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J Biol Chem, Vol. 273, Issue 46, 30680-30687, November 13, 1998
From the Glycobiology Program, La Jolla Cancer Research Center, The
Burnham Institute, La Jolla, California 92037
The aberrant expression of core 2 O-glycans on T cell surface glycoproteins has been
associated with various immunodeficient syndromes such as
Wiskott-Aldrich syndrome and AIDS. To determine the effect of this
aberrant expression of core 2 O-glycans on immune
responses, we previously generated transgenic mice overexpressing core
2 Carbohydrate structures of glycoproteins and glycolipids
dramatically change during development and differentiation (1-3). Abnormalities in those cell surface carbohydrates are often associated with malignant transformation and other pathological conditions, including immunodeficiency (1-4).
Among the various cell surface carbohydrates, the biological function
of mucin-type O-glycans, which are attached to serine or
threonine residues on cell surface glycoproteins, is not well understood. There are, however, several reports suggesting that O-glycans are involved in the modulation of cell adhesion
processes. For example P- and L-selectins bind with higher affinity to
sialyl LeX and sulfated sialyl LeX expressed in
O-glycans than those expressed in N-glycans,
possibly due to multivalency of O-glycans (5-7).
In O-glycans of blood cells, various ligand carbohydrate
structures can be formed only in a branched O-linked
hexasaccharide, NeuNAc Various pathological conditions, including certain immunodeficient
syndromes, are associated with aberrant expression of core 2 O-glycans. Examples include Wiskott-Aldrich syndrome (13, 14), leukemia (15, 16), AIDS (16-18), and malignant transformation (19). In these diseases, core 2 O-glycans are highly
expressed on resting T cells, while they are not expressed in normal
conditions. To examine the influences of aberrant expression of core 2 O-glycans on the immune system, we have previously generated
transgenic mice whose T cells overexpress core 2 O-glycans.
We demonstrated that T cells from these transgenic mice exhibit reduced
primary T cell immune responses, as assessed by delayed-type
hypersensitivity, proliferation upon stimulation, and cytokine
production (20). We demonstrated that reduced immune responses of T
cells are due to impaired interaction between T cells and
antigen-presenting cells (20). The next important question is whether
overexpression of core 2 O-glycans affects humoral immune
responses. In this study, we address this question by examining humoral
immune responses in the transgenic mice compared with those of control
mice. We demonstrate that overexpression of core 2 O-glycans
impairs humoral immune responses to thymus-dependent
antigen. We suggest that this reduced B cell immune response is due to
inhibition of the interaction between T and B cells (T-B interaction).
Transgenic Mice--
Transgenic mice (Lck-mC2GnT) were
established in the previous study (20). Briefly, the transgene, mouse
C2GnT, was cloned into pBluescript under the control of p56lck
promoter (20, 21) and injected into FVB/N mouse zygote pronuclei. Stable lines of mice were generated by breeding founders with FVB/N
mice (20). Two founder mice were generated (Lck-mC2GnT-1 and -2), and
all experiments were carried out using cells isolated from
6-8-week-old litter mates, which were generation 4 and 5. Sex- and
age-matched littermates were used as wild-type controls. The results
obtained from each founder were not designated, as no detectable
difference in the results was noted using their offspring.
Glycosyltransferase Assay--
To measure C2GnT activity,
T cells were purified from spleens with Mouse T Cell Enrichment Column
(R&D Systems, Minneapolis, MN) (22). The resultant preparations were
>95% pure T cells, as determined by flow cytometric analysis for
Western Blot Analysis--
T cells were purified from a pool of
four spleens (from two males and two females) using the procedure
described above. Whole cell lysates of purified T cells were prepared
and blotted as described previously (20). The membranes were incubated
with the anti-CD43 monoclonal antibodies as described (20), and with anti-CD44 (IM7, culture supernatant, 1:40 dilution; Ref. 25), anti-CD45RB (23G2, 10 µg/ml), and anti-CD2 (RM2-5, 0.5 µg/ml). The
antibodies except for anti-CD44 were purchased from PharMingen (San
Diego, CA).
