots indicate median, quartile 1 (Q1), and Q3, and minimum (min) and maximum (max). P values were determined with the 2-sided, paired t-test. T0, screening; T1, end of lead-in DEC; T2, end of combination IPI + DEC cycle 1; and T3, end of combination IPI + DEC cycle 2.