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

Abstract

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). 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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 <1–3% contamination with T cells (CD3+). NK cells were resuspended at a concentration of 2.5 × 106 cells/ml in RPMI 1640 medium and cultured for 12–14 h with or without the addition of 1000 units/ml recombinant human IL-2 before being used in functional assays. Details of the cytotoxicity assay have been extensively described elsewhere (31Coligan J.E. Kruisbeck A.M. Margulies D.H. Shevach E.M. Strober W. Current Protocols in Immunology. John Wiley & Sons, New York1994Google Scholar). Briefly, target cells (2 × 104 cells/well) were seeded in flat bottom 96-well plates (Becton Dickinson) and grown to confluence at 37 °C and 5% CO2 overnight. The monolayers were washed with Hanks' solution (Life Technologies, Inc.), labeled with 51Cr (2–4 μCi/well, Amersham Pharmacia Biotech) for 60 min, and then extensively washed before being used in functional assays. Effector cells were added at the desired effector:target ratio, typically 20:1 for assays using PBMCs and 10:1 for assays using purified NK cells, and brought to a final volume of 200 μl/well. After incubation for 4 h, 100 μl of supernatant was collected, and 51Cr was measured in a gamma counter Inc., are as specific lysis or of lysis. specific lysis was using the × was by 10% of lysis was using the 100 × lysis with lysis for lysis with All assays were in with a of are as the S.E. monolayers were treated with the isolated at of and 200 for 60 min at 37 °C and 5% The monolayers were washed in Hanks' solution and used in a endothelium was treated with at to complement lysis of porcine endothelium H. J.M. F. M. G. M. P. Transplantation. 1996; 62: PubMed Scopus Google Scholar). Briefly, monolayers were treated with isolated from the been or isolated from bacteria for 4 h at or The monolayers were then extensively washed with Hanks' solution and labeled and used in NK assays. of the porcine endothelium with was specific for Gal α(1,3)Gal epitopes and not carbohydrate epitopes not with not Gal α(1,3)Gal epitopes or carbohydrate epitopes as an appropriate All oligosaccharide were from NK cells were with a concentration of oligosaccharide at 37 °C for 60 min before being used in assays. COS-7 cells were grown to confluence in RPMI 1640 medium. Details of the containing the α(1,3)-galactosyltransferase gene have been described 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). Briefly, × 106 was added to culture medium (Life Technologies) with 5% (Life Technologies) and brought to a final concentration of The was for min at and then to a final volume of The transfection were added to 1 × 106 cells and at 37 °C with 5% The the transfection were and the cells were cultured for h before The porcine endothelial cell line was with the pig α(1,2)-fucosyltransferase in the expression S. E. McKenzie I.F.C. Sandrin M.S. 1996; PubMed Scopus Google and the using a technique with of and 1 of were for of by in containing using and in containing × of appropriate target cells were washed and resuspended in phosphate-buffered saline with bovine serum lectin (2 or I was added to cell suspension and for min at 4 °C. The cells were washed and in 1% was measured in a (Becton endothelial cell with α(1,3)-galactosyltransferase and surface expression of terminal Gal α(1,3)Gal residues were as for lysis by human NK and HUVECs Human NK lysis of porcine endothelium was that of human endothelium 1 with the possibility that expression of Gal α(1,3)Gal susceptibility of endothelium to lysis by human NK cells. directly the of the terminal Gal α(1,3)Gal in the susceptibility of porcine endothelium to human NK lysis, were using the lectin which to this J. Biol. Chem. Full Text PDF PubMed Google Scholar). NK lysis of porcine endothelium was reduced in the presence of in a The of to the terminal α(1,3)-linked galactose residue within the Gal α(1,3)Gal as an of porcine ligands in human NK cell lysis. of porcine endothelium with reduced NK lysis in a which with the of reduced Gal α(1,3)Gal expression the concentration of NK lysis was by a of a in cell surface expression of Gal This of NK lysis was specific to of terminal α(1,3)-linked galactose residues, because with 1 directly the of surface expression of α(1,3)-linked galactose residues on susceptibility to NK lysis, cells were with the α(1,3)-galactosyltransferase gene These cells do not express Gal α(1,3)Gal epitopes and susceptibility to lysis in the presence of human serum after transfection with α(1,3)-galactosyltransferase 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 cells with α(1,3)-galactosyltransferase, not the susceptibility to lysis by human NK cells at effector:target ratio 2 A recent using cells demonstrated of human NK cells to cells Gal α(1,3)Gal Sandrin M.S. R. Transplantation. 1997; 63: Scopus Google Scholar). results these observations and that the binding of NK cells to terminal Gal α(1,3)Gal residues by ligands on cells with α(1,3)-galactosyltransferase to of NK cell stimulatory receptors and target cell lysis. because augmented NK lysis of α(1,3)-galactosyltransferase cells was for NK cells at and after 2 these findings suggest that the stimulatory NK cell receptors that ligands containing Gal α(1,3)Gal are of porcine endothelium with the gene encoding the α(1,2)-fucosyltransferase has been to the of Gal α(1,3)Gal expression M.S. E. S. S. E. McKenzie I.F.C. Med. 1995; Scopus Google Scholar). The α(1,2)-fucosyltransferase with α(1,3)-galactosyltransferase for the Gal β(1,4)GlcNAc and of Gal α(1,3)Gal β(1,4)GlcNAc to β(1,4)GlcNAc or blood the of terminal Gal α(1,3)Gal or residues on NK lysis of porcine human NK cells were with of of the backbone and appropriate after The I NK lysis by higher the that carbohydrate binding