Expression of CD45 in CHO Cell Lines--
CD45ABC
cDNA containing the complete coding region (26) was subcloned into
pcDNAI (pcDNAI-CD45ABC). CHO cells stably
expressing leukosialin (CHO-leu) and CHO cells stably expressing
leukosialin and C2GnT (CHO-leu-C2) (27) were transiently transfected
with pcDNAI-CD45ABC using LipofectAMINE PLUS reagent
(Life Technologies, Inc.). Two days after the transfection, whole cell
lysates were prepared and subjected to Western blotting analysis. A
monoclonal antibody reactive to all isoforms of the CD45 (30-F11, 10 µg/ml; Ref. 28) and a CD45-exonB specific antibody (23G2, 10 µg/ml), purchased from PharMingen, were used. CD45ABC
cDNA was kindly provided by Dr. Matthew Thomas (Washington
University School of Medicine, St. Louis, MO).
Flow Cytometry--
Monodispersed cells (1 × 106) from mouse tissues were stained with
fluorescent-conjugated antibodies (1 µg/ml) as described previously
(20). PE-conjugated anti-CD43 activation-associated isoform (1B11),
FITC-conjugated anti-B220 (RA3-6B2), FITC-conjugated anti- Analysis of CD40 Ligand (CD40L) Expression on T
Cells--
CD4+ T cells were purified from spleens with
Mouse T Cell CD4 Subset Column Kit (R&D Systems, Minneapolis, MN) (22).
The resultant preparations were >95% pure CD4+ T cells,
as determined by flow cytometric analysis for CD4. Purified CD4+ T cells were activated for 6 h in 24-well plates
coated with anti-CD3 (142C-11, 20 µg/ml) and mouse ICAM-1-IgG fusion
protein (10 µg/ml) as described (20). In parallel, purified T cells were activated for 6 h with PMA and ionomycin as described (20). Activated T cells were subsequently harvested, washed, stained with
PE-conjugated anti-CD40 ligand, and then analyzed by flow cytometer.
B Cell Proliferation Assay--
Spleen cell suspension was
prepared from a pool of spleens from eight mice (four males and four
females). CD4+ T cells were isolated from half of a spleen
cell suspension as described above. B cells were isolated from the
other half of the cell suspension by a combination of treatment with
antibodies to Thy-1.1, CD4, and CD8 (PharMingen), followed by rabbit
complement (ICN, Irvine, CA) and incubation on plastic dishes to
deplete adherent cells (29). The resultant preparations were >95%
pure B cells, as determined by flow cytometric analysis with B220. For
B cell proliferation assays, purified B cells (2 × 105 cells/well, 200 µl) were cultured in RPMI 1640 medium
containing 10% fetal calf serum, 2-mercaptoethanol (50 µM), penicillin (100 units/ml), and streptomycin (100 µg/ml) for 72 h in 96-well flat-bottomed tissue culture plates.
In some experiments, the medium also contained 10 ng/ml IL-4
(PharMingen), 1 µg/ml lipopolysaccharide (Sigma), or 5 µg/ml
anti-CD40 (HM40-3, PharMingen). In parallel, purified B cells (1 × 105 cells/well) were co-cultured with non-activated or T
cells activated for 6 h (1 × 105 cell/well) in
the same medium as described above. The T cells were fixed with 0.4%
paraformaldehyde in PBS for 15 min at room temperature, then washed and
used to stimulate B cells (30). Cultures were pulsed with 1 µCi/well
[3H]thymidine (NEN Life Science Products) for the last
6 h of a 72-h culture period and then harvested onto a glass fiber
filter. [3H]Thymidine uptake was measured by liquid
scintillation counting.
Anti-KLH Antibodies Analysis by ELISA--
Mice were
intraperitoneally immunized with 100 µg of KLH (Sigma) in complete
Freund's adjuvant. Mouse anti-KLH IgM, IgG1, IgG2a, IgG2b, IgG3, and
IgA levels in the serum were determined by an isotype-specific ELISA.
Briefly, 96-well plates were coated with 10 µg/ml KLH for 24 h.
Plates were then washed and blocked with 1% bovine serum albumin in
PBS. Diluted serum samples were then added to wells, and plates were
incubated for 1 h at 37 °C. Anti-KLH IgM antibody titer was
determined with biotin-conjugated goat anti-mouse IgM and
streptavidin-conjugated horseradish peroxidase (PharMingen). The other
antibody titers were determined using alkaline phosphatase-conjugated
goat anti-mouse IgG1, IgG2a, IgG2b, IgG3, or IgA (PharMingen). ELISAs
were developed by using 1-Step ABTS substrate (Pierce) for peroxidase
and p-nitrophenyl phosphate substrate (PharMingen) for
alkaline phosphatase (31, 32).
