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1–3Gal1
Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02129
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1–3Gal
1–4GlcNAc-R
(Gal), synthesized by the enzyme UDP
galactose:-D-galactosyl-1,4-N-acetyl-D-glucosaminide
(1–3)galactosyltransferase or GT. Using GT knockout mice
(GT0 mice), which like humans produce serum Abs that bind
Gal, we examined the role of T cells in production of Abs specific
for Gal. GT0 mice were crossed with TCR- knockout
mice (TCR-0) to generate double-knockout mice
(GT0/TCR-0). While
GT0/TCR-+ mice exhibited an age-dependent
increase in the serum titer of natural Abs specific for Gal, a
similar increase was not observed in
GT0/TCR-0 mice, and the titer of
Gal-specific Abs in double knockouts was significantly lower than in
age-matched GT0/TCR-+ mice. Immunization
with pig cells resulted in a significant increase in the serum titer of
Gal-specific Abs in GT0/TCR-+ mice, but
had no effect on the level of Gal-specific serum Abs in
GT0/TCR-0 mice. Treatment of
GT0/TCR-+ mice with anti-CD40L Abs
before immunization with pig cells prevented sensitization to Gal.
Our data suggest that the majority of Gal-specific Abs are T cell
dependent and that production of Gal-specific Abs after
sensitization can be prevented by blocking costimulatory
pathways. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1–3Gal1–4GlcNAc-R, hereafter referred to as Gal
(1, 2, 3). This carbohydrate epitope is synthesized by the
addition of a terminal galactosyl group to a preexisting galactose
residue linked to a N-acetyl-glucosaminyl structure, and is
catalyzed by the glucosyltransferase UDP
galactose:-D-galactosyl-1,4-N-acetyl-D-glucosaminide
(1, 2, 3)galactosyltransferase (EC 2.4.1.151), or GT. As many as
106 to 107 Gal epitopes
can be expressed on the surface of pig cells and tissues
(4). In humans, Gal-specific Abs comprise 
1–8% of
circulating Ig, and it has been shown that approximately 1% of
EBV-transformed peripheral blood B cells make Abs that bind Gal
(5, 6).
Abs specific for Gal are produced in the host without intentional
immunization and are termed natural Abs. Production of natural Abs
specific for Gal is thought to occur following exposure to bacteria
that colonize the gastrointestinal tract (7, 8). The
presence of these Abs in serum and secretory fluids, such as colostrum
and saliva, suggests that these Abs have evolved to play a protective
role in primate immunity. Enveloped viruses produced in
Gal-expressing animal cells, such as lymphocytic choriomeningitis
virus, Newcastle disease virus, and vesicular stomatitis virus, as well
as C-type retroviruses, have all been shown to be susceptible to
inactivation by Gal-specific Abs in serum. Gal-modified host cell
surface proteins are incorporated into virus envelopes, which renders
the virus susceptible to elimination by Gal-specific Abs (9, 10).
In humans, Gal-specific Abs appear to be encoded for by a restricted
set of Ig VH genes from the
VH3 family (11). More recently, the
development of knockout mice lacking the enzyme UDP
galactose:-D-galactosyl-1,4-N-acetyl-D-glucosaminide
(1, 2, 3)galactosyltransferase, the enzyme that generates the Gal
epitope, has made it possible to study regulation of Gal Ab
production in a small animal model (12, 13), because GT
knockout mice (GT0 mice), like humans, produce
Abs specific for Gal (14). Recently, the analysis of a
small number of B cell hybridomas isolated from immunized
GT0 mice revealed that Abs specific for Gal
appear to exhibit restricted V region gene usage (15).
Interestingly, somatic mutations were observed in the
VH genes utilized by these hybridomas as well as
human VH genes encoding Gal-specific Abs
(11). The presence of somatic mutations is surprising
because Gal-specific Abs are thought to be largely T cell
independent due to the structural nature of the epitope on
glycoproteins and glycolipids, which should allow efficient
cross-linking of the B cell Ag receptor. Insofar as Ig somatic
hypermutation requires T cell involvement (16), the
presence of somatic mutations suggests that Abs specific for Gal may
have a T cell-dependent component.
