Single-cell profiling identifies an AIM2-dependent macrophage program amplifying T cell–mediated acute cardiac allograft rejection

OBJECTIVE To characterize macrophage subset features within cardiac grafts at single-cell resolution, with an emphasis on AIM2 inflammasome pathway activation in pro-inflammatory macrophages. METHODS We analyzed murine heart transplant scRNA-seq datasets to cluster and define graft-infiltrating macrophage subsets, focusing on subset-specific changes in interferon-associated programs and DNA-sensing PRR pathways. We integrated transcriptomic microarray data from human endomyocardial biopsies with macrophage RNA-seq datasets to assess macrophage functional states across rejection conditions and inflammatory stimulation. Key findings were validated using in vitro and in vivo experiments. RESULTS scRNA-seq identified expansion of M1-like macrophage programs in acutely rejecting grafts, marked by increased MHC-II expression, strengthened interferon-response signatures, and upregulation of AIM2 and inflammasome components. In human biopsy analyses, rejection samples showed increased M1 macrophage abundance with enhanced interferon signaling, DNA-sensing pathways, and AIM2 inflammasome gene-set activity; AIM2 activity correlated with M1 macrophage proportion. Functionally, under pro-inflammatory stimulation, AIM2 inhibition reduced CD86 and MHC-II induction and diminished macrophage-driven proliferation of naïve CD4+ and CD8+ T cells. In vivo, administration of the AIM2-inhibitory oligodeoxynucleotide A151 prolonged murine cardiac graft survival, reduced intragraft macrophage and T-cell infiltration, and decreased Th1/Tc1 frequencies in recipient lymphoid organs. CONCLUSION This study defines macrophage heterogeneity during acute cardiac allograft rejection and identifies an IFN-driven AIM2 axis in M1-like macrophages as a mechanism amplifying T cell-mediated rejection. Targeting AIM2 attenuates macrophage costimulatory activation, weakens naïve T-cell priming, and mitigates acute rejection while improving graft survival in mice, supporting AIM2 inhibition as a potential adjunct strategy in heart transplantation.