Immunocytochemical Detection of Germinal Centers--
Mice were
immunized with KLH as described above. Spleen flat sections (5 µm
thick), 7 days after the immunization, were fixed with 4%
paraformaldehyde in PBS. The sections were incubated with antibody or
lectin in a humidified box (30). The sections were suspended in 10 mM HEPES buffer containing 0.15 M NaCl, 1 mM CaCl2, 1 mM MgCl2,
and 3% fetal calf serum, and stained with biotinylated peanut
agglutinin (Vector Laboratories, Burlingame, CA) for 1 h at room
temperature (33). Parallel sections were suspended in PBS supplemented
with 3% fetal calf serum and stained with biotinylated goat anti-mouse
IgM (R6-60.2, PharMingen). Biotinylated peanut agglutinin and
anti-mouse IgM were stained with PE-conjugated streptavidin
(PharMingen). The slides were evaluated and photographed under a Zeiss
Axioplan fluorescence microscope.
Generation of Transgenic Mice Overexpressing Branched
Hexasaccharides on T Cells--
As shown previously, we have generated
transgenic mice that express the mouse C2GnT gene under the control of
the proximal promoter of p56lck. This promoter allowed
increased activity of C2GnT in lymphoid tissues where T cells are
present (20). However, the previous results were obtained on a mixture
of T and B lymphocytes, and we could not exclude the possibility that B
lymphocytes also contained increased C2GnT activity in the transgenic
mice. To determine whether only T cells contain increased C2GnT
activity, T and B cells were purified from spleens, and
glycosyltransferases were assayed in cell lysates of purified T and B
cells. As shown in Table I, T cells
derived from the transgenic mice (Lck-mC2GnT) contained C2GnT activity
approximately 50 times higher than did T cells derived from control
mice. In contrast, the activities of
In the above experiments, as well as those that follow, we found that
results obtained from two independently generated transgenic lines
(Lck-mC2GnT-1 and -2) were practically identical. Therefore, we
conclude that clonal variations play little or no role in
determining the phenotype of either line.
T Cell C2GnT Overexpression Resulted in a Change in
Glycosylation of T Cell Surface Glycoproteins--
CD43 is one of the
most abundant T cell surface glycoproteins. There are two distinct
glycoforms of CD43: the 115-kDa form seen on resting T cells, which
carries simple tetrasaccharides and can be detected by S7 antibody, and
a 130-kDa glycoform seen on activated T cells, which carries branched
hexasaccharides and can be detected by the 1B11 antibody (34).
Previously, we have shown that CD43 expressing the hexasaccharide was
increased in the whole cell populations of the transgenic mice (20). As
shown in Fig. 1, the population of T
cells (
Fig. 2 shows the results of Western
blotting of T cell extracts. On the T cells from control mice, most of
CD43 was the 115-kDa tetrasaccharide form, while on T cells from
Lck-mC2GnT transgenic mice, most CD43 was the 130-kDa hexasaccharide
form carrying core 2 O-glycans (Fig. 2A). Similar
results were obtained for other O-glycosylated proteins such
as CD44 and CD45RB. CD44 was detected as an 85-kDa protein on T cells
from control mice, whereas T cells from the transgenic mice displayed
CD44 as an 88-kDa band (Fig. 2A). CD45RB is an isoform of
CD45. Western blotting using a monoclonal antibody, 23G2, identified
CD45RB as a 200-kDa band on T cells from control mice, whereas on T
cells from the transgenic mice, this 200-kDa band was faint (Fig.
2A). The antibody 23G2 reacts with a peptide portion that is
heavily glycosylated with O-glycans (35, 36). It is likely
that 23G2 cannot react efficiently with the same polypeptide when it is
glycosylated with core 2 O-glycan containing
oligosaccharides. In fact, the reactivity of 23G2 was weaker against
CD45 expressed on CHO-leu·C2 cells, which express core 2 O-glycans (27), than it was against the parent CHO cells,
which do not express core 2 O-glycans (lanes 5 and 6 in Fig. 2B). In contrast,
almost equal reactivity was observed when another antibody, 30F-11,
which reacts with all isoforms of CD45 (28), was used (lanes
3 and 4 in Fig. 2B). On the other
hand, mouse C2GnT overexpression did not affect CD2, because CD2 does
not contain O-glycans (Fig. 2A). These results strongly suggest that overexpression of mouse C2GnT in Lck-mC2GnT transgenic mice causes these T cell surface glycoproteins to carry substantially more core 2 O-glycans than do glycoproteins
derived from control mice. These observations are consistent with the results obtained by Western blotting of C2GnT-overexpressing cell lines (37).
