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

John H. ArtripDagger §, Pawel KwiatkowskiDagger , Robert E. MichlerDagger , Shu-Feng WangDagger , Sorina TuguleaDagger , Jan AnkersmitDagger , Larisa Chisholm, Ian F. C. McKenzie, Mauro S. Sandrin, and Silviu ItescuDagger parallel

From the Dagger  College of Physicians and Surgeons, Columbia University, New York, New York 10032 and  Austin Research Institute, Heidelberg, Victoria 3084, Australia

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Susceptibility of porcine endothelial cells to human natural killer (NK) cell lysis was found to reflect surface expression of ligands containing Gal alpha (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 alpha (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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha (1,3)Gal epitopes present in various cell surface structures on porcine endothelium (1-6). In contrast to pigs, humans and Old World monkeys do not express Gal alpha (1,3)Gal in their tissues, because the gene encoding the alpha (1,3)-galactosyltransferase, which links a terminal galactose residue to Gal beta (1,4)GlcNAc oligosaccharide backbone structures, is inactive in these species (7,8).

Anti-Gal alpha (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 alpha (1,3)-linked galactose residues in their cell walls (9,10). Consequently, there exists a window period in which these IgM antibodies are not present in neonatal primates (11). The absence of preformed IgM anti-Gal alpha (1,3)Gal antibodies in neonatal primates enables porcine cardiac xenografts transplanted heterotopically into unmedicated newborn baboons to survive beyond the hyperacute period (12); 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)1 cells, macrophages, and deposition of induced IgG antibodies (13-17). 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 (18), 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 (19,20). 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 (21, 22). 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 (23, 24). 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 (25-27). 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-gamma . Costimulatory signals provided by the NK cells, together with the effects of NK cell-derived IFN-gamma on B cell differentiation, isotype switching, and immunoglobulin secretion, ultimately result in augmentation of the IgG humoral response against the T cell-independent antigen (28-30). 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 alpha (1,3)Gal, we addressed the possibility that receptors on human NK cells may also react with ligands containing terminal Gal alpha (1,3)Gal residues, leading to augmented natural cytotoxicity as well as IgG humoral activity against porcine endothelium.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Preparation of Target Cells

Pig Aortic Endothelial Cells (PAECs)-- 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.

Human Umbilical Vein Endothelial Cells (HUVECs)-- 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 Cells-- 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.

Preparation of Effector Cells

Peripheral Blood Mononuclear Cells (PBMCs)-- 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.

Purified NK Cells-- 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.

Cytotoxicity Assay

Details of the cytotoxicity assay have been extensively described elsewhere (31). 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 (Clinigamma 1272; Wallac Inc., Gaithersburg, MD). Data are presented as percent specific lysis or percent inhibition of lysis. Percent specific lysis was calculated using the formula (experimental cpm - spontaneous cpm)/(maximum cpm - spontaneous cpm) × 100; where maximum cpm was determined by adding 10% Triton X-100 (Sigma). Percent inhibition of lysis was calculated using the formula 100 × (% lysis observed with control condition - % lysis observed for each experimental condition)/% lysis observed with control condition. All assays were done in triplicate with a minimum of three donors. Results are presented as the mean ± S.E.

Treatment of PAECs with IB4

51Cr-Labeled PAEC monolayers were treated with the plant isolectin b4 (IB4 Sigma) isolated from Bandeiraea (Griffonia) simplicifolia at concentrations of 2.0, 20, and 200 µg/ml for 60 min at 37 °C and 5% CO2. The monolayers were washed in Hanks' solution and used in a standard lytic assay.

Enzymatic Treatment of PAECs

Porcine endothelium was treated with alpha -galactosidase at concentrations shown to reduce antibody-directed complement lysis of porcine endothelium (32). Briefly, PAEC monolayers were treated with either alpha -galactosidase isolated from the Green coffee been (Sigma) or beta -galactosidase isolated from Escherichia coli bacteria (Sigma) for 4 h at pH 6.0 or 7.3, respectively. The monolayers were then extensively washed with Hanks' solution and labeled with 51Cr and used in standard NK lytic assays. Treatment of the porcine endothelium with alpha -galactosidase was specific for Gal alpha (1,3)Gal epitopes and did not affect other carbohydrate epitopes (data not shown). Furthermore, treatment with beta -galactosidase did not remove Gal alpha (1,3)Gal epitopes or other carbohydrate epitopes serving as an appropriate negative control.