Using GT0 mice, we examined the requirement for
T cells in the production of Gal-specific Abs. To this end,
GT0 mice were mated to TCR--deficient mice
(17) to generate double-knockout mice
(GT0/TCR-0). We then
compared the ability of
GT0/TCR-0 and
GT0/TCR-+ mice to
produce Gal-specific natural Abs. Our data suggest that the majority
of Gal-specific natural Abs that develop over time and Abs produced
following sensitization to pig cells are T cell dependent. Furthermore,
production of Gal-specific Abs following immunization with pig cells
can be prevented by blocking the interaction of CD40 and CD40L.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The breeding of GT0 mice for these studies
is described previously (18). TCR- knockout mice on the
C57BL/6 background were obtained from The Jackson Laboratory (Bar
Harbor, ME) and mated with H-2b
GT0 mice. All experiments were conducted using
mice between 6 and 8 wk of age. C57BL/6SnJ mice were used as
GT+ controls, and were obtained from The Jackson
Laboratory. All mice were housed in autoclaved microisolator conditions
and maintained on irradiated feed and autoclaved acidified drinking
water.
Immunization
Heparinized pig blood was collected from miniature swine from
the Massachusetts General Hospital herd housed at the Tufts University
School of Veterinary Medicine (North Grafton, MA) (19).
PBMC were purified from pig blood, as described previously
(20). Mice were immunized i.p. with
107 irradiated (2500 rad) pig PBMC
(pPBMC).3 Immunization
with Streptococcus pneumoniae strain R36A was conducted as
described previously (21). The multivalent polymer
Gal1–3Gal1–4GlcNAc-polyacrylamide (Gal-PAA) was used as a
T cell-independent type 2 Ag in this study, and was purchased from
GlycoTech (Rockville, MD). Gal-PAA consists of a multivalent PAA
polymer of approximately 30 kDa containing 20% carbohydrate on a molar
basis. Mice were immunized i.p. with 100 µg Gal-PAA dissolved in
normal saline at approximately 8 wk of age. Mice were immunized 3 wk
later, and the serum titer of Gal-specific Abs was determined by
ELISA at days 5 and 10 following the second immunization.
Flow cytometry
Cell surface staining of peripheral blood, flow cytometry, and
analyses were performed as described previously (22).
Gal epitopes were detected using the Gal-specific IB4 lectin from
Bandeiraea simplicifolia (BS-I isolectin
B4) (23). T cells were detected
using the mAb H57-597, which recognizes all TCRs
(24)
Enzyme-linked immunosorbent assay
ELISAs were conducted as described previously (22).