No Difference in CD40 Ligand and CD40 Expression Levels
between Lck-mC2GnT Transgenic and Control Mice--
We then asked
whether overexpression of core 2 O-glycans affects T-B
interaction. To address this question, we first examined whether
overexpression of core 2 O-glycans affects expression levels
of CD40 ligand (CD40L), since T-B interaction is mainly mediated by
CD40L-CD40 interaction (26, 27, 38-40). When T cells were incubated
without activators, no CD40L expression was observed in either
transgenic or control mice (Fig.
3A, top panel). When T cells
were activated for 6 h with anti-CD3 and mouse ICAM-1-IgG fusion
protein, T cells from the transgenic mice expressed CD40L on the cell
surface at a level equivalent to that of control mice (Fig.
3A, middle panel, 13-17% and 13-15%,
respectively). T cells activated with PMA and ionomycin from either the
transgenic or control mice expressed similar amounts of CD40L as well
(Fig. 3A, bottom panel, 64-69% and 63-69%,
respectively). These results show that similar amounts of CD40L were
induced on in vitro activated CD4+ T cells
derived from the transgenic mice and control mice (Fig. 3A).
In addition, B cells purified from both the transgenic and control mice
expressed comparable amounts of CD40 (Fig. 3B).
Activated T cells from Lck-mC2GnT Transgenic Mice Inefficiently
Induce B Cell Proliferation Compared with Control--
To further
examine whether overexpression of core 2 O-glycans affects
T-B interaction, we assayed T cell-dependent B cell proliferation in vitro. This assay is critical because
CD40-CD40L interaction is too weak to be detected by adhesion (41). T
cells were activated for 6 h with anti-mouse CD3 and a mouse
ICAM-1-IgG fusion protein shown in Fig. 3A. B cells from the
transgenic mice (closed symbols) were cocultured
with activated T cells from control mice (shown as C) or the
transgenic mice (shown as Lck). B cell proliferation was
induced more by activated T cells from wild-type mice than by those
from the transgenic mice (shown as Lck). The proliferative
responses of B cells from the transgenic mice were enhanced by addition
of IL-4. However, T cells derived from the transgenic mice yielded a
reduced proliferative response even in the presence of IL-4 (Fig.
4A).
Similarly, B cells derived from control mice were cocultured with T
cells from the transgenic and control mice (open
symbols in Fig. 4A). T cells derived from the
transgenic mice produced a reduced proliferative response of control B
cells both in the absence and presence of IL-4 (Fig. 4A).
These results indicate that T cells derived from the transgenic mice
induced an impaired T-B interaction, regardless of whether the B cells
were derived from wild-type or the transgenic mice.
In contrast, B cells from the control and transgenic mice showed
comparable proliferative responses to lipopolysaccharide, to
lipopolysaccharide plus IL-4, or to anti-CD40 treatment (Fig. 4B), where T-B cell interaction was not required. These
results indicate that overexpression of core 2 O-glycans on
T cell surface glycoproteins inhibits T-B interaction.
Isotype Switching Is Impaired in Lck-mC2GnT Transgenic
Mice--
To investigate how overexpression of core 2 O-glycans in T cells influences antibody production, we
tested primary and secondary antibody responses of the transgenic mice.
IgM antibody is produced in primary immune responses, and production
switches to other immunoglobulin subtypes, such as IgG, in secondary
responses. As shown in Fig. 5, the
production of anti-KLH IgG1, IgG2a, and IgG2b responses was
significantly lower in Lck-mC2GnT transgenic mice (closed
symbols) than in control mice (open
symbols). In addition, the amount of IgM product was higher
in transgenic mice than in the control mice due to impaired switching
to other immunoglobulin isotypes (Fig. 5). The production of anti-KLH
IgG3 and IgA in the transgenic mice was similar to that of control
mice. These results, as a whole, indicate that isotype switching was
impaired in Lck-mC2GnT transgenic mice.
Impaired Germinal Center Formation in Lck-mC2GnT Transgenic
Mice--
Germinal center formation is dependent on the interaction
between CD40L and CD40 (30, 31, 42). We thus tested if germinal center
formation in Lck-mC2GnT transgenic mice is reduced, since CD40L-CD40
interaction is impaired in transgenic mice as shown in Fig.
4A. Mice were immunized with KLH on day 0. Spleens from immunized mice were taken on day 7, the peak time of germinal center
formation. Germinal centers from control mice detected by either
anti-IgM antibody or peanut agglutinin were larger and denser than
those from the transgenic mice (Fig. 6).
