🎗Epigenetic Erasure of Pro-inflammatory Programs by VISTA Reshapes Macrophage Fate to Mitigate Acute Cardiac Allograft Rejection

Introduction: Early activation of macrophages plays a pivotal role in initiating acute solid organ allograft rejection, yet macrophage-targeted immunosuppressive strategies remain elusive. VISTA (Vsir) is a negative immune checkpoint that distinctly regulates myeloid quiescence. However, its mechanistic role in solid organ transplantation is poorly understood. This study aims to investigate the therapeutic potential and underlying epigenetic mechanisms of macrophage-targeted VISTA modulation in acute cardiac allograft rejection.
Methods: A murine abdominal heterotopic heart transplantation model (C57BL/6 to C3H) was established. To specifically overexpress Vsir in recipient macrophages, an adeno-associated virus-2 (AAV-2) vector driven by the F4/80 promoter was injected intravenously 3 weeks prior to transplantation. Graft-infiltrating immune cells were analyzed utilizing single-cell RNA sequencing (scRNA-seq) and flow cytometry. The antigen-presenting capacity was assessed via an in vitro OT-II CD4⁺ T cell and macrophage co-culture system. To elucidate the downstream epigenetic mechanisms, we performed an integrative multi-omics analysis combining CUT&Tag (targeting the active promoter mark H3K4me3) with bulk RNA-seq.
Results: scRNA-seq and immunofluorescence revealed a precipitous decline of VISTA expression in graft-infiltrating macrophages during early acute rejection. Targeted in vivo overexpression of Vsir in recipient macrophages significantly prolonged allograft survival, attenuated myocardial damage, and curtailed CD4⁺ and CD8⁺ T cell infiltration. Pseudotime trajectory analysis of scRNA-seq data demonstrated that acute rejection drove a near-complete (98.3%) shift toward a terminal M1-like inflammatory state. Strikingly, VISTA overexpression actively reversed this trajectory, averting terminal pro-inflammatory differentiation while rescuing the tissue-reparative (M2-like) macrophage pool. Functionally, VISTA impaired the antigen-presenting capacity of macrophages by downregulating MHC-II and CD80, which profoundly suppressed donor-specific T cell proliferation. Mechanistically, integrative multi-omics demonstrated that VISTA orchestrated this phenotypic reprogramming via H3K4me3-dependent epigenetic silencing. VISTA selectively stripped active H3K4me3 marks from the promoters of critical inflammatory hubs (e.g., TRAF5 and CHDH), fundamentally dampening the NF-κB and MAPK signaling cascades at the transcriptional initiation stage.
Conclusion: VISTA mitigates acute allograft rejection through a dual-tiered regulatory network: it epigenetically erases pro-inflammatory macrophage programs via H3K4me3 landscape remodeling, and subsequently severs the antigen presentation crosstalk with effector T cells. Macrophage-targeted VISTA modulation highlights a novel and highly promising therapeutic strategy for inducing durable immune tolerance in solid organ transplantation.