Defining and Directing T Cell Subsets Involved in Rejection of Vascularized Composite Allografts
Veronika Malek, BSc; Georg Furtmüller, MD; Byoungchol Oh, MD, PhD; Damon S. Cooney, MD, PhD; Gerald Brandacher, MD
Johns Hopkins University, Baltimore, MD
Introduction: Vascularized Composite Allotransplantation (VCA) has emerged as a viable treatment option for complex tissue defects. Thus far, over 100 hand allograft transplantations have been performed worldwide, yielding highly encouraging functional results. Still, the need for long-term immunosuppression hampers wider utilization of VCA. Barriers to more targeted immunosuppression in VCA include (1) limited knowledge of the cells and cytokines mediating acute skin rejection, (2) high antigenicity of the VCA skin component, and (3) lack of strategies to promote allograft tolerance. We aimed to address these challenges by more fully characterizing the rejection process, possibly identifying novel targets for intervention. We focused on CD4+ Th17 cells due to their involvement in dermatologic autoimmune diseases and allograft rejection in solid organ transplantation.
Materials & Methods: Allogeneic orthotopic hindlimb transplants were performed in the following mouse strains: C57BL6 wild type, RORgT -/- (lacking Th17 cells), Tbet -/- (lacking Th1 cells), IL-4 -/- (lacking Th2 cells) and Foxp3DTR with diphtheria toxin treatment (lacking Treg cells). Animals were assessed daily for clinical signs of rejection and tissue biopsies were obtained on post-operative day 8 to assess rejection kinetics. Tissue digestion and T cell extraction for flow analysis were performed on POD 8 to analyze T cell phenotypes within the rejecting graft. H & E histology and Immunohistochemistry were performed as confirmation.
Results: Rejection kinetics varied between groups with wild type animals reaching grade 4 rejection between days 8-9 and Foxp3DTR mice between days 6-7, while RORgT -/- mice did not fully reject until POD 17. Flow analysis revealed a predominantly CD4+ Th1+ cell population in rejecting skin and muscle tissue in wild type mice. Curiously, a lack of either Th2 or Th1 cells resulted in a primarily CD4+ Th17+ cell infiltrate within the rejecting graft. Additionally, the ratio of regulatory T cells to effector T cells was significantly higher in Tbet -/- mice (4.9), lacking Th1 cells, than in any other group (<0.8 for all others).
Conclusions: Allogeneic orthotopic hindlimb transplantions revealed distinct rejection patterns in different knock-out mice. Graft rejection occurred in all groups, suggesting redundancy of T cell effector mechanisms. In wild type animals, CD4+ Th1+ cells were the driving rejection. However, the dominance of Th17 cells in the absence of Th1 cells is particularly relevant in the context of immunosuppression, where eliminating Th1 cells could still result in graft rejection.
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