Moreover, significantly fewer germinal centers were found in the
transgenic mice than in controls (Table
II). There are no obvious differences in
numbers and ratios of T and B cells between the transgenic and control
mice (Fig. 1). These observations are consistent with the phenotypes of
CD40L-deficient or CD40-deficient mice. Mice carrying null mutations in
either gene do not display abnormalities in the number and ratios of T
and B cells, although germinal center formation is defective in
both mice (30, 31, 42).
This study demonstrated that B cells derived from the transgenic
mice overexpressing core 2 oligosaccharides in T cells exhibit reduced
humoral immune responses to thymus-dependent antigen, which
depend on T-B interaction. B cells derived from the transgenic mice
exhibited delayed immunoglobulin isotype switching (Fig. 5) and
impaired germinal center formation (Fig. 6). Moreover, T cells derived
from the transgenic mice inefficiently stimulated B cell proliferation,
regardless of whether B cells were derived from wild-type or the
transgenic mice (Fig. 4A). Overexpression of core 2 O-glycans on various surface glycoproteins of T cells apparently renders T-B interaction inefficient, resulting in reduced humoral immune responses in B cells.
B cell proliferation is induced by T-B interaction, which is mainly
mediated by CD40L and CD40 (29-31, 38-40). Binding of CD40L, a member
of the tumor necrosis factor gene family, to CD40, a protein related to
tumor necrosis factor receptor, is thought to trigger signal
transduction in B cells (43). A soluble CD40-IgG fusion protein was
shown to reduce T cell-dependent B cell proliferation by
inhibiting the interaction between T and B cells (39). No significant
difference was detected in the expression levels of CD40L and CD40
induced in vitro on CD4+ T cells derived from
the transgenic and control mice (Fig. 3). This observation was
supported by the fact that anti-CD40 antibody treatment induced almost
identical proliferation responses in B cells from the transgenic and
control mice (Fig. 4B). CD40L expression is very rapid and
transient. CD40L expression reaches maximal levels 6-8 h after
activation and then returns to resting levels in 24 h (40). In
contrast, B cell proliferation stimulated by T cells from the
transgenic mice, assayed after a 72-h incubation, was decreased
compared with that stimulated by T cells from control mice (Fig.
4A). CD40L contains few if any O-glycans (44).
Core 2 O-glycans overexpressed on various glycoproteins on
the T cell surface interfere with the interaction between T and B cells
most likely in later stages, resulting in reduced T
cell-dependent B cell proliferation (Fig. 4). These
results, taken together, suggest that T cell activation in early stages
is not impaired, but T-B interaction in later stages, which may be
mediated by other than CD40-CD40L interaction, is impaired in the
transgenic mice.
Previously, CD40L-deficient mice were shown to lack completely the
ability to undergo immunoglobulin isotype switching in response to KLH
(31). Similarly, B cells from CD40-deficient mice were unable to
undergo isotype switching in response to thymus-dependent antigens (30, 42), and no germinal centers were found in CD40L or
CD40-deficient mice (30, 31, 42). In CD40L- and CD40-deficient mice,
the impairment in immune responses such as isotype switching or
germinal center formation is complete. In contrast, delayed immunoglobulin isotype switching and reduced formation of germinal centers were observed in the transgenic mice overexpressing core 2 O-glycans (Figs. 5 and 6). The results strongly suggest that the overexpression of core 2 O-glycans interferes with the
interaction between T and B cells and CD40L-CD40 interaction is reduced
but not abolished in the transgenic mice.
IL-4 and IFN- The results obtained in this present study are consistent with those of
other reports demonstrating the effect of glycosylation on cell-cell
interaction. In particular, cell adhesion of A375 melanoma cells was
disrupted after cells were induced to express ASGP-1 mucin
glycoprotein, a protein similar to leukosialin (CD43) (45). In
contrast, it was reported that T cells derived from the
N-acetylglucosaminyltransferase V null mice produced
hyperimmune responses to T cell mitogens (46). Since the side chain
synthesized by N-acetylglucosaminyltransferase V is further
modified to produce poly-N-acetyllactosamines, the results
strongly suggest that bulky N-glycans are essential to
weaken unnecessary hyperimmune responses. These, and the findings
presented here, indicate that the complexity and amount of
O-glycans and N-glycans play critical roles in
cell adhesion.
