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INTRODUCTION |
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
(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
(1,3)Gal in their tissues, because the gene encoding the
(1,3)-galactosyltransferase, which links a terminal galactose residue to Gal
(1,4)GlcNAc oligosaccharide backbone structures, is
inactive in these species (7,8).
Anti-Gal
(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
(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
(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-
. Costimulatory signals provided
by the NK cells, together with the effects of NK cell-derived IFN-
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
(1,3)Gal, we addressed the possibility that
receptors on human NK cells may also react with ligands containing terminal Gal
(1,3)Gal residues, leading to augmented natural cytotoxicity as well as IgG humoral activity against porcine endothelium.
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EXPERIMENTAL PROCEDURES |
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
-galactosidase at
concentrations shown to reduce antibody-directed complement lysis of
porcine endothelium (32). Briefly, PAEC monolayers were treated with
either
-galactosidase isolated from the Green coffee been (Sigma) or
-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
-galactosidase was specific for Gal
(1,3)Gal epitopes and did not affect other carbohydrate epitopes
(data not shown). Furthermore, treatment with
-galactosidase did not remove Gal
(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
(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
(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).
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RESULTS |
Susceptibility of Porcine Endothelium to Human NK Lysis Correlates
with Expression of Gal
(1,3)-Gal--
Two endothelial cell lines
with opposing
(1,3)-galactosyltransferase activities and surface
expression of terminal Gal
(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
(1,3)Gal increases susceptibility
of xenogeneic endothelium to lysis by human NK cells. To more directly
examine the role of the terminal Gal
(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
(1,3)-linked galactose residue within the Gal
(1,3)Gal
structure as an essential component of porcine ligands involved in
triggering human NK cell lysis. Enzymatic treatment of porcine
endothelium with
-galactosidase reduced NK lysis in a
concentration-dependent manner, which correlated with the
level of reduced Gal
(1,3)Gal expression (Fig. 1, d and
e). At the highest concentration of
-galactosidase used, 20 units/ml, NK lysis was inhibited by a mean of 35% accompanying a
44% reduction in cell surface expression of Gal
(1,3)Gal. This inhibition of NK lysis was specific to cleavage of terminal
(1,3)-linked galactose residues, because enzymatic treatment with
-galactosidase had no effect (Fig. 1f).

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Fig. 1.
a, expression of Gal (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 (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 (1,3)Gal on PAECs after treatment with -galactosidase.
PAECs were treated with 0.2-20 units/ml -galactosidase, and
expression of Gal (1,3)Gal was measured by direct flow cytometry
using FITC-conjugated IB4. e, reduction in NK
lysis of PAEC targets after treatment with -galactosidase.
IL-2-activated human PBMCs were used as effector cells against
-galactosidase-treated PAEC targets. f, specificity of
-galactosidase treatment of PAECs for reduction in NK lysis.
IL-2-activated human PBMCs were used as effector cells against - or
-galactosidase (gal'ase)-treated PAEC targets.
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Expression of Gal
(1,3)-Gal in COS Cells Increases
Susceptibility to NK Lysis--
To directly demonstrate the effect of
surface expression of
(1,3)-linked galactose residues on
susceptibility to NK lysis, COS cells were transfected with the murine
(1,3)-galactosyltransferase gene (Fig.
2a). These cells do not
normally express Gal
(1,3)Gal epitopes and acquire susceptibility to
complement-mediated lysis in the presence of human serum after
transfection with
(1,3)-galactosyltransferase (4). In the present
study, COS cells transfected with
(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
(1,3)Gal (35). Our results extend these observations and show that
the increased binding of NK cells to terminal Gal
(1,3)Gal residues
expressed by ligands on cells transfected with
(1,3)-galactosyltransferase leads to activation of NK cell
stimulatory receptors and causes increased target cell lysis. Moreover,
because augmented NK lysis of
(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
(1,3)Gal are constitutively expressed.

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Fig. 2.
a, induced surface expression of Gal
(1,3)Gal on COS cells after transfection with
(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
(1,3)-galactosyltransferase gene or the plasmid alone. After
(1,3)-galactosyltransferase transfection, 28.2% of COS cells
expressed Gal (1,3)Gal; in contrast, COS cells transfected with
control vector failed to demonstrate any surface expression of Gal
(1,3)Gal. b and c, COS cells transfected with
the (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 (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
(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 (1,3)-galactosyltransferase was ~30%
irrespective of whether the NK cells were resting or
IL-2-activated.
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Inhibition of Human NK Cell Lysis of Porcine Endothelium by Soluble
Gal
(1,3)-Gal or Fuc
(1,2)-Gal Oligosaccharide
Derivatives--
Transfection of porcine endothelium with the gene
encoding the
(1,2)-fucosyltransferase enzyme has been shown to
reduce the level of Gal
(1,3)Gal expression (36). The
(1,2)-fucosyltransferase competes with
(1,3)-galactosyltransferase for the acceptor substrate Gal
(1,4)GlcNAc and diverts synthesis of Gal
(1,3)Gal
(1,4)GlcNAc to Fuc
(1,2)Gal
(1,4)GlcNAc ("H substance" or blood type O
phenotype). To compare the effect of terminal Gal
(1,3)Gal or Fuc
(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
(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
(1,3)Gal are bound by receptors on
human NK cells. The addition of a terminal Fuc
(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
(1,3)Gal and Fuc
(1,2)Gal residues.