Briefly, ELISA plates (Corning, Corning, NY) were coated overnight at
4°C with either Gal conjugated to BSA or lactosamine conjugated to
BSA (Lac-BSA; V-Labs, Covington, LA) in carbonate buffer (pH 9.5), and
then washed with PBS containing 0.05% Tween 20 (PBS-Tween). Lac-BSA
shares all determinants with Gal-BSA, except for the terminal
galactose structure, and serves as a specificity control. The wells
were blocked with 1% BSA in PBS-Tween for 1 h at room temperature
and then washed. Serum samples were serially diluted in PBS-Tween,
added to the plates, and incubated for 1 h at 37°C. The plates
were then washed extensively with PBS-Tween, and bound Abs were
detected using HRP-conjugated goat anti-mouse IgM. The plates were
incubated for 1 h at 37°C and then washed five times with
PBS-Tween. A total of 0.01 mg/ml o-phenylenediamine
dihydrochloride in substrate buffer was then added for 20 min at room
temperature to develop the assays. The reaction was terminated by
adding H2SO4 to each well,
and absorbency was read at 492 nm. Background values obtained from
Lac-BSA-coated plates were subtracted from those obtained using
Gal-BSA-coated plates. Assays were performed in duplicate. The titer of
phosphorylcholine (PC)-specific Abs following immunization with
S. pneumoniae was determined as described previously
(21).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells in production of Gal-specific
natural Abs
To determine the role of T cells in production of Gal-specific
natural Abs, GT0 mice were crossed with TCR-
knockout mice (17). Offspring were then intercrossed to
generate GT-TCR- double knockouts
(GT0/TCR-0 mice). Double
knockouts identified by staining of peripheral blood cells for
expression of Gal and mature T cells, followed by flow
cytometry, were then intercrossed to establish a colony of
GT0/TCR-0 mice (Fig. 1
). Starting at 5 wk of age,
GT0/TCR-0,
GT0/TCR-+, as well as
GT+/TCR-+ normal
controls were bled every other week, and sera were analyzed for the
presence of Abs capable of binding to gal(1, 3)gal-conjugated
to BSA (Gal-BSA) by ELISA. Binding of serum Abs to Lac-BSA, an Ag
that shares all determinants with Gal-BSA except for the terminal
galactose, was examined in parallel to confirm that Ab binding to
Gal-BSA was specific. IgM Abs were studied because we have observed
that in GT0 mice IgM Abs make up the majority of
Gal-specific Abs (our unpublished observation). At 5 wk of
age, low levels of Gal-specific serum Abs were detectable in the
sera of GT0/TCR-0 and
GT0/TCR-+ mice (Fig. 2). As expected, Gal-specific Abs were
not detected in
GT+/TCR-+ or
GT+/TCR-0 (TCR
knockouts) at any time analyzed (not shown). While
GT0/TCR-+ mice exhibited
an age-dependent increase in the serum titer of Gal-specific Abs
apparent by 14 wk of age, a similar increase was not observed in
GT0/TCR-0 mice (Fig. 2).
Similar results were observed at 22 wk of age (not shown). These data
suggest that an age-dependent increase in the level of Gal-specific
serum natural Ab is largely T cell dependent.
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Gal-specific Abs following pig cell immunization
is T cell dependent
We next examined the role of T cells in the production of
Gal-specific Abs following exposure to pig cells. To this end,
8-wk-old GT0/TCR-0,
GT0/TCR-+, and
GT+/TCR-+ mice were
immunized i.p. with 107 irradiated pPBMC. Ten
days later, the mice were reimmunized, and 2 wk after the second
immunization the mice were bled and sera were analyzed for the
presence of Gal-specific Abs by ELISA. As expected based on our
previous work (22), following immunization with pPBMC,
GT0/TCR-+ exhibited a
significant increase in Gal-specific Ab titer, while Gal-specific
Abs were undetectable in the serum of immunized
GT+/TCR-+ controls (Fig. 3). In contrast, immunization of
GT0/TCR-0 did not lead
to a significant increase in serum Gal-specific Ab titer (Fig. 3).
The level of Gal-specific Ab in
GT0/TCR-0 was
essentially unchanged from that observed before immunization. These
data suggest that anti-Gal Ab production following exposure to
pig cells is T cell dependent.
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0 mice with T
cell-independent type 2 Ags containing Gal structures
To directly determine the extent to which T cell-independent Abs
can contribute to a response to Gal, age-matched
GT0/TCR-0 and
GT0/TCR-+ mice were
immunized i.p. with a T cell-independent type 2 Ag, Gal conjugated
to polyacrylamide (Gal-PAA). Repeated immunization of either
GT0/TCR-0 or
GT0/TCR-+ mice with
Gal-PAA led to only a modest increase in the serum titer of
Gal-specific Abs (Fig. 4A).
Similar results were obtained following i.v. immunization (not
shown).
