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Target Cell Susceptibility to Lysis by Human Natural Killer Cells Is Augmented by α(1,3)-Galactosyltransferase and Reduced by α(1,2)-Fucosyltransferase

1999; Elsevier BV; Volume: 274; Issue: 16 Linguagem: Inglês

10.1074/jbc.274.16.10717

1083-351X

John H. Artrip, Paweł Kwiatkowski, Robert E. Michler, Shu-Feng Wang, Sorina Tugulea, Hendrik Jan Ankersmit, Larisa Chisholm, Ian F. C. McKenzie, Mauro S. Sandrin, Silviu Itescu,

T-cell and B-cell Immunology

Susceptibility of porcine endothelial cells to human natural killer (NK) cell lysis was found to reflect surface expression of ligands containing Gal α(1,3)GlcNAc, the principal antigen on porcine endothelium recognized by xenoreactive human antibodies. Genetically modifying expression of this epitope on porcine endothelium by transfection with the α(1,2)-fucosyltransferase gene reduced susceptibility to human NK lysis. These results indicate that surface carbohydrate remodeling profoundly affects target cell susceptibility to NK lysis, and suggest that successful transgenic strategies to limit xenograft rejection by NK cells and xenoreactive antibodies will need to incorporate carbohydrate remodeling. Susceptibility of porcine endothelial cells to human natural killer (NK) cell lysis was found to reflect surface expression of ligands containing Gal α(1,3)GlcNAc, the principal antigen on porcine endothelium recognized by xenoreactive human antibodies. Genetically modifying expression of this epitope on porcine endothelium by transfection with the α(1,2)-fucosyltransferase gene reduced susceptibility to human NK lysis. These results indicate that surface carbohydrate remodeling profoundly affects target cell susceptibility to NK lysis, and suggest that successful transgenic strategies to limit xenograft rejection by NK cells and xenoreactive antibodies will need to incorporate carbohydrate remodeling. Target cell susceptibility to lysis by human natural killer cells is augmented by α(1,3)-galactosyltransferase and reduced by α(1,2)-fucosyltransferase.Journal of Biological ChemistryVol. 274Issue 21PreviewPage 10717, abstract, line 3:"Gal α(1,3)GlcNAc" should be "Gal α(1,3)Gal β(1,4)GlcNAc." Full-Text PDF Open Access The severe shortage of human organs has focused recent investigation into cross-species transplantation. Pigs are an appropriate donor source, because their organs have similar physiology and size to human organs, they can be bred in large numbers, and they are relatively free of pathogens capable of causing infection in humans. However, porcine xenografts transplanted into primate recipients undergo hyperacute rejection within minutes to hours of engraftment. The process is mediated by host complement and preformed IgM antibodies directed against Gal α(1,3)Gal epitopes present in various cell surface structures on porcine endothelium (1Galili U. Macher B.A. Buehler J. Shohet S.B. J. Exp. Med. 1985; 162: 573-582Crossref PubMed Scopus (367) Google Scholar, 2Platt J.L. Lindman B.J. Chen H. Spitalnik S.L. Bach F.H. Transplantation. 1990; 50: 817-822Crossref PubMed Scopus (167) Google Scholar, 3Parker W.R. Bruno D. Holzknecht Z.E. Platt J.L. J. Immunol. 1994; 153: 3791-3803PubMed Google Scholar, 4Sandrin M.S. Vaughan H.A. Dabkowski P.L. McKenzie I.F.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11391-11395Crossref PubMed Scopus (556) Google Scholar, 5Vaughan H.A. Loveland B.E. Sandrin M.S. Transplantation. 1994; 58: 879-882Crossref PubMed Scopus (96) Google Scholar, 6Sandrin M.S. McKenzie I.F.C. Immunol. Rev. 1994; 141: 169-190Crossref PubMed Scopus (269) Google Scholar). In contrast to pigs, humans and Old World monkeys do not express Gal α(1,3)Gal in their tissues, because the gene encoding the α(1,3)-galactosyltransferase, which links a terminal galactose residue to Gal β(1,4)GlcNAc oligosaccharide backbone structures, is inactive in these species (7Galili U. Shohet S.B. Kobrin E. Stults C.M. Macher B.A. J. Biol. Chem. 1988; 263: 17755-17762Abstract Full Text PDF PubMed Google Scholar,8Larsen R.D. Rivera-Marrero C.A. Ernst L.K. Cummings R.D. Lowe J.B. J. Biol. Chem. 1990; 265: 7055-7061Abstract Full Text PDF PubMed Google Scholar). Anti-Gal α(1,3)Gal antibodies develop in humans and higher primates within the first months of life, in parallel with the colonization of the gastrointestinal tract with bacteria containing α(1,3)-linked galactose residues in their cell walls (9Galili U. Mandrell R.E. Hamahdeh R.M. Shohet S.B. Griffis J.M. Infect. Immun. 