Macrophage VISTA Restrains Inflammatory and Antigen-Presenting Programs During Alloimmune Tissue Injury [Cut&Tag]

Macrophage inflammatory plasticity is a central determinant of immune-mediated tissue injury, yet the intrinsic checkpoint mechanisms that restrain inflammatory macrophage programming remain incompletely defined. Here, using acute cardiac allograft rejection as an alloimmune tissue-injury model, we found that Vsir, encoding V-domain Ig suppressor of T-cell activation (VISTA), was preferentially expressed in graft macrophages but declined as macrophages acquired inflammatory and antigen-presenting states. Single-cell RNA sequencing and pseudotime analysis revealed that VISTA downregulation accompanied macrophage progression from reparative/resident-like states toward inflammatory programs. In vitro, VISTA overexpression intrinsically restrained pro-inflammatory macrophage polarization, reduced inflammatory cytokine expression, and limited the induction of antigen-presenting molecules including MHC-II and CD80, thereby attenuating macrophage-driven CD4⁺ T-cell proliferation. Integrative CUT&Tag and RNA-seq analyses further showed that VISTA overexpression was associated with reduced H3K4me3 enrichment at inflammatory regulatory loci, including TRAF5 and CHDH, together with transcriptional repression of NF-κB-, TNF-, MAPK-, and IL-17-associated inflammatory programs. In vivo F4/80 promoter-directed Vsir restoration shifted intragraft macrophages away from inflammatory polarization, reduced T-cell accumulation, and attenuated early rejection-associated tissue injury. Together, these findings identify VISTA as an intrinsic regulator of macrophage inflammatory programming and point to H3K4me3-associated chromatin remodeling as a regulatory layer linked to VISTA-mediated inflammatory restraint.