Rejection of Synthetic Human Skin Can Be Triggered by Donor Endothelial Cell Presentation of Alloantigen

Esen Sefik – Yale University; Lingfeng Qin – Yale University; Gwendolyn Davis – Yale University; Richard A. Flavell – Yale University; W.Mark Saltzman – Yale University; Jordan S. Pober – Yale University

Abstract Text: In contrast to vascularized skin, avascular synthetic skin constructs containing human fibroblasts and keratinocytes allogeneic to the recipient are not rejected clinically or by adoptively transferred allogeneic human PBMCs in immunodeficient mice. The presence of graft endothelial cell (EC)-lined microvessels may function to activate T cells or simply provide a conduit into the tissue. We generated 3D-printed human skin using fibroblasts, pericytes, keratinocytes and ECs from a single donor using ECs that were either unmodified or deleted of class I and II HLA antigens and implanted them on adult MISTRG6 mouse that had received adult hematopoietic stem cells (HSCs) as neonates, Mice were inoculated 2 weeks following skin grafting with PBMCs from the same donor as the HSCs. HSC-engrafted mice developed circulating myeloid and lymphoid cells; circulating human T cells dramatically increased after PBMC inoculation. Mice engrafted with HSCs-only did not reject grafts. Grafts with HLA+ ECs became infiltrated by human macrophages, CD4+ and CD8+ T cells progressively over three weeks following PBMC inoculation. CD8+ cells expressed granzyme B and grafts displayed both EC activation (E-selectin and VCAM-1 expression) and vascular injury indicative of rejection. In contrast, grafts formed using HLA- ECs were not rejected and lymphocyte infiltration was minimal despite having comparable vascularization. We conclude HLA+ presentation by ECs is necessary and sufficient to trigger human allograft rejection in the absence of passenger leukocytes. Our model can be used to study the roles of specific human graft cells and molecules in rejection.