1988; 56: 1730-1737Crossref PubMed Google Scholar,10Xu H. Edwards N.M. Chen J.M. Xu D. Michler R.E. J. Thorac. Cardiovasc. Surg. 1995; 110: 1023-1029Abstract Full Text PDF PubMed Scopus (15) Google Scholar). Consequently, there exists a window period in which these IgM antibodies are not present in neonatal primates (11Xu H. Edwards N.M. Kwiatkowski P.A. Rosenberg S.E. Michler R.E. Transplantation. 1995; 59: 1189-1194Crossref PubMed Scopus (29) Google Scholar). The absence of preformed IgM anti-Gal α(1,3)Gal antibodies in neonatal primates enables porcine cardiac xenografts transplanted heterotopically into unmedicated newborn baboons to survive beyond the hyperacute period (12Kaplon R.J. Michler R.E. Xu H. Kwiatkowski P.A. Edwards N.M. Platt J.L. Transplantation. 1995; 59: 1-6Crossref PubMed Scopus (73) Google Scholar); making this an appropriate model for studying the subsequent immunological barriers to xenotransplantation. In these recipients, a second primate anti-pig immunological response occurs after 3–4 days, resulting in graft loss accompanied by dense xenograft infiltration with natural killer (NK) 1The abbreviations used are: NK, natural killer; MHC, major histocompatibility complex; PAEC, pig aortic endothelial cell; HUVEC, human umbilical vein endothelial cell; PBMC, peripheral blood mononuclear cell; IL, interleukin; FITC, fluorescein isothiocyanate. cells, macrophages, and deposition of induced IgG antibodies (13Michler R.E. 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Because similar findings have been demonstrated in guinea pig-to-rat cardiac xenotransplantation in which the recipients were treated with cobra venom factor to inactivate the host complement system (18Blakely M.L. Van Der Werf W.J. Berndt M.C. Dalmassso A.P. Bach F.H. Hancock W.H. Transplantation. 1994; 58: 1059-1066Crossref PubMed Scopus (241) Google Scholar), these observations suggest that a T cell-independent delayed rejection process, mediated largely by NK cells, occurs in widely disparate transplant combinations, including pig to primate. NK cell lysis is regulated by a balance of intracellular signals transmitted via stimulatory and inhibitory cell surface receptors after specific binding to their respective target cell ligands (19Yokoyama W.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3081-3085Crossref PubMed Scopus (98) Google Scholar,20Gumperz J.E. Parham P. Nature. 1995; 378: 245-248Crossref PubMed Scopus (231) Google Scholar). Inhibitory receptors on NK cells have carbohydrate binding domains with specificity for target cell glycoprotein ligands encoded by certain major histocompatibility complex (MHC) class I genes (21Daniels B.F. Nakamura M.C. Rosen S.D. Yokoyama W.M. Seaman W.E. Immunity. 1994; 1: 785-792Abstract Full Text PDF PubMed Scopus (107) Google Scholar, 22Brennan J. Takei F. Wong S. Mager D.L. J. Biol. Chem. 1995; 270: 9691-9694Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Stimulatory receptors on NK cells also have carbohydrate binding domains within C-type lectin structures; however, their target cell glycoprotein ligands have not been well-defined (23Bezouska K. Vlahas G. Horvath O. Jinochova G. Fiserova A. Giorda R. Chambers W.H. Feizi T. Pospisil M. J. Biol. Chem. 1994; 269: 16945-16952Abstract Full Text PDF PubMed Google Scholar, 24Bezouska K. Yuen C.T. O'Brien J. Childs R.A. Chai W. Lawson A.M. Drbal K. Fiserova A. Pospisil M. Feizi T. Nature. 1994; 372: 151-157Crossref Scopus (260) Google Scholar). Recent evidence suggests that NK cells and a subset of B cells may belong to an innate immunological system designed to combat frequently encountered carbohydrate antigens, such as those in the cell walls of bacterial pathogens (25Mond J.J. Lees A. Snapper C.M. Annu. Rev. Immunol. 1995; 13: 655-692Crossref PubMed Scopus (708) Google Scholar, 26Mond J.J. Vos Q. Lees A. Snapper C.M. Curr. Opin. Immunol. 1995; 7: 349-354Crossref PubMed Scopus (218) Google Scholar, 27Snapper C.M. Mond J.J. J. Immunol. 