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

Park, K-W.;Yoo, J.Y.;Choi, K.M.;Yang, B.S.;Im, G.S.;Seol, J.G.

  • Received : 2008.01.23
  • Accepted : 2008.07.24
  • Published : 2009.01.01


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 I. 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-I 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${\pm}$15.1% compared to the control (59.8${\pm}$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.


Cytotoxic T Lymphocyte (CTL) cells;Swine Leukocyte Antigen (SLA)-I;Unique Short (US) 2;Xenotransplantation


  1. Galili, U. 1993. Interaction of the natural anti-Gal antibody with $\alpha$-galactosyl epitopes: A major obstacle for xenotransplantation in humans. Immunol. Today 14:480-482
  2. Sandrin, M. S., H. A. Vaughan, P. L. Dabkowsky and I. F. C. McKenzie. 1993. Anti-pig IgM antibodies in human serum react predominantly with Gal ($\alpha$1-3) Gal epitopes. Proc Natl. Acad. Sci. USA 90:11391-11395
  3. Tanemura, M., A. S. Chong, V. J. DiSesa and U. Galili. 2002. Direct killing of xenograft cells by CD8+ T cells of discordant xenograft recipients. Transplantation. 74:1587-1595
  4. Yi, S., X. Feng and W. Hawthorne. 2000. CD8+ T cells are capable of rejecting pancreatic islet xenografts. Transplantation 70:896-906
  5. Forte, P., B. C. Baumann, E. H. Weiss and J. D. Seebach. 2005. HLA-E expression on porcine cells: Protection from human NK cytotoxicity depends on peptide loading. Am. J. Transplant 5:2085-2093
  6. Good, A. H., D. K. C. Cooper and A. J. Malcolm. 1992. Identification of carbohydrate structures that bind human antiporcine antibodies: implications for discordant xenografting in humans. Transplant Proc. 24:559-562
  7. Hegde, N. R. and D. C. Johson. 2003. Human cytomegalovirus US2 causes similar effects on both major histocompatibility complex class I and II proteins in epithelial and glial cells. J. Virol. 77:9287-9294
  8. Yamada, K., K. Yazawa and A. Shimizu. 2005. Marked prolongation of porcine renal xenograft survival in baboons through the use of α 1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue. Nat. Med. 11:32-34
  9. Murray, A. G., M. M. Khodadoust, J. S. Pober and A. L. M. Bothwell. 1994. Porcine aortic endothelial cells activated human T cells: direct presentation of MHC antigens and costimulation by ligands for human CD2 and CD28. Immunity 1:57-63
  10. Shishido, S., B. Naziruddin, T. Howard and T. Mohanakumar. 1997. Recognition of porcine major histocompatibility complex class I antigens by human CD8+ cytolytic T cell clones. Transplantation 64:340-346
  11. Morgan, C. and J. H. Lee. 2001. Current status of xenotransplantation. Asian-Aust. J. Anim. Sci. 14:1497-1504
  12. Hengel, H., J. O. Koopmann, T. Flohr, W. Muranyi, E. Goulmy, G. J. Hammerling, U. H. Koszinowski and F. Momburg. 1997. A viral ER-resident glycoprotein inactivates the MHC-encoded peptide transporter. Immunity 6:623-632
  13. Kuwaki, K., Y. L. Tseng and F. J. M. F. Dor. 2005. Heart transplantation in baboons using α1,3-galactosyltransferase gene-knockout pigs as donors: initial experience. Nat. Med. 11:29-31
  14. Wiertz, E. J., D. Tortorella, M. Bogyo, J. Yu, W. Mothes, T. R. Jones, T. A. Rapoport and H. L. Ploegh. 1996. Sec61-mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction. Nature 384:432-438
  15. Del, V. M., H. Hengel, H. Hacker, U. Hartlaub, T. Ruppert, P. Lucin and U. H. Koszinowski. 1992. Cytomegalovirus prevents antigen presentation by blocking the transport of peptide-loaded major histocompatibility complex class I molecules into the medial-Golgi compartment. J. Exp. Med. 176:729-738
  16. Tanemura, M., D. Yin, A. S. Chong and U. Galili. 2000. Differential immune responses to α-gal epitopes on xenografts and allografts: implications for accommodation in xenotransplantation. J. Clin. Invest. 105:301
  17. Phelps, C. J., C. Koike and T. D. Vaught. 2003. Production of $\alpha$1,3-Galactosyltransferase-deficient pigs. Sci. 299:411-414
  18. Valdes-Gonzalez, R. A., L. M. Dorantes, G. N. Garibay, E. Bracho-Blanchet, A. J. Mendez, R. Davila-Perez, R. B. Elliott, L. Teran and D. J. White. 2005. Xenotransplantation of porcine Figure 4. Cytotoxicity assay of human cytotoxic T Lymphocytes against minipig's fetal fibroblast cells transfected with US2. In vitro cytotoxicity assay cultured CD8+ T lymphocyte from the healthy volunteers. Effector cell:target cell = 15:1 (open circles), effector cell:target cell = 10:1 (closed circles). neonatal islets of Langerhans and Sertoli cells: a 4-year study. Eur. J. Endocrinol. 153:419-427
  19. Lai, L., D. Kolber-Simonds and K. W. Park. 2002. Production of $\alpha$-1,3-Galactosyltransferase knockout pigs by nuclear transfer cloning. Sci. 295:1089-1092
  20. Zhan, Y., J. L. Brady, R. M. Sutherland and A. M. Lew. 2001. Without CD4 help, CD8 rejection of pig xenografts requires CD28 costimulation but not perforin killing. J. Immunol. 167:6279-6285
  21. O'connell, P. 2002. Pancreatic islet xenotransplantation. Xenotransplantation 9:367-371
  22. Wiertz, E. J., T. R. Jones, L. Sun, M. Bogyo, H. J. Geuze and H. L. Ploegh. 1996. The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol. Cell 84:769-779