Leukocytes from patients with immunodeficient syndromes such as
Wiskott-Aldrich syndrome (WAS) (13, 14, 47) and AIDS (16-18) show
increased activity of C2GnT and levels of core 2 O-glycans. C2GnT activity of peripheral blood lymphocytes from WAS patients (0.4 nmol/h/mg of protein) is 8-fold higher than that of normal individuals
(0.05 nmol/h/mg of protein) (13). This difference is similar to that
seen in peripheral blood lymphocytes of the Lck-mC2GnT transgenic mice
(0.46 nmol/h/mg of protein) compared with control mice (0.09 nmol/h/mg
of protein) (20). Moreover, it has been shown that immunoglobulin
isotype switching and IL-4 and INF- We thank Dr. Robert Hyman at the Salk
Institute for anti-CD44 monoclonal antibody, Dr. Matthew Thomas at
Washington University School of Medicine for CD45ABC
cDNA, Dr. Shu-ichi Matsuzawa at our institute for helpful advice on
immunological assays, Dr. Maria Granovsky at the Samuel Lunenfeld Research Institute, Toronto, for useful discussions, Jacqueline Avis
and Rhonda Jenkins for excellent technical support to establish and
maintain the transgenic mice colony, Dr. Stanislaw Krajewski for
assistance in making tissue sections, Drs. Edgar Ong and Elise Lamar
for critical reading of the manuscript, and Susan Greaney for
organizing the manuscript.
*
This work was supported by a MERIT award (R37CA33000) from
the National Cancer Institute.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.
The abbreviations used are:
C2GnT, core 2 2
M.-M. Huang, S. Tsuboi, M. Fukuda, K. Weinberg,
and D. B. Kohn, manuscript in preparation.
Overexpression of Branched O-Linked Oligosaccharides
on T Cell Surface Glycoproteins Impairs Humoral Immune Responses in
Transgenic Mice*
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-1,6-N-acetylglucosaminyltransferase (C2GnT) in T
cells, and demonstrated that T cell primary immune responses mediated through interaction between T cells and antigen-presenting cells are
impaired in the transgenic mice (Tsuboi, S., and Fukuda, M. (1997)
EMBO J. 16, 6364-6373). In this study, we determined
whether overexpression of core 2 oligosaccharides on T cells leads to impaired humoral immune responses by B cells using the same transgenic mice. When T cells were activated, both T and B cells from the transgenic and control mice expressed an equivalent amount of CD40L and
CD40, which are, respectively, the receptor and counter-receptor for
the interaction between T and B cells. However, activated T cells from
the transgenic mice induced B cell proliferation less efficiently than
those from control mice, regardless of whether B cells were isolated
from control or the transgenic mice. This suggests that overexpression
of core 2 O-glycans on T cell surface glycoproteins renders
T cell-B cell interaction inefficient. Moreover, in the transgenic mice
both immunoglobulin isotype switching and germinal center formation
were also impaired. Taken together, these results indicate that
aberrant expression of core 2 O-glycans on T cell surface
glycoproteins results in impaired humoral immune responses due to an
impaired interaction between T and B cells.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
2
3Gal13(NeuNAc23Gal14GlcNAc16)GalNAc1Ser/Thr, called a core 2 O-glycan. Core 2 -1,6-N-acetylglucosaminyltransferase (C2GnT)1 is essential to form
core 2 O-glycans (8, 9). In T cell development, the
expression of core 2 O-glycans is restricted to cortical
thymocytes, and their expression is barely detectable in medullary
thymocytes (10). This down-regulation of core 2 O-glycan
synthesis is apparently critical for apoptotic processes in T cell
development (11). In peripheral T cells, resting T cells express a
simple tetrasaccharide,
NeuNAc23Gal13(NeuNAc26)GalNAc1Ser/Thr. When
T cells are activated by mitogens, the expression of C2GnT is induced,
and as a result, O-glycans are converted to the core 2 O-glycans,
NeuNAc23Gal13(NeuNAc23Gal14GlcNAc16)GalNAc1Ser/Thr (12).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
T cell receptor. B cells were purified from spleens by flow
cytometric sorting. The resultant preparations were >99% positive for
B220 by flow cytometric analysis. Purified T or B cells were
homogenized in 0.1 ml of an extraction solution (0.4% Triton X-100,
150 mM NaCl), and the assay was performed essentially as
described using Gal13GalNAc1p-nitrophenol as a
substrate (9). Similarly,
UDP-Gal:-GlcNAc-1,4-galactosyltransferase and
UDP-Gal:-GalNAc-1,3-galactosyltransferase were assayed using p-nitrophenylglycosides (23, 24). In all of these assays, resultant products were adsorbed to a disposable C18 cartridge (Waters,
MA) and eluted by 70% methanol in water as described (9).
