Background: Hyperacute xenograft rejection (HXR) previously represented the major immunological barrier to overcome in successful xenotransplantation. The generation of genetically modified pigs null for xenoantigens has drastically reduced the incidence of HXR in pig-to-nonhuman primate (NHP) xenotransplantation models. Despite these advancements, acute xenograft rejection (AXR) remains a major obstacle to clinical xenotransplantation. Natural killer (NK) cells play an important role in mediating acute allograft rejection, and recent literature has shown that NK cells also contribute to AXR. Dissecting the mechanisms of NK cell-mediated AXR is thus essential to developing approaches to overcome this barrier to clinical xenotransplantation.

Methods: A literature review has been performed for the role of NK cells in the development of AXR. In order to understand an in-depth characterization of NK cell-mediated xenograft rejection, the role of NK cells in normal human physiology, infection, cancer, and allograft rejection has been carefully reviewed. The key receptor-ligand interactions responsible for tuning NK cell education have also been reviewed with a focus on the key activating (NKp44 and NKG2D) and inhibitory (KIR2DL1-4, NKG2A, and LIR-1) NK cell receptors.

Results: Natural mechanisms of NK cell tolerance are identified and characterized in pregnancy, alpha-herpesvirus infection, and cancer. Increased expression of the human MHC I molecules HLA-C, -E, and -G on trophoblast cell membranes at the maternal-fetal interface in early pregnancy leads to amplified inhibitory signaling to decidual (dNK) and circulating peripheral NK cells (pNK) through the KIR2DL1-3, NKG2A, and KIR2DL4 receptors, respectively. Aggressive cancers similarly upregulate cell-surface expression of the non-classical MHC I molecules HLA-E and -G, leading to increased NK cell tolerance. Expression of the signal peptide UL40 and the decoy ligand gpUL18 in CMV-infected cells mediates NK cell tolerance by increased inhibitory signaling via NKG2A and LIR-1, respectively. Successful approaches to reduce NK cell activation include removing the major xenoantigens (αGal, Neu5Gc, and Sda) to reduce antibody-dependent cellular cytotoxicity and expressing the human MHC class I molecules HLA-C, -E, and -G to reduce cell-mediated cytotoxicity (Figure 1). Additional barriers, including NK cell recruitment via VLA-4, VCAM-1, and CD99, self-recognition via NKG2A and activation via foreign interactions (e.g. NKG2D-ULBP and other unknown ligands to NKp44, CD2, and vCD28) should be explored (Figure 1). Time-efficient methods to screen, identify, and evaluate promising xenograft genetic modifications are reviewed, including the use of immortalized porcine endothelial cell lines (ipLDECs) and spheroid models.

Conclusions: Promising future directions to address NK cell-mediated AXR are proposed, with an emphasis on potential genetic modifications to porcine xenografts.