*Purpose: “Off the shelf” tissue-engineered organs will require use of allogeneic cell sources. Allogeneic human endothelial cells (ECs), necessary for organ perfusion, can initiate graft rejection in absence of professional antigen presenting cells. ECs express both class I and class II MHC molecules that can both bind donor-specific antibody and activate alloreactive T effector memory (T_EM) cells. Here we employ CRISPR/Cas9 ablation of MHC molecule expression to engineer ECs that evade alloimmune rejection and retain core EC characteristics including the ability to form microvessels in vivo.

*Methods: Human cord-blood derived ECs were subjected to CRISPR/Cas9-mediated biallelic ablation of both β₂-microglobulin and CIITA. Clones of ablated and control ECs, before and after cytokine activation, were analyzed by FACS for surface protein expression and by RNA sequencing for transcriptional profiling. Human alloantibody binding was also assessed by FACS and complement-induced signaling was assayed by immunoblotting. The ability of ECs to activate allogeneic CD4⁺ and CD8⁺ T_EM cells in vitro was assayed by CFSE-dilution and CTL- and NK cell-mediated cytolysis was assayed by calcein AM release. The ability to form perfused microvessels in vivo was assessed by implantation of collagen gel-suspended ECs into SCID/bg mice and immunoevasion was tested by quantifying microvessels with and without introduction of allogeneic human peripheral blood mononuclear cells (PBMCs) into mice with implants.

*Results: Cultured β₂-microglobulin^null/CIITA^null human ECs lack both class I and class II MHC molecule expression before and after IFN-γ treatment. RNA sequencing of β₂-microglobulin^null/CIITA^null cells does not reveal significant changes in expression of genes unrelated to MHC molecule expression. Normal cultured EC characteristics are also maintained, such as formation of tight monolayers with junctional expression of CD31 and CD144, thrombin or TNF-α-induced leakiness, and normal TNF-α-dependent upregulation of E-selectin and ICAM-1. Importantly, these cells no longer bind human alloantibodies, have markedly diminished capacity to activate allogeneic CD8+ and CD4+ T_EM cells and are resistant to killing by CD8+ alloreactive cytotoxic T lymphocytes in vitro. β₂-microglobulin^null/CIITA^null cells do not trigger NK cell activation or cytolysis. When suspended in a collagen gel and implanted into an immunodeficient mouse, β₂-microglobulin^null/CIITA^null ECs form perfused microvessels and, unlike control ECs, are protected from destruction following inoculation of allogeneic PBMCs.

*Conclusions: These data suggest that tissue engineered constructs can be perfused through vessels lined by allogeneic MHC-absent ECs that likely will be significantly less prone to rejection.

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