*Purpose: Myeloid cells are involved in early alloactivation in response to organ allografts and can initiate acute rejection. The heterogeneity of myeloid populations and the modulation of surface markers during the immune response make it difficult to delineate between subsets using traditional myeloid lineage markers and pairwise gating. We therefore performed high-dimensional unsupervised analysis using single cell mass cytometry (Cytometry by Time-Of-Flight; CyTOF) to characterize myeloid cells in acute rejection using a panel of 46 metal-conjugated antibodies.

*Methods: We developed a fully MHC mismatched (BALB/c onto C57Bl/6) murine vascularized composite allotransplant (VCA) model with heterotopic transplantation of donor limb onto recipient mice to investigate how composite tissue allografts impact myeloid immunity. In this VCA model, rejection is characterized by scarring and color change of the graft by day 3-5 post-transplant, and rejection is complete by day 8-10. We collected splenocytes on days 3, 5, and 7 from groups (n=4) of syngeneic and allogeneic graft recipients. All samples (n=24) were barcoded and stained with an antibody panel against 46 surface antigens and analyzed by CyTOF.

*Results: The antibody panel was designed to identify macrophages, classical and non-classical monocytes, granulocytes, type 1 and type 2 conventional dendritic cells (cDC1 and cDC2), plasmacytoid dendritic cells, and monocyte-derived dendritic cells. By day 3 of alloimmunity, classical monocytes have 10-fold elevated expression of CCR2, a chemokine receptor that enables monocyte trafficking. By day 5, the proportion of CCR2+ classical monocytes was increased significantly (p < 0.05) in allograft recipients. In addition, a subset of CCR2+ monocytes upregulated MHCII and PDL1 was upregulated on a subset of Ly6G+/Ly6C- neutrophils. By day 7 post-transplant we observed a sustained, increased proportion of CCR2+ monocytes along with elevated CLEC10a on both cDC2 and MHCII+ monocytes.

*Conclusions: In summary, we developed a dynamic myeloid map of early VCA acute rejection representing multiple myeloid populations, protein markers, and time-points. Our systems level high-dimensional analysis is the groundwork for characterizing pathways critical for acute rejection, identifying biomarkers that distinguish rejecting from non-rejecting grafts, and developing therapeutic strategies that specifically target alloreactive myeloid cells

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