1996; 157: 2229-2233PubMed Google Scholar). Carbohydrate antigens can induce T cell-independent B cell antibody responses and can directly stimulate NK cells, without previous antigen sensitization or MHC restriction, to initiate lysis and to produce IFN-γ. Costimulatory signals provided by the NK cells, together with the effects of NK cell-derived IFN-γ on B cell differentiation, isotype switching, and immunoglobulin secretion, ultimately result in augmentation of the IgG humoral response against the T cell-independent antigen (28Snapper C.M. Paul W.E. Science. 1987; 236: 944-947Crossref PubMed Scopus (1632) Google Scholar, 29Becker J.C. Kolanus W. Lonnemann C. Schmidt R.E. Scand. J. Immunol. 1990; 32: 153-162Crossref PubMed Scopus (36) Google Scholar, 30Snapper C.M. Yamaguchi H. Moorman M.A. Sneed R. Smoot D. Mond J.J. J. Immunol. 1993; 151: 5251-5260PubMed Google Scholar). Because the T cell-independent process of delayed xenograft rejection involves NK cells and IgG antibodies, and the principal antigen on porcine endothelium recognized by xenoreactive human antibodies is the carbohydrate epitope Gal α(1,3)Gal, we addressed the possibility that receptors on human NK cells may also react with ligands containing terminal Gal α(1,3)Gal residues, leading to augmented natural cytotoxicity as well as IgG humoral activity against porcine endothelium. Fresh pig aortas were treated for 1 h with 0.5% collagenase (Type IV, Sigma), lightly washed with Hanks' solution, and gently raked with a plastic cell scraper. The liberated endothelial cells were added to tissue culture vessels in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and 1% penicillin-streptomycin (Life Technologies). The cells were grown to confluence and then transferred to T25 flasks (Becton Dickinson, Franklin Lakes, NJ) in fresh medium. HUVECs were purchased from the American Type Culture Collection (Rockville, MD; cell line CRL-1730), transferred to T25 flasks with fresh medium, and grown to confluence. COS-7 cells were purchased from the American Type Culture Collection (cell line CRL-1651), transferred to T25 flasks, and grown to confluence. All cells were used between the third and seventh passages. Human PBMCs were isolated from heparinized whole blood using Isopaque-Ficoll (Gallard Schlesinger Co., Carle Place, NY) and suspended at a concentration of 2.5 × 106cells/ml in augmented RPMI 1640 medium. Depending on the assay condition, the cells were cultured for 12–14 h with or without the addition of 1000 units/ml recombinant human interleukin 2 (IL-2; Peprotech, Rocky Hill, NJ) before being used in functional assays. PBMCs were suspended at a concentration of 2.0 × 106 cells/ml in phosphate-buffered saline (Life Technologies) with 1% bovine serum albumin (Life Technologies). A mixture of magnetic beads conjugated with antibody directed against T cells (CD3), B cells (CD19), and monocytes (CD14) (Dynal, Inc., Lake Success, NY) was added to the cell suspension (at a ratio of 10 beads/cell) and electronically stirred for 60 min at 4 °C. The Magnet Particle Concentrator (MPC-1, Dynal) was used to isolate the beads containing CD3-, CD19-, and CD14-positive cells. The suspension was collected, washed, and stained for the presence of CD56- and CD16-positive cells. This technique reliably isolated a population of cells that was >80–85% NK cells (CD56+, CD16+) with 2-fold greater than that of allogeneic human endothelium (Fig. 1 b), consistent with the possibility that expression of Gal α(1,3)Gal increases susceptibility of xenogeneic endothelium to lysis by human NK cells. To more directly examine the role of the terminal Gal α(1,3)Gal structure in the heightened susceptibility of xenogeneic porcine endothelium to human NK lysis, inhibition experiments were performed using the plant lectin IB4, which specifically binds to this structure (34Hayes C.E. Goldstein I.J. J. Biol. Chem. 1974; 249: 1904-1914Abstract Full Text PDF PubMed Google Scholar). NK lysis of porcine endothelium was markedly reduced in the presence of IB4 in a concentration-dependent manner (Fig.1 c). The next set of experiments sought to identify the terminal α(1,3)-linked galactose residue within the Gal α(1,3)Gal structure as an essential