Inhibition of NK Lysis by Soluble Oligosaccharides

All oligosaccharide derivatives were obtained from Dextra Laboratories (Reading, UK). Purified NK cells were incubated with a 10-5 M concentration of each oligosaccharide derivative at 37 °C for 60 min before being used in standard lytic assays.

Transfection of COS-7 Cells

COS-7 cells were grown to confluence in RPMI 1640 medium. Details of the CDM8 plasmid containing the murine alpha (1,3)-galactosyltransferase gene have been previously described (4). Briefly, vector DNA (7-10 µg/1 × 106 targeted cells) was added to Optimem culture medium (Life Technologies) with 5% LipofectAMINE (Life Technologies) and brought to a final concentration of 5-10 µg/ml. The DNA was incubated for 20 min at room temperature and then diluted to a final volume of 7-10 ml. The transfection media were added to 1 × 106 COS cells and incubated overnight at 37 °C with 5% CO2. The following morning the transfection media were aspirated, and the cells were cultured for 48 h before use.

Transfection of Porcine Endothelial Cells

The porcine endothelial cell line was cotransfected with the pig alpha (1,2)-fucosyltransferase cDNA in the expression vector pCDNA-1 (pHT plasmid; Ref. 33) and the pcDNA-1-neo plasmid (Invitrogen, Carlsbad, CA) using a standard calcium phosphate technique with 20 µg of pHT plasmid and 1 µg of pcDNA-1-neo plasmid. Cells were selected for stable integration of transfected DNA by selection in media containing G418, cloned using limiting dilution, and maintained in media containing G418.

Flow Cytometry

5 × 105 of appropriate target cells were washed and resuspended in phosphate-buffered saline with 0.1% bovine serum albumin. Fluorescein isothiocyanate (FITC)-conjugated lectin (2 µg/ml), IB4, or Ulex europaeus agglutinin, type I (Sigma) was added to each cell suspension and incubated for 45 min at 4 °C. The cells were washed and fixed in 1% paraformaldehyde (Sigma). Mean channel fluorescence was measured in a FACScan flow cytometer (Becton Dickinson).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Susceptibility of Porcine Endothelium to Human NK Lysis Correlates with Expression of Gal alpha (1,3)-Gal-- Two endothelial cell lines with opposing alpha (1,3)-galactosyltransferase activities and surface expression of terminal Gal alpha (1,3)Gal residues were selected as initial targets for lysis by human NK cells: PAECs and HUVECs (Fig. 1a). Human NK lysis of xenogeneic porcine endothelium was >2-fold greater than that of allogeneic human endothelium (Fig. 1b), consistent with the possibility that expression of Gal alpha (1,3)Gal increases susceptibility of xenogeneic endothelium to lysis by human NK cells. To more directly examine the role of the terminal Gal alpha (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 (34). NK lysis of porcine endothelium was markedly reduced in the presence of IB4 in a concentration-dependent manner (Fig. 1c). The next set of experiments sought to identify the terminal alpha (1,3)-linked galactose residue within the Gal alpha (1,3)Gal structure as an essential component of porcine ligands involved in triggering human NK cell lysis. Enzymatic treatment of porcine endothelium with alpha -galactosidase reduced NK lysis in a concentration-dependent manner, which correlated with the level of reduced Gal alpha (1,3)Gal expression (Fig. 1, d and e). At the highest concentration of alpha -galactosidase used, 20 units/ml, NK lysis was inhibited by a mean of 35% accompanying a 44% reduction in cell surface expression of Gal alpha (1,3)Gal. This inhibition of NK lysis was specific to cleavage of terminal alpha (1,3)-linked galactose residues, because enzymatic treatment with beta -galactosidase had no effect (Fig. 1f).