structures on human NK cells may have a for ligands containing I of a terminal Gal α(1,3)Gal residue specific NK lysis of porcine endothelium by higher the backbone with previous that ligands containing Gal α(1,3)Gal are by receptors on human NK cells. The addition of a terminal residue also of NK lysis of porcine endothelium by higher the backbone human NK cells can Gal α(1,3)Gal and the of cell surface carbohydrate remodeling on susceptibility to human NK lysis, porcine endothelial cells were with α(1,2)-fucosyltransferase and were that demonstrated expression widely of the expression of Gal α(1,3)Gal was to that of the of for Gal by the in surface expression of Gal α(1,3)Gal reduced susceptibility of porcine endothelial cells to lysis by human NK cells 4 susceptibility in parallel with in surface of Gal α(1,3)Gal, human NK lysis not be reduced by with of Gal α(1,3)Gal This of human NK lysis that with endothelium. Because the process of delayed xenograft rejection involves NK cells and IgG antibodies, we addressed the possibility that human NK cells may also react with ligands containing terminal Gal α(1,3)Gal principal antigen on porcine endothelium recognized by xenoreactive antibodies. of porcine endothelium with reduced human NK lysis of porcine which with the of Gal α(1,3)Gal a primate cell line with the α(1,3)-galactosyltransferase gene the susceptibility to lysis by human NK cells, with the expression of Gal These results suggest that receptors on human NK cells can directly Gal α(1,3)Gal epitopes on target cells, leading to of the NK cell and target cell transgenic strategies have been to hyperacute rejection of porcine xenografts to preformed IgM antibodies directed against Gal α(1,3)Gal of porcine endothelium against the effects of human complement by expression of human complement inhibitory W. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar, A.M. J. Transplantation. 1995; 59: PubMed Google Scholar, S.B. Platt J.L. Transplantation. 1996; PubMed Scopus Google and in the of Gal α(1,3)Gal expression on porcine endothelium by expression of the α(1,2)-fucosyltransferase M.S. E. S. S. E. McKenzie I.F.C. Med. 1995; Scopus Google Scholar). The second is on the that α(1,3)-galactosyltransferase and α(1,2)-fucosyltransferase the Gal to of Gal α(1,3)Gal β(1,4)GlcNAc and β(1,4)GlcNAc or blood are into cells, α(1,2)-fucosyltransferase that Gal α(1,3)Gal expression is in the presence of M.S. E. S. S. E. McKenzie I.F.C. Med. 1995; Scopus Google Scholar). This is the result of the of of these with α(1,2)-fucosyltransferase to the Gal β(1,4)GlcNAc because of specific in which target to within the McKenzie I.F.C. E. Sandrin M.S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). oligosaccharide to human NK cell lysis, we found that the addition of a terminal Gal α(1,3)Gal or terminal residue to their respective carbohydrate human NK lysis of porcine endothelium. These results suggest that Gal α(1,3)Gal and residues are to human NK cells and the possibility that of a terminal residue for a terminal α(1,3)-linked residue to the Gal backbone not target cell susceptibility to human NK cell lysis. directly this possibility using porcine endothelial cells that were with α(1,2)-fucosyltransferase in surface expression of Gal α(1,3)Gal reduced susceptibility of porcine endothelial cells to lysis by human NK cells in parallel with in surface of Gal However, NK lysis not be with of Gal α(1,3)Gal that may to the human NK cell response to porcine endothelium. between ligands on porcine endothelium and stimulatory receptors on human NK cells between MHC class I and inhibitory receptors on human NK cells. These suggest that carbohydrate remodeling of porcine endothelium by expression of α(1,2)-fucosyltransferase susceptibility to human NK lysis by a of Gal α(1,3)Gal residues within porcine endothelial cell which to stimulatory receptors on human NK and an of residues within porcine endothelial cell which or inhibitory receptors on human NK cells. In human and MHC class I structures, a is at P. J.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar), to residues of the which the epitopes recognized by inhibitory NK cell receptors W.J. R.D. J. R.E. P. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, M. G. M. J.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: PubMed Scopus Google Scholar, M. A. J.L. M. J. Exp. Med. 1994; PubMed Scopus Google Scholar). In the oligosaccharide structures at this are various MHC class I and terminal residues J.E. J.H. J.C. Parham P. J. Immunol. 1996; Google Scholar). Because endothelial cell expression of α(1,2)-fucosyltransferase terminal A. P. R. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, Chen E. H. Galili U. Transplantation. 1997; PubMed Scopus Google Scholar), because of with for the is that the of a terminal residue to the oligosaccharide at of the MHC class I may binding of the MHC to human NK cell inhibitory This possibility is the of investigation in the of transgenic pig organs to hyperacute the subsequent immunological by these xenografts on into primate recipients will be that NK cells and In this we have that primate NK cells react with the principal on porcine endothelium that is recognized by xenoreactive antibodies, the between NK cells and B cells within an innate of the system that is T expression of which binding of xenoreactive antibodies, porcine endothelium against lysis by human NK cells. Because the transgenic for hyperacute rejection is to induce expression of human complement inhibitory to porcine endothelium against the effects of human organs in this will to be to a process of delayed xenograft rejection mediated by NK cells and induced IgG antibodies with ligands Gal α(1,3)Gal suggests that successful transgenic strategies for xenotransplantation will need to incorporate carbohydrate remodeling to limit xenograft rejection by a T cell-independent and humoral for