component of porcine ligands involved in triggering human NK cell lysis. Enzymatic treatment of porcine endothelium with α-galactosidase reduced NK lysis in a concentration-dependent manner, which correlated with the level of reduced Gal α(1,3)Gal expression (Fig. 1, d ande). At the highest concentration of α-galactosidase used, 20 units/ml, NK lysis was inhibited by a mean of 35% accompanying a 44% reduction in cell surface expression of Gal α(1,3)Gal. This inhibition of NK lysis was specific to cleavage of terminal α(1,3)-linked galactose residues, because enzymatic treatment with β-galactosidase had no effect (Fig. 1 f). To directly demonstrate the effect of surface expression of α(1,3)-linked galactose residues on susceptibility to NK lysis, COS cells were transfected with the murine α(1,3)-galactosyltransferase gene (Fig.2 a). These cells do not normally express Gal α(1,3)Gal epitopes and acquire susceptibility to complement-mediated lysis in the presence of human serum after transfection with α(1,3)-galactosyltransferase (4Sandrin M.S. Vaughan H.A. Dabkowski P.L. McKenzie I.F.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11391-11395Crossref PubMed Scopus (556) Google Scholar). In the present study, COS cells transfected with α(1,3)-galactosyltransferase, but not the vector alone, showed increased susceptibility to lysis by human NK cells at every effector:target ratio tested (Fig. 2 b). A recent study using similarly transfected COS cells demonstrated enhanced adhesion of human NK cells to COS cells expressing Gal α(1,3)Gal (35Inverardi L. Clissi B. Stolzer A.L. Bender J.R. Sandrin M.S. Pardi R. Transplantation. 1997; 63: 318-330Crossref Scopus (125) Google Scholar). Our results extend these observations and show that the increased binding of NK cells to terminal Gal α(1,3)Gal residues expressed by ligands on cells transfected with α(1,3)-galactosyltransferase leads to activation of NK cell stimulatory receptors and causes increased target cell lysis. Moreover, because augmented NK lysis of α(1,3)-galactosyltransferase transfected cells was observed for both NK cells at rest and after cytokine activation (Fig. 2 c), these findings suggest that the stimulatory NK cell receptors that bind ligands containing Gal α(1,3)Gal are constitutively expressed. Transfection of porcine endothelium with the gene encoding the α(1,2)-fucosyltransferase enzyme has been shown to reduce the level of Gal α(1,3)Gal expression (36Sandrin M.S. Fodor W.L. Mouhtouris E. Osman N. Cohney S. Rollins S. Guilmette E.R. Setter E. Squinto S.P. McKenzie I.F.C. Nat. Med. 1995; 12: 1261-1267Crossref Scopus (283) Google Scholar). The α(1,2)-fucosyltransferase competes with α(1,3)-galactosyltransferase for the acceptor substrate Gal β(1,4)GlcNAc and diverts synthesis of Gal α(1,3)Gal β(1,4)GlcNAc to Fuc α(1,2)Gal β(1,4)GlcNAc ("H substance" or blood type O phenotype). To compare the effect of terminal Gal α(1,3)Gal or Fuc α(1,2)Gal residues on NK lysis of porcine endothelium, human NK cells were incubated with two pairs of soluble oligosaccharides, each pair consisting of the tetrasaccharide backbone and its appropriate derivative after glycosyltransferase catalysis (Fig.3, a–d). The type I tetrasaccharide lacto-N-tetra inhibited NK lysis by 2.1-fold higher levels than the type II tetrasaccharide lacto-N-neo-tetra (Fig. 3 e), suggesting that carbohydrate binding structures on human NK cells may have a preference for ligands containing type I structures. Addition of a terminal Gal α(1,3)Gal residue inhibited specific NK lysis of porcine endothelium by 3.3-fold higher levels than the lacto-N-neo-tetra backbone structure (Fig. 3 e), consistent with our previous data that ligands containing Gal α(1,3)Gal are bound by receptors on human NK cells. The addition of a terminal Fuc α(1,2)Gal residue also increased inhibition of NK lysis of porcine endothelium by levels 2.5-fold higher than the lacto-N-tetra backbone structure (Fig. 3 e). Thus, human NK cells can bind both Gal α(1,3)Gal and Fuc α(1,2)Gal residues. To investigate the effect of cell surface carbohydrate remodeling on susceptibility to human NK lysis, porcine endothelial cells were transfected with α(1,2)-fucosyltransferase cDNA, and lines were derived that