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 1.   a, expression of Gal alpha (1,3)Gal on PAECs and HUVECs. Flow cytometric analysis of PAECs and HUVECs was performed using the FITC-conjugated lectin IB4, which specifically binds to Gal alpha (1,3)Gal. b, specific NK lysis of PAEC and HUVEC targets. Standard 51Cr release lytic assays were performed using human PBMCs as effector cells with PAEC or HUVEC targets. Freshly isolated PBMCs demonstrated significantly increased NK lysis of PAECs compared with HUVECs, which was augmented after activation of the PBMCs with IL-2. IL-2 activation increased NK lysis of PAECs and HUVECs by 1.5- and 1.7-fold, respectively, suggesting that the effects of cytokine activation are not target cell-dependent. c, inhibition of NK lysis of porcine endothelium by IB4. IL-2-activated human PBMCs were used as effector cells against PAEC targets incubated with increasing concentrations of the lectin IB4. d, reduction of Gal alpha (1,3)Gal on PAECs after treatment with alpha -galactosidase. PAECs were treated with 0.2-20 units/ml alpha -galactosidase, and expression of Gal alpha (1,3)Gal was measured by direct flow cytometry using FITC-conjugated IB4. e, reduction in NK lysis of PAEC targets after treatment with alpha -galactosidase. IL-2-activated human PBMCs were used as effector cells against alpha -galactosidase-treated PAEC targets. f, specificity of alpha -galactosidase treatment of PAECs for reduction in NK lysis. IL-2-activated human PBMCs were used as effector cells against alpha - or beta -galactosidase (gal'ase)-treated PAEC targets.

Expression of Gal alpha (1,3)-Gal in COS Cells Increases Susceptibility to NK Lysis-- To directly demonstrate the effect of surface expression of alpha (1,3)-linked galactose residues on susceptibility to NK lysis, COS cells were transfected with the murine alpha (1,3)-galactosyltransferase gene (Fig. 2a). These cells do not normally express Gal alpha (1,3)Gal epitopes and acquire susceptibility to complement-mediated lysis in the presence of human serum after transfection with alpha (1,3)-galactosyltransferase (4). In the present study, COS cells transfected with alpha (1,3)-galactosyltransferase, but not the vector alone, showed increased susceptibility to lysis by human NK cells at every effector:target ratio tested (Fig. 2b). A recent study using similarly transfected COS cells demonstrated enhanced adhesion of human NK cells to COS cells expressing Gal alpha (1,3)Gal (35). Our results extend these observations and show that the increased binding of NK cells to terminal Gal alpha (1,3)Gal residues expressed by ligands on cells transfected with alpha (1,3)-galactosyltransferase leads to activation of NK cell stimulatory receptors and causes increased target cell lysis. Moreover, because augmented NK lysis of alpha (1,3)-galactosyltransferase transfected cells was observed for both NK cells at rest and after cytokine activation (Fig. 2c), these findings suggest that the stimulatory NK cell receptors that bind ligands containing Gal alpha (1,3)Gal are constitutively expressed.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2.   a, induced surface expression of Gal alpha (1,3)Gal on COS cells after transfection with alpha (1,3)-galactosyltransferase (gal'txase) cDNA. Flow cytometric analysis using FITC-conjugated IB4 was performed on COS cells transfected with either a plasmid containing the murine alpha (1,3)-galactosyltransferase gene or the plasmid alone. After alpha (1,3)-galactosyltransferase transfection, 28.2% of COS cells expressed Gal alpha (1,3)Gal; in contrast, COS cells transfected with control vector failed to demonstrate any surface expression of Gal alpha (1,3)Gal. b and c, COS cells transfected with the alpha (1,3)-galactosyltransferase gene have increased susceptibility to NK lysis. b, human PBMCs were activated with IL-2 and used as effector cells against transfected COS cell targets. COS cells transfected with alpha (1,3)-galactosyltransferase showed increased susceptibility to NK lysis compared with COS cells transfected with the control vector. c, freshly isolated and IL-2-activated purified human NK cells were used as effector cells. Lysis of alpha (1,3)-galactosyltransferase-transfected COS cells was greater than COS cells transfected with the control vector, which was also observed after NK activation with IL-2. The relative increase in NK lysis of COS cells transfected with alpha (1,3)-galactosyltransferase was ~30% irrespective of whether the NK cells were resting or IL-2-activated.

