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Xenotransplantation of pig organs into humans is a potential solution for the shortage of donor organs for transplantation. However, multiple immune barriers preclude its clinical application. In particular, the initial type of rejection in xenotransplantation is an acute cellular rejection by host CD8 + cytotoxic T lymphocyte (CTL) cells that react to donor major histocompatibility complex (MHC) class 1. The human cytomegalovirus (HCMV) glycoprotein Unique Short (US) 2 specifically targets MHC class I heavy chains to relocate them from the endoplasmic reticulum (ER) membrane to the cytosol, where they are degraded by the proteasome. In this study we transfected the US2 gene into minipig fetal fibroblasts and established four US2 clonal cell lines. The integration of US2 into transgenic fetal cells was confirmed using PCR and Southern blot assay. The reduction of Swine Leukocyte Antigen (SLA)-I by US2 was also detected using Flow cytometry assay (FACS). The FACS analysis of the US2 clonal cell lines demonstrated a substantial reduction in SLA-I surface expression. The level (44% to 76%) of SLA-1 expression in US2 clonal cell lines was decreased relative to the control. In cytotoxicity assay the rate of CD8 + T cell-mediated cytotoxicity was significantly reduced to 23.8±15.1% compared to the control (59.8±8.4%, p<0.05). In conclusion, US2 can directly protect against CD8 + -mediated cell lysis. These results indicate that the expression of US2 in pig cells may provide a new approach to overcome the CTL-mediated immune rejection in xenotransplantation.

asian-australasian journal of animal sciences

Inhibition of Human CD8+ Cytotoxic T Lymphocyte (CTL) -mediated Cytotoxicity in Porcine Fetal Fibroblast Cells by Overexpression of Human Cytomegalovirus Glycoprotein Unique Short (US) 2 Gene