demonstrated stable expression but widely divergent levels of the H substance (Fig. 4, aandb). Surface expression of Gal α(1,3)Gal β(1,4)Glc was inversely proportional to that of Fuc α(1,2)Gal β(1,4)Glc, reflecting the degree of competition for Gal β(1,4)Glc substrate by the glycosyltransferases. Reduction in surface expression of Gal α(1,3)Gal significantly reduced susceptibility of porcine endothelial cells to lysis by human NK cells (Fig. 4 c). Although lytic susceptibility decreased in direct parallel with reduction in surface levels of Gal α(1,3)Gal, human NK lysis could not be reduced by >55% even with >80% reduction of Gal α(1,3)Gal expression. This level of human NK lysis approaches that seen with allogeneic endothelium. Because the process of delayed xenograft rejection involves NK cells and inducible IgG antibodies, we addressed the possibility that human NK cells may also react with ligands containing terminal Gal α(1,3)Gal residues—the principal antigen on porcine endothelium recognized by xenoreactive antibodies. Enzymatic treatment of porcine endothelium with α-galactosidase reduced human NK lysis of porcine endothelium, which specifically correlated with the level of Gal α(1,3)Gal expression. Furthermore, transfecting a primate cell line with the murine α(1,3)-galactosyltransferase gene increased the susceptibility to lysis by human NK cells, again correlating with the expression of Gal α(1,3)Gal. These results suggest that receptors on human NK cells can directly recognize Gal α(1,3)Gal epitopes on target cells, leading to activation of the NK cell lytic machinery and target cell death. Two transgenic strategies have been proposed to overcome complement-mediated hyperacute rejection of porcine xenografts attributable to preformed IgM antibodies directed against Gal α(1,3)Gal epitopes: 1) protection of porcine endothelium against the effects of human complement by expression of human complement inhibitory proteins (37Fodor W.L. Williams B.L. Matis L.A. Madri J.A Rollins S.A. Knight J.W. Velander W. Squinto S.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11153-11157Crossref PubMed Scopus (310) Google Scholar, 38Rosengard A.M. Cary N.R. Langford G.A. Tucker A.W. Wallwork J. White D.J. Transplantation. 1995; 59: 1325-1333Crossref PubMed Google Scholar, 39Diamond L.E. McCurry K.R. Martin M.J. McClellen S.B. Oldham E.R. Platt J.L. Logan J.S. Transplantation. 1996; 61: 1241-1249Crossref PubMed Scopus (141) Google Scholar) and 2) reduction in the level of Gal α(1,3)Gal expression on porcine endothelium by high level expression of the enzyme α(1,2)-fucosyltransferase (36Sandrin M.S. Fodor W.L. Mouhtouris E. Osman N. Cohney S. Rollins S. Guilmette E.R. Setter E. Squinto S.P. McKenzie I.F.C. Nat. Med. 1995; 12: 1261-1267Crossref Scopus (283) Google Scholar). The second strategy is predicated on the knowledge that both α(1,3)-galactosyltransferase and α(1,2)-fucosyltransferase use the same acceptor substrate, Gal β(1,4)GlcNAc, to direct synthesis of Gal α(1,3)Gal β(1,4)GlcNAc and Fuc α(1,2)Gal β(1,4)GlcNAc (H substance or blood type O phenotype), respectively. When both enzymes are cotransfected into COS cells, α(1,2)-fucosyltransferase dominates over α(1,3)-galactosyl transferase so that Gal α(1,3)Gal expression is almost completely suppressed in the presence of Fuc α(1,2)Gal (36Sandrin M.S. Fodor W.L. Mouhtouris E. Osman N. Cohney S. Rollins S. Guilmette E.R. Setter E. Squinto S.P. McKenzie I.F.C. Nat. Med. 1995; 12: 1261-1267Crossref Scopus (283) Google Scholar). This effect is the result of the temporal order of action of these enzymes, with α(1,2)-fucosyltransferase having preferential access to the Gal β(1,4)GlcNAc acceptor substance because of specific amino acid sequences in its cytoplasmic domain, which target its localization to particular compartments within the Golgi apparatus (40Osman N. McKenzie I.F.C. Mouhtouris E. Sandrin M.S. J. Biol. Chem. 1996; 271: 33105-33109Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Using soluble oligosaccharide derivatives to competitively inhibit human NK cell lysis, we found that the addition of a terminal Gal α(1,3)Gal or terminal Fuc α(1,2)Gal residue to their respective carbohydrate derivative further inhibited human NK lysis