Inhibition of Human NK Cell Lysis of Porcine Endothelium by Soluble Gal alpha (1,3)-Gal or Fuc alpha (1,2)-Gal Oligosaccharide Derivatives-- Transfection of porcine endothelium with the gene encoding the alpha (1,2)-fucosyltransferase enzyme has been shown to reduce the level of Gal alpha (1,3)Gal expression (36). The alpha (1,2)-fucosyltransferase competes with alpha (1,3)-galactosyltransferase for the acceptor substrate Gal beta (1,4)GlcNAc and diverts synthesis of Gal alpha (1,3)Gal beta (1,4)GlcNAc to Fuc alpha (1,2)Gal beta (1,4)GlcNAc ("H substance" or blood type O phenotype). To compare the effect of terminal Gal alpha (1,3)Gal or Fuc alpha (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. 3e), suggesting that carbohydrate binding structures on human NK cells may have a preference for ligands containing type I structures. Addition of a terminal Gal alpha (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. 3e), consistent with our previous data that ligands containing Gal alpha (1,3)Gal are bound by receptors on human NK cells. The addition of a terminal Fuc alpha (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. 3e). Thus, human NK cells can bind both Gal alpha (1,3)Gal and Fuc alpha (1,2)Gal residues.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 3.   Inhibition of NK lysis of PAECs with soluble oligosaccharide derivatives containing terminal Gal alpha (1,3)Gal or Fuc alpha (1,2)Gal structures. Depicted are the core structures lacto-N-neo-tetra (LNNT; a) and lacto-N-tetra (LNT; c), along with their respective alpha (1,3)-galactosylated or alpha (1,2)-fucosylated derivative Gallili pentasaccharide (GLI; b) and H5 (d). e, purified human NK cells were incubated with a 10-5 M concentration of each oligosaccharide and used in a standard lytic assay with PAEC targets. The addition of a terminal alpha (1,3)-galactose residue to lacto-N-neo-tetra and the addition of a terminal alpha (1,2)-fucose residue to lacto-N-tetra increased the percent inhibition of NK lysis.

Expression of H Substance in Porcine Endothelium Reduces Target Cell Susceptibility to NK Lysis-- To investigate the effect of cell surface carbohydrate remodeling on susceptibility to human NK lysis, porcine endothelial cells were transfected with alpha (1,2)-fucosyltransferase cDNA, and lines were derived that demonstrated stable expression but widely divergent levels of the H substance (Fig. 4, a and b). Surface expression of Gal alpha (1,3)Gal beta (1,4)Glc was inversely proportional to that of Fuc alpha (1,2)Gal beta (1,4)Glc, reflecting the degree of competition for Gal beta (1,4)Glc substrate by the glycosyltransferases. Reduction in surface expression of Gal alpha (1,3)Gal significantly reduced susceptibility of porcine endothelial cells to lysis by human NK cells (Fig. 4c). Although lytic susceptibility decreased in direct parallel with reduction in surface levels of Gal alpha (1,3)Gal, human NK lysis could not be reduced by >55% even with >80% reduction of Gal alpha (1,3)Gal expression. This level of human NK lysis approaches that seen with allogeneic endothelium.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4.   a and b, surface expression of Fuc alpha (1,2)Gal and Gal alpha (1,3)Gal on porcine endothelial cells after transfection with alpha (1,2)-fucosyltransferase cDNA. Flow cytometric analysis was performed on porcine endothelial cells transfected with alpha (1,2)-fucosyltransferase cDNA using FITC-conjugated IB4 and UEA-I, which recognize Gal alpha (1,3)Gal (34) and Fuc alpha (1,2)Gal residues (48), respectively. Log fluorescence is shown relative to unstained control cell lines 20.3 (a) and 20.1 (b). As surface expression of Fuc alpha (1,2)Gal increased, surface expression of Gal alpha (1,3)Gal progressively decreased, consistent with the known dominant effect of alpha (1,2)-fucosyltransferase over alpha (1,3)-galactosyltransferase. c, porcine endothelial cells transfected with alpha (1,2)-fucosyltransferase cDNA have reduced susceptibility to human NK lysis. Purified human NK cells were used against porcine endothelial cell targets transfected with alpha (1,2)-fucosyltransferase cDNA. NK lysis decreased in parallel with the reductions in Gal alpha (1,3)Gal expression.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha (1,3)Gal residues---the principal antigen on porcine endothelium recognized by xenoreactive antibodies. Enzymatic treatment of porcine endothelium with alpha -galactosidase reduced human NK lysis of porcine endothelium, which specifically correlated with the level of Gal alpha (1,3)Gal expression. Furthermore, transfecting a primate cell line with the murine alpha (1,3)-galactosyltransferase gene increased the susceptibility to lysis by human NK cells, again correlating with the expression of Gal alpha (1,3)Gal. These results suggest that receptors on human NK cells can directly recognize Gal alpha (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 alpha (1,3)Gal epitopes: 1) protection of porcine endothelium against the effects of human complement by expression of human complement inhibitory proteins (37-39) and 2) reduction in the level of Gal alpha (1,3)Gal expression on porcine endothelium by high level expression of the enzyme alpha (1,2)-fucosyltransferase (36). The second strategy is predicated on the knowledge that both alpha (1,3)-galactosyltransferase and alpha (1,2)-fucosyltransferase use the same acceptor substrate, Gal beta (1,4)GlcNAc, to direct synthesis of Gal alpha (1,3)Gal beta (1,4)GlcNAc and Fuc alpha (1,2)Gal beta (1,4)GlcNAc (H substance or blood type O phenotype), respectively. When both enzymes are cotransfected into COS cells, alpha (1,2)-fucosyltransferase dominates over alpha (1,3)-galactosyl transferase so that Gal alpha (1,3)Gal expression is almost completely suppressed in the presence of Fuc alpha (1,2)Gal (36). This effect is the result of the temporal order of action of these enzymes, with alpha (1,2)-fucosyltransferase having preferential access to the Gal beta (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 (40).