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Fig. 3.
Inhibition of NK lysis of PAECs with soluble
oligosaccharide derivatives containing terminal Gal (1,3)Gal or Fuc
(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 (1,3)-galactosylated or (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 (1,3)-galactose residue to lacto-N-neo-tetra and
the addition of a terminal (1,2)-fucose residue to
lacto-N-tetra increased the percent inhibition of NK
lysis.
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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
(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
(1,3)Gal
(1,4)Glc was
inversely proportional to that of Fuc
(1,2)Gal
(1,4)Glc,
reflecting the degree of competition for Gal
(1,4)Glc substrate by
the glycosyltransferases. Reduction in surface expression of Gal
(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
(1,3)Gal, human NK lysis could not be reduced by
>55% even with >80% reduction of Gal
(1,3)Gal expression. This
level of human NK lysis approaches that seen with allogeneic
endothelium.

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Fig. 4.
a and b, surface expression
of Fuc (1,2)Gal and Gal (1,3)Gal on porcine endothelial cells
after transfection with (1,2)-fucosyltransferase cDNA. Flow
cytometric analysis was performed on porcine endothelial cells
transfected with (1,2)-fucosyltransferase cDNA using
FITC-conjugated IB4 and UEA-I, which recognize Gal
(1,3)Gal (34) and Fuc (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
(1,2)Gal increased, surface expression of Gal (1,3)Gal
progressively decreased, consistent with the known dominant effect of
(1,2)-fucosyltransferase over (1,3)-galactosyltransferase.
c, porcine endothelial cells transfected with
(1,2)-fucosyltransferase cDNA have reduced susceptibility to
human NK lysis. Purified human NK cells were used against porcine
endothelial cell targets transfected with (1,2)-fucosyltransferase
cDNA. NK lysis decreased in parallel with the reductions in Gal
(1,3)Gal expression.
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DISCUSSION |
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
(1,3)Gal residues
the principal antigen on porcine endothelium recognized by xenoreactive antibodies. Enzymatic treatment of porcine
endothelium with
-galactosidase reduced human NK lysis of porcine
endothelium, which specifically correlated with the level of Gal
(1,3)Gal expression. Furthermore, transfecting a primate cell line
with the murine
(1,3)-galactosyltransferase gene increased the
susceptibility to lysis by human NK cells, again correlating with the
expression of Gal
(1,3)Gal. These results suggest that receptors on
human NK cells can directly recognize Gal
(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
(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
(1,3)Gal expression on porcine endothelium by high level expression
of the enzyme
(1,2)-fucosyltransferase (36). The second strategy is
predicated on the knowledge that both
(1,3)-galactosyltransferase
and
(1,2)-fucosyltransferase use the same acceptor substrate,
Gal
(1,4)GlcNAc, to direct synthesis of Gal
(1,3)Gal
(1,4)GlcNAc and Fuc
(1,2)Gal
(1,4)GlcNAc (H substance or blood
type O phenotype), respectively. When both enzymes are cotransfected
into COS cells,
(1,2)-fucosyltransferase dominates over
(1,3)-galactosyl transferase so that Gal
(1,3)Gal expression is
almost completely suppressed in the presence of Fuc
(1,2)Gal (36).
This effect is the result of the temporal order of action of these
enzymes, with
(1,2)-fucosyltransferase having preferential access to
the Gal
(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
(1,3)Gal or terminal Fuc
(1,2)Gal residue to their respective carbohydrate derivative further inhibited human NK lysis of porcine endothelium. These results suggest that both Gal
(1,3)Gal and Fuc
(1,2)Gal residues are able to bind human NK cells and raise the
possibility that substitution of a terminal
(1,2)-linked fucosyl
residue for a terminal
(1,3)-linked galactosyl residue to the Gal
(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
(1,2)-fucosyltransferase cDNA. Reduction in surface expression of Gal
(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
(1,3)Gal. However, NK lysis could
not be fully eliminated even with almost complete reduction of Gal
(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
(1,2)-fucosyltransferase decreases
susceptibility to human NK lysis by two possible mechanisms: 1) a
reduction of Gal
(1,3)Gal residues within porcine endothelial cell
ligands, which bind to stimulatory receptors on human NK cells; and 2)
an increase of Fuc
(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
-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
(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
(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
(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
(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.