of porcine endothelium. These results suggest that both Gal α(1,3)Gal and Fuc α(1,2)Gal residues are able to bind human NK cells and raise the possibility that substitution of a terminal α(1,2)-linked fucosyl residue for a terminal α(1,3)-linked galactosyl residue to the Gal β(1,4)Glc backbone structure would not reduce target cell susceptibility to human NK cell lysis. We directly investigated this possibility using porcine endothelial cells that were transfected with α(1,2)-fucosyltransferase cDNA. Reduction in surface expression of Gal α(1,3)Gal significantly reduced susceptibility of porcine endothelial cells to lysis by human NK cells in direct parallel with reduction in surface levels of Gal α(1,3)Gal. However, NK lysis could not be fully eliminated even with almost complete reduction of Gal α(1,3)Gal expression, suggesting that additional factors may contribute to the human NK cell response to porcine endothelium. Possible additional mechanisms include interactions between noncarbohydrate ligands on porcine endothelium and stimulatory receptors on human NK cells and/or incompatibility between swine MHC class I molecules and inhibitory receptors on human NK cells. These data suggest that carbohydrate remodeling of porcine endothelium by high level expression of α(1,2)-fucosyltransferase decreases susceptibility to human NK lysis by two possible mechanisms: 1) a reduction of Gal α(1,3)Gal residues within porcine endothelial cell ligands, which bind to stimulatory receptors on human NK cells; and 2) an increase of Fuc α(1,2)Gal residues within porcine endothelial cell ligands, which bind nonactivating or inhibitory receptors on human NK cells. In both human and murine MHC class I structures, a conservedN-linked glycosylation site is located at Asn-86 (41Parham P. Alpert B.N. Orr H.T. Strominger J.L. J. Biol. 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Immunol. 1996; 156: 3275-3284PubMed Google Scholar). Because high level endothelial cell expression of α(1,2)-fucosyltransferase reduces terminal sialylation (46Sepp A. Skacel P. Lindstedt R. Lechler R.I. J. Biol. Chem. 1997; 272: 23104-23110Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 47Shinkel T.A. Chen C.G. Salvaris E. Henion T.R. Barlow H. Galili U. Pearse M.J. D'Apice A.J.F. Transplantation. 1997; 64: 197-204Crossref PubMed Scopus (64) Google Scholar), presumably because of competition with sialytransferases for the lactosamine substrate, it is possible that the substitution of a terminal α(1,2)-linked fucose residue to the oligosaccharide chain at Asn-86 of the swine MHC class I structure may enhance binding of the MHC molecule to human NK cell inhibitory receptors. This possibility is currently the subject of investigation in our laboratory. With the development of transgenic pig organs resistant to complement-mediated hyperacute rejection, the subsequent immunological barrier confronted by these genetically modified xenografts on transplantation into primate recipients will be that comprising NK cells and macrophages. In this report, we have shown that primate NK cells react prominently with the same principal xenoantigen on porcine endothelium that is recognized by naturally occurring xenoreactive antibodies, confirming the relationship between NK cells and B cells within an innate compartment of the immune system that is T cell-independent. High level expression of α(1,2)-fucosyltransferase, which reduces binding of xenoreactive antibodies, protected porcine endothelium against lysis by human NK cells. Because the alternative transgenic strategy for overcoming complement-mediated hyperacute rejection is to induce expression of human complement inhibitory proteins to protect porcine endothelium against the effects of human complement, organs modified in this manner will continue to be susceptible to a process of delayed xenograft rejection mediated by NK cells and induced IgG antibodies reactive with ligands expressing Gal α(1,3)Gal epitopes. Our study suggests that successful transgenic strategies for pig-to-primate xenotransplantation will need to incorporate carbohydrate remodeling to limit xenograft rejection by a T cell-independent cellular and humoral process. We thank Raul Cortes for technical assistance.