Using soluble oligosaccharide derivatives to competitively inhibit human NK cell lysis, we found that the addition of a terminal Gal alpha (1,3)Gal or terminal Fuc alpha (1,2)Gal residue to their respective carbohydrate derivative further inhibited human NK lysis of porcine endothelium. These results suggest that both Gal alpha (1,3)Gal and Fuc alpha (1,2)Gal residues are able to bind human NK cells and raise the possibility that substitution of a terminal alpha (1,2)-linked fucosyl residue for a terminal alpha (1,3)-linked galactosyl residue to the Gal beta (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 alpha (1,2)-fucosyltransferase cDNA. Reduction in surface expression of Gal alpha (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 alpha (1,3)Gal. However, NK lysis could not be fully eliminated even with almost complete reduction of Gal alpha (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 alpha (1,2)-fucosyltransferase decreases susceptibility to human NK lysis by two possible mechanisms: 1) a reduction of Gal alpha (1,3)Gal residues within porcine endothelial cell ligands, which bind to stimulatory receptors on human NK cells; and 2) an increase of Fuc alpha (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 conserved N-linked glycosylation site is located at Asn-86 (41), adjacent to residues 74-83 of the alpha -chain, which encode the polymorphic epitopes recognized by inhibitory NK cell receptors (42-44). In humans, the oligosaccharide structures at this site are remarkably uniform among various MHC class I allotypes and generally contain terminal sialic acid residues (45). Because high level endothelial cell expression of alpha (1,2)-fucosyltransferase reduces terminal sialylation (46, 47), presumably because of competition with sialytransferases for the lactosamine substrate, it is possible that the substitution of a terminal alpha (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 alpha (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 alpha (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.

    ACKNOWLEDGEMENT

We thank Raul Cortes for technical assistance.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by National Institutes of Health National Research Service Award HL09744.

parallel To whom correspondence should be addressed: Dept. of Surgery, Columbia-Presbyterian Medical Center, 630 W. 168th St., PH 14-1485, New York, NY 10032. Tel.: 212-305-7176; Fax: 212-305-8145; E-mail: si5{at}columbia.edu.

