W186 - High Dimensional Proteomic Analysis of Immune Cell Populations Following Vascularized Composite Allotransplantation in Mice

Shuling Han – Department of Surgery, Division of Abdominal Transplantation – Stanford University School of Medicine; Wenming Zhang – Department of Surgery, Division of Abdominal Transplantation – Stanford University School of Medicine; Andrea Reitsma – Department of Surgery, Division of Abdominal Transplantation – Stanford University School of Medicine; Olivia Martinez – Professor, Department of Surgery, Division of Abdominal Transplantation, Stanford University School of Medicine; Sheri Krams – Professor, Department of Surgery, Division of Abdominal Transplantation, Stanford University School of Medicine

Abstract Text: Vascularized composite tissue allotransplantation (VCA) represents a promising therapeutic approach for individuals that have incurred debilitating injuries to the face or limbs. However, the complexity of tissues within VCA allografts presents unique challenges to our understanding of the host immune response. To address this, syngeneic (C57BL/6 to C57BL/6) and allogeneic (BALB/c to C57BL/6) hind limb transplants were performed in mice, and spleen cells collected on post-transplant days 1, 3, 5, and 7 (n=4-6 mice/per group/day). Custom panels of metal-conjugated antibodies to characterize myeloid populations (54 markers), T cell subsets and cytokines (24 markers) were established and validated. Cells were stained with antibodies and analyzed by mass cytometry. By UMAP dimensionality reduction, we identified seven myeloid cell clusters including a F4/80+Ly6ChiCD11b+CCR2+ subset, that rapidly expanded in the spleen of allogeneic hind limb recipients and was significantly elevated compared to syngeneic recipients by day 5 (P < 0.001). Three distinct CD8+ TCRa/b+ effector/effector memory subsets (Tem) were elevated in the allogeneic group. In particular, the CD8+CD62lowCD44hiCD25hiCCR7- Tem subset was evident by day 3 and was significantly increased in the allogeneic group compared to the syngeneic group on days 5 and 7 (both P < 0.001). This Tem subset expressed the highest levels of cytokines (IFN-g, TNF-a, IL-6, and IL-10), granzyme B, and perforin. In summary, we identified both specific myeloid and Tem populations associated with graft rejection in VCA. Deep profiling and high dimensional analysis of VCA resulted in unique targets that will lead to more targeted and effective immunosuppression.