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0 and
GT0/TCR-+ mice to
respond to T cell-independent type 2 Ags, mice were immunized with
heat-killed S. pneumoniae strain R36A, and the ability to
make Abs specific for PC was examined by ELISA. Following immunization
with S. pneumoniae, both
GT0/TCR-0 and
GT0/TCR-+ mice were able
to make a robust response to PC (Fig. 4
B). Therefore,
GT0/TCR-0 and
GT0/TCR-+ mice are
capable of making Ab responses to T cell-independent Ags. Insofar as
immunization with Gal-PAA did not lead to a significant increase in
the titer of Gal-specific serum Abs, while pig cell immunization
did, we suggest that Gal-specific Abs are predominantly T cell
dependent.
Costimulatory blockade prevents sensitization to Gal
The results of our experiments strongly suggested that production
of Gal-specific Abs is predominantly T cell dependent. We therefore
sought to examine whether inhibiting T cell responses using
costimulatory blockade could affect the ability of
GT0/TCR-+ mice to make a
response to Gal following immunization with pig cells. To this end,
GT0/TCR-+ mice were
treated with 0.1 mg anti-CD154 mAb (MR1 (25)) in
saline on days -3 and -1 relative to immunization with
106 irradiated pPBMC. The mice received a third
dose on day 3 after immunization. As expected, control mice treated
with saline alone exhibited a significant increase in the serum titer
of Gal-specific Abs following pig cell immunization (Fig. 5). In contrast,
GT0/TCR-+ mice receiving
anti-CD154 mAb failed to exhibit a significant increase in the
serum titer of Gal-specific Abs following pig cell immunization
(Fig. 5). Thus, costimulatory blockade prevents production of
Gal-specific Abs following exposure to pig cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Gal represent the major immunological hurdle
to successful transplantation of pig organs and tissues into humans,
and are thought to play a role in host immunity to various pathogens.
Yet, very little is known about how production of Abs specific for
Gal is regulated. Using GT0 mice, we examined
the role of T cells in the production of Gal-specific Abs.
Immunization with pig cells was able to elicit production of
Gal-specific Abs in GT0 mice only when
T cells were present. Immunization with T cell-independent Ags
containing the Gal epitope had little effect on the serum level of
Gal-specific Abs. Furthermore, costimulatory blockade was able to
completely inhibit production of Gal-specific Abs following
sensitization with pig cells. Together, these data suggest that the
majority of natural Abs produced in GT0 mice are
T cell dependent.
Based on the structural nature of the Gal Ag on glycoproteins and
glycolipids, and the observation that IgM Gal Ab is the predominant
Ig involved in in vitro cytotoxicity to pig cells as well as hyperacute
rejection (reviewed in Ref. 26), it has been thought that
T cell-independent Abs make up a significant fraction of the response
to Gal. However, the observation that somatic mutations are present
in VH genes utilized by hybridomas making
Gal-specific IgM Abs isolated from GT0 mice
(15), and human VH genes encoding
Gal-specific Abs (11), has called for a reexamination
of the T cell independence of these Abs because Ig somatic
hypermutation requires T cell involvement (16). Recent
evidence in rodent models suggesting a role of T cells in production of
Gal-specific Abs has been indirect, inferred based on the isotype
composition of elicited Abs following sensitization, and the effect of
nondepleting anti-CD4 Abs on survival of Gal expressing allo-
and xenogeneic hearts (27, 28). The genetic approach
presented in this work suggests that IgM Abs specific for Gal are T
cell dependent.
Although the majority of Gal-specific Ab is T cell dependent, we
were able to observe a small amount of Gal-specific natural Ab in T
cell-deficient GT0 mice. While production of this
Ab is clearly independent of T cells in these mice, it is not
clear whether 
T cells could be involved in its production.
