Macrophage—Treg immunopathology in mucosal chronic graft-versus-host disease defined by single-cell and spatial profiling 2309654

Abstract Introduction Chronic graft-versus-host disease (cGVHD) is an alloimmune complication of allogeneic hematopoietic stem cell transplantation driven by donor—recipient antigen mismatch. The oral mucosa is a frequent target; however, the cellular circuitry and spatial architecture that sustain oral cGVHD remain incompletely defined. Methods Using single-cell RNA sequencing, we observed a marked enrichment of macrophage subsets in cGVHD oral mucosa. Spatial transcriptomics mapped CD68+ macrophages preferentially along the epithelial basement membrane. Ultrahigh-plex spatial phenotyping further delineated a significant expansion of proinflammatory (M1-like) macrophages, defined by HLA-DR, CD86 expression, accompanied by elevated IL-17 within lesional regions. Results These data suggest an IL-17—driven axis in M1 macrophage recruitment and activation that may amplify mucosal inflammation. Spatial proximity analyses demonstrated juxtaposition of M1 macrophages with CD4+ regulatory T cells (Tregs) in cGVHD tissue, yet TIGIT+ Tregs were comparatively reduced, consistent with impaired regulatory—myeloid crosstalk. Ligand—receptor inference identified upregulation of JAK-STAT signaling, including increased CXCL10—CXCR3 interactions, suggesting chemokine-guided positioning of effector cells. Clinically, strategies that expand Tregs or antagonize CSF1R to limit macrophage activity have shown benefit in cGVHD, underscoring the therapeutic relevance of these pathways. Co-culture assays with macrophage-Treg cells are needed to probe reciprocal effects on cytokine production, Treg suppressive capacity, and macrophage polarization with verification in cGVHD patients. Conclusion Collectively, these studies may define macrophage—Treg crosstalk as an important determinant of immune disequilibrium and tissue injury in oral cGVHD. Funding Source Intramural program of the NIDCR, NIH Topic Categories Transplantation Immunology (TRAN)