T
cell receptor (H57-597) or anti-CD40 (HM40-3), and PE-conjugated anti-CD40 ligand (MR1) were purchased from PharMingen. Anti-mouse CD32/16 (PharMingen) was included in all incubations to block antibody
binding to Fc receptors. Analyses were done with a FACSort flow
cytometer using the CellQuest program (Becton-Dickinson, San Jose, CA).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-1,3-galactosyltransferase,
which forms core 1 in O-glycans, and
-1,4-galactosyltransferase, which forms
N-acetyllactosamines, were almost equal in control and the
transgenic mice (Table I). Moreover, no difference in C2GnT activities
was found in B cells between the transgenic and control mice (Table I).
These results indicate that C2GnT is specifically increased in T cells
of the transgenic mice without an accompanying change in the activities of other glycosyltransferases.
Glycosyltransferase activities in purified T cells and B cells
T cell receptor positive cells) displaying the
CD43-hexasaccharide, which carries core 2 O-glycans,
increased in both spleens and lymph nodes derived from the transgenic
mice (Fig. 1). In contrast, the number of B cells, which are positive
for B220 staining, was similar in control (49-52%) and the transgenic
(47-50%) mice (Fig. 1).
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Fig. 1.
Mouse C2GnT overexpression resulted in a
change in glycosylation of T cell surface glycoproteins. The
expression of a CD43-hexasaccharide form carrying core 2 O-glycans in spleen and lymph node was analyzed by flow
cytometry. Splenocytes and lymph node cells were stained with
PE-anti-CD43-hexasaccharide form carrying core 2 O-glycans
(activation-associated form, 1B11) and FITC-conjugated anti- T
cell receptor (TcR) (H57-597). Splenocytes were also
stained with FITC-conjugated anti-B220 antibody (RA3-6B2). Spleens and
lymph nodes were isolated from two mice (one male and one female), and
the analysis was independently performed three times using two
different transgenic lines. The numbers indicate the ranges
of positive cells in percentages.
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Fig. 2.
Western blot analysis of whole-cell extracts
of purified T cells (A) and transfected CHO cells
(B). A, monoclonal antibodies specific to the
CD43-tetrasaccharide form (CD43-tetra, S7, 2 µg/ml),
CD43-hexasaccharide form (CD43-hexa, activation-associated
form, 1B11, 2 µg/ml), CD44 (IM7, culture supernatant, 1:40 dilution),
CD45RB (23G2, 10 µg/ml), and CD2 (RM2-5, 0.5 µg/ml) were used. The
results were obtained on lysates (100 µg of protein) of T cells
derived from four spleens. B, the parent CHO cells
(lanes 3 and 5) and CHO-leu·C2
(lanes 4 and 6) were transfected with
pcDNAI-CD45ABC. Forty-eight h after transfection, cell
lysates were subjected to Western blotting with 23G2 (lanes
5 and 6) and 30-F11 (lanes
3 and 4) antibodies. Lanes
1 and 2, respectively, represent the Western blot
of mock-transfected parent CHO and CHO-leu·C2 cells with 30-F11
antibody. The same amount of protein (200 µg) was applied to each
lane.
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Fig. 3.
Expression level of CD40 ligand and CD40 in
Lck-mC2GnT transgenic and control mice. A, analysis of
CD40 ligand expression on T cells. CD4+ T cells were
purified from spleens, activated in vitro for 6 h, then
stained with PE-anti-CD40 ligand (MR1). T cells were incubated without
activators (top panel) or activated with anti-CD3
(145-2C11, 20 µg/ml) and mouse ICAM-1-IgG fusion protein (10 µg/ml) (middle panel) or PMA (10 ng/ml) and ionomycin (0.5 µg/ml) (bottom panel). B, analysis of CD40
expression on B cells. B cells were purified from spleens and stained
with FITC-conjugated anti-CD40 (HM40-3). Open histograms
indicate the negative staining control. In both A and
B, cells isolated from spleens of three mice (one male and
two females) were pooled and analyzed. In A, T cell
activation and flow cytometric analysis were independently performed
three times using offspring from Lck-mC2GnT-1 and Lck-mC2GnT-2. The
numbers indicate the ranges of the percentages of the
CD40L+ cells.
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Fig. 4.
