TIM3-Galectin9 axis drives CSF-1R–independent senescent macrophage and TSCM crosstalk in chronic graft-versus-host disease

Abstract Background: Chronic graft-versus-host disease (cGVHD) remains a major cause of morbidity post-allogeneic hematopoietic cell transplantation. Despite CSF-1R blockade depleting pro-fibrogenic macrophages, ~30% of cGVHD patients remain refractory, highlighting an urgent need to identify resistance mechanisms and translational targets. We identify a CSF-1R–independent macrophage population that escapes anti-CSF-1R therapy and collaborates with stem-like memory CD8+T cells (Tscm) to drive cGVHD pathogenesis. Methods Using single-cell RNA sequencing, spatial transcriptomics, and flow cytometry, we analyzed human cGVHD skin and murine cGVHD target organs. Functional assays, and CellChat analysis elucidated interactions between macrophages and TCF1+Tscm. TIM3-Fc fusion protein was therapeutically tested. Results We observed that in cGVHD patient, hypoxic niches fostered tertiary lymphoid structure (TLS)-like aggregates enriched for CD68+CD14+ macrophages and TCF1+ Tscm colocalized in the skin lesion. In an MHC-mismatched murine cGVHD model (C57BL/6 donor to BALB/c recipients), P21+CSF-1R– macrophages and Tscm (Tcf1+IL-7Rα+CD27+Bhlhe40+Runx3+) significantly expanded in cGVHD target organs liver and lung, both exhibiting senescence-associated secretory phenotype (SASP). P21+CSF-1R– macrophages secreted IL-6, IL-1β, ROS, TGF-β and collagen IV, while Tscm produced IFN-γ and TNF-α. CellChat analysis identified robust interactions between Galectin-9+P21+CSF-1R– macrophages and TIM3+Tscm. Galectin-9 and TIM3 interaction activated MAPK/ERK signaling pathway in P21+CSF-1R– macrophage. Coculture these two subsets amplified IFN-γ and TNF-α in Tscm and SASP in macrophages, sustaining a feedforward loop. Critically, TIM3-Fc administration significantly reduced SASP factors (IL-6, IL-1β and TGF-β), disrupted senescent macrophage-Tscm crosstalk and ameliorated established fibrosis in lung and liver. Conclusions We define a novel pathogenic axis where TIM3-Galectin9 mediates senescence amplification between CSF-1R- macrophages and Tscm, driving cGVHD progression. Targeting this axis breaks the pathogenic loop, providing a clinical translational strategy for CSF-1R blockade-resistant cGVHD.