Human endothelial cells (ECs) derived from cord blood progenitors have significant replicative potential and are thus well suited for use in tissue engineering of vascularized organs. However, like other human ECs, these cells readily express both class I and class II MHC molecules and can initiate rejection by direct presentation to host alloreactive T effector memory (T_EM) cells. Moreover, in tissue engineered grafts (lacking passenger leukocytes), ECs may be the only cells capable of initiating graft rejection through direct presentation. We previously showed that lentiviral transduction of Cas9 and guide strands directed towards class II transactivator (CIITA) abrogated expression of class II MHC antigens and eliminated the capacity of EC to be recognized by allogeneic CD4 T_EM but not CD8 T_EM cells. Here we used the same approach to ablate β₂-microglobulin expression, causing loss of class I MHC expression. Class I MHC-negative ECs retain expression of constitutive EC surface markers CD31 and Blood group H antigen and show normal TNF-α-dependent upregulation of E-selectin and ICAM-1 as well as normal IFN-γ-dependent upregulation of ICAM-1 and PD-L1. Cas9-mediated dual ablation of β₂-microglobulin and CIITA in EC largely abrogated proliferation of both allogeneic CD8+ and CD4+ T_EM cells in vitro. As allograft rejection is dependent upon T cell activation, these studies suggest that tissue engineered constructs that incorporate allogeneic (to the host) Cas9-modified EC lacking MHC molecules will be significantly less prone to rejection.

CITATION INFORMATION: Merola J, Anderson C, Xu H, Manes T, Pober J. Modification of Human Endothelial Cells by CRISPR/Cas9-Mediated Ablation of Class I and II Major Histocompatibility Complex Antigen Expression. Am J Transplant. 2017;17 (suppl 3).

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