Activated T cells from Lck-mC2GnT transgenic
mice inefficiently induce B cell proliferation. A,
CD4+ T cells were purified from spleens, derived from
control mouse (C) or the transgenic mouse (Lck),
activated with anti-CD3 and mouse ICAM-1-IgG, and fixed with
paraformaldehyde. B cells, purified from control mice (open
symbols) or the transgenic mice (closed
symbols), were cocultured with activated CD4+ T
cells for 72 h in 96-well tissue culture plates with or without
exogenous IL-4 (10 ng/ml). B, purified B cells were cultured
for 72 h with or without addition of lipopolysaccharide
(LPS, 1 µg/ml), lipopolysaccharide plus IL-4 (10 ng/ml),
or anti-CD40. Each symbol indicates a different set of experiments
using T and B cells derived from a pool of three spleens.
Bars indicate the average numbers. The experiments were independently
performed three times using two different transgenic lines
(Lck-mC2GnT-1 and -2), and representative results in one experiment are
shown.
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Fig. 5.
Isotype switching is impaired in Lck-mC2GnT
transgenic mice. Mice were immunized with KLH in complete
Freund's adjuvant (100 µg, intraperitoneally), boosted 14 days
later, and bled at the indicated time after the first injection. Mouse
anti-KLH IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA levels were determined
by isotype-specific ELISA. Sera were diluted 1:10,000 to assay IgM and
IgG1 and diluted 1:1000 to assay IgG2a, IgG2b, IgG3, and IgA.
Open symbols, control mice; closed
symbols, Lck-mC2GnT transgenic mice. Each symbol represents
three different sets of experiments using three different controls and
transgenic mice. The experiments were independently repeated three
times using offspring derived from Lck-mC2GnT-1 (four females and three
males) and Lck-mC2GnT-2 (two females), respectively. A representative
result from one experiment is shown here. The differences observed in
the amount of IgM at 7 days and IgG1, IgG2a, and IgG2b at 20 days
obtained in three separate experiments were statistically significant
(p < 0.05).
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Fig. 6.
Germinal center formation is impaired in
Lck-mC2GnT transgenic mice. Spleen flat sections were prepared
from control mice (A-D) and the transgenic mice
(E-H) 7 days after injection of KLH. The sections were
stained with biotinylated peanut agglutinin (PNA), followed
by PE-streptavidin (A, C, E, and
G). Parallel sections were stained with biotinylated goat
anti-mouse IgM (anti-IgM), followed by PE-streptavidin
(B, D, F, and H). Sections
seen in A and B were prepared from a different
mouse than those in C and D. The sections in
E and F were also prepared from different
transgenic lines than those in G and H. Slides
were evaluated by a Zeiss Axioplan fluorescence microscope and
photographed. Bar = 50 µm.
Number of germinal centers in spleens from control and Lck-mC2GnT
transgenic mice.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
produced in activated T cells also play critical roles
in B cell differentiation and immunoglobulin isotype switching (41). In
Lck-mC2GnT transgenic mice, both in vivo and in
vitro T cell activation was reduced, resulting in reduction of
IL-4 and IFN- production (20). However, the difference in T
cell-dependent B cell proliferation between the transgenic
and control mice was observed even after addition of IL-4 (Fig.
4A). We conclude that overexpression of core 2 O-glycans on T cell surface glycoproteins results in
impaired T-B cell interaction and reduced production of cytokines,
leading to impaired humoral immune responses in Lck-mC2GnT transgenic mice.
production are impaired in WAS
patients (48, 49). These results suggest that overexpression of core 2 O-glycans on cell surface glycoproteins may be a cause of
impaired B cell immune responses in WAS patients. The gene defective in
WAS has been identified (50), and that gene product interacts with both the cytoskeleton and intracellular signaling molecules (51). Our
preliminary studies indicate that introduction of a normal WAS gene
into lymphocytes from WAS patients results in dramatically reduced
levels of core 2 oligosaccharides.2 In future
studies, it will be important to determine whether the mutated WAS gene
promotes constitutive activation of the C2GnT gene, resulting in
aberrant expression of core 2 O-glycans in T cells of those patients.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Glycobiology Program,
La Jolla Cancer Research Center, The Burnham Institute, 10901 North
Torrey Pines Road, La Jolla, CA 92037. Tel: (619)646-3144; Fax:
(619)646-3193; E-mail: minoru{at}burnham-inst.org.
-1,6-N-acetylglucosaminyltransferase; CD40L, CD40 ligand; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PMA, phorbol 12-myristate 13-acetate; KLH, keyhole
limpet hemocyanin; WAS, Wiskott-Aldrich syndrome; T-B interaction, T
cell-B cell interaction; CHO, Chinese hamster ovary; IL, interleukin; IFN, interferon; ELISA, enzyme-linked immunosorbent assay.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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