Dual‐Targeting Nanovesicles Carrying CSF1/CD47 Identified from Single‐Cell Transcriptomics of Innate Immune Cells in Heart Transplant for Alleviating Acute Rejection

Abstract Although immunosuppressive drugs for targeting T cells are the standard of care in acute transplantation rejection, the role of innate immune cells should not be ignored. Here, single‐cell RNA sequencing (scRNA‐seq) and flow cytometry are performed to reveal the dynamic changes of innate immune cells within the acute rejection time and find a significantly‐increased presence of Ly6G − Ly6C + inflammatory macrophages and decreased presence of neutrophils among all types of immune cells. Next, to further explore potential targets regulating Ly6G − Ly6C + inflammatory macrophages, scRNA‐seq is used to analyze the reciprocal signaling of both neutrophils and macrophages, along with the surface genes of macrophages. It is found that activating colony‐stimulating factor 1/ colony‐stimulating factor 1 receptor (CSF1/CSF1R) andcluster of differentiation 47/signal regulatory protein α (CD47/SIRPα) signaling may serve as a strategy to relieve Ly6G − Ly6C + inflammatory macrophage‐mediated early graft rejection. To investigate this hypothesis, CSF1/CD47 dual‐targeting nanovesicles (NVs) derived from IFN‐γ‐stimulated induced pluripotent stem cell‐derived mesenchymal stem cells ( iPSC‐MSCs )are designed and constructed. It is confirmed that CSF1/CD47 NVs synergistically induce the differentiation of Ly6G − Ly6C − M2 inhibitory macrophages by the CSF1/CSF1R pathway, and inhibit the phagocytosis of inflammatory macrophages and inflammatory response by the CD47/SIRPα pathway, ultimately relieving immune rejection. This study highlights the power of dual‐targeting CSF1/CD47 NVs as an immunosuppressant against early innate immune responses with the potential for broad clinical applications.