    ABBREVIATIONS

The 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Galili, U., Macher, B. A., Buehler, J., and Shohet, S. B. (1985) J. Exp. Med. 162, 573-582[Abstract]
  2. Platt, J. L., Lindman, B. J., Chen, H., Spitalnik, S. L., and Bach, F. H. (1990) Transplantation 50, 817-822[Medline] [Order article via Infotrieve]
  3. Parker, W. R., Bruno, D., Holzknecht, Z. E., and Platt, J. L. (1994) J. Immunol. 153, 3791-3803[Abstract/Free Full Text]
  4. Sandrin, M. S., Vaughan, H. A., Dabkowski, P. L., and McKenzie, I. F. C. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11391-11395[Abstract]
  5. Vaughan, H. A., Loveland, B. E., and Sandrin, M. S. (1994) Transplantation 58, 879-882[Medline] [Order article via Infotrieve]
  6. Sandrin, M. S., and McKenzie, I. F. C. (1994) Immunol. Rev. 141, 169-190[Medline] [Order article via Infotrieve]
  7. Galili, U., Shohet, S. B., Kobrin, E., Stults, C. M., and Macher, B. A. (1988) J. Biol. Chem. 263, 17755-17762[Abstract/Free Full Text]
  8. Larsen, R. D., Rivera-Marrero, C. A., Ernst, L. K., Cummings, R. D., and Lowe, J. B. (1990) J. Biol. Chem. 265, 7055-7061[Abstract/Free Full Text]
  9. Galili, U., Mandrell, R. E., Hamahdeh, R. M., Shohet, S. B., and Griffis, J. M. (1988) Infect. Immun. 56, 1730-1737[Medline] [Order article via Infotrieve]
  10. Xu, H., Edwards, N. M., Chen, J. M., Xu, D., and Michler, R. E. (1995) J. Thorac. Cardiovasc. Surg. 110, 1023-1029[Abstract/Free Full Text]
  11. Xu, H., Edwards, N. M., Kwiatkowski, P. A., Rosenberg, S. E., and Michler, R. E. (1995) Transplantation 59, 1189-1194[Medline] [Order article via Infotrieve]
  12. Kaplon, R. J., Michler, R. E., Xu, H., Kwiatkowski, P. A., Edwards, N. M., and Platt, J. L. (1995) Transplantation 59, 1-6[Medline] [Order article via Infotrieve]
  13. Michler, R. E., Xu, H., O'Hair, D. P., Shah, A., Kwiatkowski, P. A., Minanov, O., and Itescu, S. (1996) Transplant. Proc. 28, 651-652[Medline] [Order article via Infotrieve]
  14. Itescu, S., Kwiatkowski, P. A., Wang, S. F., Blood, T., Minanov, O. P., Rose, S., and Michler, R. E. (1996) Transplantation 62, 1927-1933[Medline] [Order article via Infotrieve]
  15. Minanov, O. P., Itescu, S., Neethling, F. A, Morganthau, A. S., Kwiatkowski, P. A., Cooper, D. K. C., and Michler, R. E. (1997) Transplantation 63, 182-186[Medline] [Order article via Infotrieve]
  16. Itescu, S., Kwiatkowski, P., Artrip, J. H., Wang, S. F., Ankersmit, J., Minanov, O. P., and Michler, R. E. (1998) Hum. Immunol. 59, 275-286[CrossRef][Medline] [Order article via Infotrieve]
  17. Minanov, O. P., Artrip, J. H., Szabolcs, M., Kwiatkowski, P. A., Galili, U., Itescu, S., and Michler, R. E. (1998) J. Thorac. Cardiovasc. Surg. 115, 998-1006[Abstract/Free Full Text]
  18. Blakely, M. L., Van Der Werf, W. J., Berndt, M. C., Dalmassso, A. P., Bach, F. H., and Hancock, W. H. (1994) Transplantation 58, 1059-1066[Medline] [Order article via Infotrieve]
  19. Yokoyama, W. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3081-3085[Free Full Text]
  20. Gumperz, J. E., and Parham, P. (1995) Nature 378, 245-248[CrossRef][Medline] [Order article via Infotrieve]
  21. Daniels, B. F., Nakamura, M. C., Rosen, S. D., Yokoyama, W. M., and Seaman, W. E. (1994) Immunity 1, 785-792[Medline] [Order article via Infotrieve]
  22. Brennan, J., Takei, F., Wong, S., and Mager, D. L. (1995) J. Biol. Chem. 270, 9691-9694[Abstract/Free Full Text]
  23. Bezouska, K., Vlahas, G., Horvath, O., Jinochova, G., Fiserova, A., Giorda, R., Chambers, W. H., Feizi, T., and Pospisil, M. (1994) J. Biol. Chem. 269, 16945-16952[Abstract/Free Full Text]
  24. Bezouska, K., Yuen, C. T., O'Brien, J., Childs, R. A., Chai, W., Lawson, A. M., Drbal, K., Fiserova, A., Pospisil, M., and Feizi, T. (1994) Nature 372, 151-157