T cells have been implicated in providing help to B cells,
particularly after certain types of infections are initiated
(29). Thus, it is possible that in the absence of T
cells, infections involving stimulation of T cells could lead to
B cell activation and production of Gal-specific Abs, as observed in
other systems (30). The mice used in this study were
housed in microisolator conditions, which affects their exposure to
various flora and may influence production of Abs specific for Gal.
We are currently examining the role of T cells in the production
of Gal-specific Abs after infection, and whether exposure to various
pathogens can affect the T cell dependence of Gal-specific
Abs.
Inhibition of the interaction of CD40 and CD154 completely prevented
production of Gal-specific Abs in vivo after immunization with pig
cells. Given that the interaction of CD40 and CD154 is required for T
cell-dependent immune responses (31), these results
support our hypothesis that Gal-specific Ab production is T cell
dependent. These results also have implications for
xenotransplantation, because sensitization to Gal following exposure
to pig tissues could be overcome by blocking the interaction of CD40
and CD154. Consistent with this notion, it has been shown in baboons
that preconditioning with a combination of whole body and thymic
irradiation, extracorporeal immunoadsorption of Gal Ab,
mycophenolate mofetil, antithymocyte globulin, and anti-CD154 leads
to humoral hyporesponsiveness following infusion of cytokine-mobilized
pig blood cells (32). Indeed, the observation that
Gal-specific Ab production is T cell dependent suggests that T cell
immunosuppression will be a major part of host preparation for
xenotransplantation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. John Iacomini, Transplantation Biology Research Center, Massachusetts General Hospital, MGH-East, Building 149, 13th Street, Boston, MA 02129.
3 Abbreviations used in this paper: pPBMC, pig PBMC; Lac, lactosamine; PAA, polyacrylamide; PC, phosphorylcholine.
Received for publication August 8, 2001. Accepted for publication November 20, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1–3)Gal epitopes. Proc. Natl. Acad. Sci. USA 90:11391.-galactosyl epitopes on nucleated cells. J. Biol. Chem. 263:17755.1,3)galactose epitope. Transplantation 64:501.[Medline]
-galactosyl immunoglobulin G and bacteria of the human flora. Infect. Immun. 56:1730.1–3Gal epitope as a host modification factor eliciting natural humoral immunity to enveloped viruses. J. Virol. 72:4650.1–3)galactosyltransferase. Nature 379:85.[Medline]
1,3Gal epitopes implicated in sperm adhesion to the zona pellucida glycoprotein ZP3 are not required for fertilization in the mouse. J. Biol. Chem. 270:21437.-1,3-galactosyltransferase knockout mouse: implications for xenotransplantation. Transplantation 61:13.[Medline]
1,3-Galactosyltransferase deficient mice produce cytotoxic natural anti-Gal antibodies. Transplant. Proc. 28:561.[Medline]
1,3galactosyltransferase knock out mice. Mol. Immunol. 37:455.[Medline]
and block thymocyte development at different stages. Nature 360:225.[Medline]
T cell receptors. J. Immunol. 142:2736.[Abstract]
-gal epitopes, and xenotransplantation. U. Galili, and J. L. Avila, eds. -Gal and Anti-Gal: 1,3-Galactosyltransferase, -Gal Epitopes, and the Natural Anti-Gal Antibody 229. Kluwer Academic/Plenum Publishers, New York.
(1, 3) galactose antibody production in 1,3-galactosyltransferase gene knockout mice after xeno and allo transplantation. Transplant Immunol. 8:129.[Medline]
1,3-galactosyltransferase knockout (GT-Ko) mice. Xenotransplantation 7:275.[Medline]
T-cell help in responses to pathogens and in the development of systemic autoimmunity. Immunol. Res. 16:229.[Medline]
T-cell-independent immunoprotective response towards gut coccidia is supported by cells. Immunology 101:325.[Medline]
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and •) and
GT0/TCR-
and 
) mice at 5 (• and
) and
GT0/TCR-
) and
GT0/TCR-
) mice
(n = 5 per group) after immunization with
