Single-cell multiomics reveals macrophage-derived IL-23 and CXCL9/10 drive pathogenic IFNG + IL17 + T cells in immunotherapy-related colitis

Background Immune checkpoint blockade (ICB) therapy, while transformative in cancer treatment, is frequently complicated by immune-related colitis (irColitis), driven by poorly understood mechanisms. Methods An integrated analysis of single-cell RNA sequencing (scRNA-seq), T cell receptor (TCR) sequencing, and spatial transcriptomics was conducted to identify immune cell populations and associated signaling pathways driving irColitis. In vivo murine models of irColitis, along with flow cytometry and qPCR, were used to further validate these findings. To assess the functional roles of specific signaling pathways and cell types in driving irColitis, in vivo blockade of IL-23 and CXCL9/10 signaling was performed using neutralizing antibodies, and intestinal macrophages were depleted using clodronate liposomes. Additionally, the potential impact of these immune cells and signaling pathways on ICB therapy was evaluated through the integration of scRNA-seq and TCR sequencing with qPCR and flow cytometry. Results A pathogenic IFNG + IL17 + CD4 + T cell subset with dual Th1/Th17 features, high clonal expansion, and differentiation trajectories from tissue-resident memory T cells (TRM) was identified in human irColitis lesions and murine models. IL-23 blockade reduced the frequency of IFNG + IL17 + CD4 + T cells and mitigated colitis severity. Mechanistically, IL-23 promoted the expansion and survival of pathogenic Th17 (pTh17) precursors, while CXCL9/10–CXCR3 signaling facilitated their differentiation into IFN-γ-secreting effector cells. Intestinal macrophages were identified as major producers of IL-23, CXCL9, and CXCL10. Macrophage depletion markedly alleviated colitis and reduced pathogenic T cells. Crucially, IFNG + IL17 + CD4 + T cells showed no association with antitumor immunity in colorectal cancer immunotherapy responders, suggesting their targeting would not affect ICB efficacy. Conclusions IFNG + IL17 + CD4 + T cells and CXCL9/10-producing macrophages are key mediators of irColitis. Targeting IL-23 signaling and intestinal macrophages represents a promising strategy to alleviate gut immunopathology without compromising the efficacy of ICB therapy.