*Purpose: Alloimmune responses in acute rejection are complex, involving multiple interacting cell types. Flow cytometry with biased pairwise gating is the gold standard to phenotype cell populations; however, the number of parameters that can be examined in a sample is limited by spectral overlap. The ability to simultaneously measure multiple parameters on single cells is critical to comprehensively define cell populations in alloimmunity. Here, we utilize the high-dimensional capacity of single-cell mass cytometry (Cytometry by Time-Of-Flight; CyTOF) using 44 metal-conjugated antibodies to extensively profile the alloimmune response in acute rejection.

*Methods: We profiled both murine cardiac allotransplant, and a novel vascularized composite allotransplant (VCA), models. Splenocytes were collected on multiple days post-transplant from recipients of syngeneic and allogeneic transplants. Splenocytes from no-transplant controls were also included. All samples were barcoded and stained with a metal-conjugated antibody panel against 44 different markers and analyzed by CyTOF. We performed multiparametric characterization and unsupervised analysis of cells using hierarchical clustering and dimensionality reduction.

*Results: In allogeneic transplant recipients, there is marked phenotypic heterogeneity within the effector CD8⁺ T cell and NK cell populations that expand during acute rejection. Three distinct T cell, and five NK cell subsets, were identified and characterized as specifically responding to alloantigen. Contrastingly, the only effector CD4⁺ T cells associated with rejection are uniquely CD40L⁺. The myeloid compartment has a significant increase in Ly6C^hi inflammatory monocytes, and strikingly is only observed in the spleen at day 5 post-transplant but not on day 3. Finally, we report a relatively conserved alloimmune profile between heart and VCA transplants; however, effector CD8⁺ T cells arise earlier (day 3) in VCA.

*Conclusions: Alloimmunity is characterized by several distinct cell populations acquiring combinatorial shifts in phenotype as graft rejection progresses. Using CyTOF to deconvolve and visualize complex systems in an unsupervised way greatly enhances the capacity to simultaneously capture both global and subtle changes across multiple cell types and phenotypic markers. This systems-level approach lays the foundation for a more comprehensive roadmap of the alloimmune response, which can be used to further interrogate specific cell populations and/or inform direction of targeted therapeutic strategies that can delay or prevent acute rejection.

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