  25. Mond, J. J., Lees, A., and Snapper, C. M. (1995) Annu. Rev. Immunol. 13, 655-692[CrossRef][Medline] [Order article via Infotrieve]
  26. Mond, J. J., Vos, Q., Lees, A., and Snapper, C. M. (1995) Curr. Opin. Immunol. 7, 349-354[CrossRef][Medline] [Order article via Infotrieve]
  27. Snapper, C. M., and Mond, J. J. (1996) J. Immunol. 157, 2229-2233[Abstract]
  28. Snapper, C. M., and Paul, W. E. (1987) Science 236, 944-947[Medline] [Order article via Infotrieve]
  29. Becker, J. C., Kolanus, W., Lonnemann, C., and Schmidt, R. E. (1990) Scand. J. Immunol. 32, 153-162[Medline] [Order article via Infotrieve]
  30. Snapper, C. M., Yamaguchi, H., Moorman, M. A., Sneed, R., Smoot, D., and Mond, J. J. (1993) J. Immunol. 151, 5251-5260[Abstract/Free Full Text]
  31. Coligan, J. E., Kruisbeck, A. M., Margulies, D. H., Shevach, E. M., and Strober, W. (1994) Current Protocols in Immunology, John Wiley & Sons, New York
  32. Watier, H., Guillaumin, J. M., Piller, F., Lacord, M., Thibault, G., Lebranchhu, Y., Monsigny, M., and Bardos, P. (1996) Transplantation 62, 105-113[Medline] [Order article via Infotrieve]
  33. Cohney, S., Mouhtouris, E., McKenzie, I. F. C., and Sandrin, M. S. (1996) Immunogenetics 44, 76-79[CrossRef][Medline] [Order article via Infotrieve]
  34. Hayes, C. E., and Goldstein, I. J. (1974) J. Biol. Chem. 249, 1904-1914[Abstract/Free Full Text]
  35. Inverardi, L., Clissi, B., Stolzer, A. L., Bender, J. R., Sandrin, M. S., and Pardi, R. (1997) Transplantation 63, 318-330
  36. Sandrin, M. S., Fodor, W. L., Mouhtouris, E., Osman, N., Cohney, S., Rollins, S., Guilmette, E. R., Setter, E., Squinto, S. P., and McKenzie, I. F. C. (1995) Nat. Med. 12, 1261-1267
  37. Fodor, W. L., Williams, B. L., Matis, L. A., Madri, J. A, Rollins, S. A., Knight, J. W., Velander, W., and Squinto, S. P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11153-11157[Abstract/Free Full Text]
  38. Rosengard, A. M., Cary, N. R., Langford, G. A., Tucker, A. W., Wallwork, J., and White, D. J. (1995) Transplantation 59, 1325-1333[Medline] [Order article via Infotrieve]
  39. Diamond, L. E., McCurry, K. R., Martin, M. J., McClellen, S. B., Oldham, E. R., Platt, J. L., and Logan, J. S. (1996) Transplantation 61, 1241-1249[Medline] [Order article via Infotrieve]
  40. Osman, N., McKenzie, I. F. C., Mouhtouris, E., and Sandrin, M. S. (1996) J. Biol. Chem. 271, 33105-33109[Abstract/Free Full Text]
  41. Parham, P., Alpert, B. N., Orr, H. T., and Strominger, J. L. (1977) J. Biol. Chem. 252, 7555-7567[Medline] [Order article via Infotrieve]
  42. Storkus, W. J., Salter, R. D., Alexander, J., Ward, F. E., Ruiz, R. E., Cresswell, P., and Dawson, J. R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5989-5992[Abstract]
  43. Colonna, M., Borsellino, G., Falco, M., Ferrara, G. B., and Strominger, J. L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 12000-12004[Abstract]
  44. Cella, M., Longo, A., Ferrara, G. B., Strominger, J. L., and Colonna, M. (1994) J. Exp. Med. 180, 1235-1242[Abstract]
  45. Barber, L. D., Patel, T. P., Percival, L., Gumperz, J. E., Lanier, L. L., Phillips, J. H., Bigge, J. C., Wormald, M. R., Parekh, R. B., and Parham, P. (1996) J. Immunol. 156, 3275-3284[Abstract]
  46. Sepp, A., Skacel, P., Lindstedt, R., and Lechler, R. I. (1997) J. Biol. Chem. 272, 23104-23110[Abstract/Free Full Text]
  47. Shinkel, T. A., Chen, C. G., Salvaris, E., Henion, T. R., Barlow, H., Galili, U., Pearse, M. J., and D'Apice, A. J. F. (1997) Transplantation 64, 197-204[Medline] [Order article via Infotrieve]
  48. Matsumoto, I., and Osawa, T. (1969) Biochim. Biophys. Acta 194, 180-189[Medline] [Order article via Infotrieve]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.