(Received for publication, December 28, 1995; and in revised form, February 5, 1996)
From the
T cell surface sialylation changes during maturation in the
thymus. We have previously demonstrated increased expression of mRNA
encoding the Gal1, 3GalNAc
2,3-sialyltransferase in mature
medullary human thymocytes, compared with immature cortical thymocytes.
For this enzyme, increased expression of transferase mRNA correlated
with increased sialylation of O-glycans. We have now examined
the pattern of expression in the human thymus of two additional
sialyltransferases, the Gal
1,4GlcNAc
2,6-sialyltransferase
(ST6N) and the Gal
1,3/4GlcNAc
2,3-sialyltransferase (ST3N).
The patterns of mRNA expression were compared with the pattern of
binding of two sialic acid-specific plant lectins, Sambucus nigra agglutinin and Maackia amurensis agglutinin, which
preferentially recognize
2,6- and
2,3-linked sialic acids,
respectively, on N-glycans. By in situ hybridization,
mRNA encoding ST3N was detected uniformly throughout the thymus. All
thymocytes bound M. amurensis agglutinin, demonstrating a
direct correlation between the level of ST3N mRNA expression and
cell-surface glycosylation. In contrast, mRNA encoding ST6N was also
expressed uniformly throughout the thymus; however, only mature
(CD3
) medullary thymocytes bound S. nigra agglutinin. On mature thymocytes, S. nigra agglutinin
appeared to bind primarily to the cell-surface glycoprotein CD45; since
only the mature thymocytes expressed the CD45RA isoform, while both
mature and immature populations expressed the CD45R0 isoform, CD45RA
may be a preferred substrate for ST6N. These results demonstrate that
glycoprotein sialylation is tightly regulated during T cell development
and that the developmentally regulated expression of specific
oligosaccharide structures on the cell surface may be influenced by
expression of both the relevant glycosyltransferase and specific
acceptor substrates.
The expression of specific sialyltransferase genes has been shown to be regulated in a cell type-specific manner(1, 2, 3) . In the thymus, the site of maturation and selection of T cells, a number of reports have demonstrated that the pattern of cell-surface sialylation changes as thymocytes mature(4, 5, 6, 7) . The presence or absence of specific sialyloligosaccharides on various types of cells may result from differential expression of sialyltransferases(8, 9, 10) , from differential expression of specific glycoprotein acceptors utilized by these transferases(11) , or from competition among glycosyltransferases for various acceptors(12, 13, 14, 15) .
Our
laboratory has demonstrated that, in the human thymus, regulated
expression of two glycosyltransferases, both of which modify O-glycans, correlates directly with cell-surface glycosylation
phenotype. Expression of the core 2 Gal1, 3GalNAc
1,6GlcNAc-transferase was highest in subcapsular and cortical
thymocytes, and the level of enzyme expression correlated precisely
with the ability of thymocytes to bind a monoclonal antibody that
recognizes core 2 O-glycans created by this
enzyme(16) . Similarly, the Gal
1,3GalNAc
2,3-sialyltransferase was highly expressed by subcapsular and
medullary thymocytes, but not by cortical thymocytes, and expression of
this enzyme correlated with the loss of cell-surface binding sites for
the plant lectin peanut agglutinin(17) . Sialylation of
cell-surface glycoproteins by the Gal
1,3GalNAc
2,3-sialyltransferase blocked the oligosaccharide sequence
recognized by peanut agglutinin.
Variation in the level of
sialylation of N-glycans has been reported among lymphocyte
subsets(11, 18) . In the thymus, murine medullary
thymocytes have increased binding sites for the 2,6-linked sialic
acid-specific lectin CD22, compared with cortical thymocytes (19, 20) . In addition, Lau and co-workers (21) have demonstrated that subsets of B lymphocytes differ in
the pattern of expression of ST6N. (
)However, little is
known about the pattern of expression of sialyltransferases that modify N-glycans in developing T cells.
The addition of sialic
acid to cell-surface glycoproteins is potentially an efficient
mechanism for modulating cell-cell interactions during thymocyte
development. Endogenous lectins such as
galectin-1(16, 22) , CD22(23) ,
CD23(24) , and L-selectin (25) appear to participate in
T cell maturation, activation, or trafficking, and each of these
lectins recognizes unique oligosaccharide ligands that require (CD22
and L-selectin) or are inhibited by (galectin-1 and CD23) the presence
of sialic acid. To characterize the pattern of sialylation of
developing thymocytes, we examined human thymocytes for the ability to
bind two sialic acid-specific lectins, Sambucus nigra agglutinin (SNA) (26) and Maackia amurensis agglutinin (MAA) (27) , which preferentially bind to
2,6- and
2,3-linked sialic acids, respectively, and compared
the pattern of lectin reactivity with the pattern of expression of the
relevant sialyltransferase enzymes (28, 29) during
thymocyte maturation.
Thymocytes and PBMC (100 µl at 5
10
cells/ml) in 0.01 M sodium phosphate, 0.15 M NaCl, 1% BSA, and 0.01% sodium azide (PBA buffer) were
combined with 50 µl of lectin or antibody at the appropriate
dilution in PBA buffer and incubated on ice for 60 min. For
single-color analysis, cells were washed twice with cold PBA buffer and
analyzed directly with conjugated antibodies or were resuspended in 50
µl of PBA buffer containing FITC-labeled avidin (diluted 1:300 in
PBA buffer) for SNA and MAA. For two-color analysis, cells were
incubated with biotin-conjugated lectin in the first step and then with
FITC-labeled avidin in the second step and with the PE-conjugated CD3
reagent in the third step. Control samples containing biotin-labeled
BSA or an isotype-matched irrelevant murine antibody were included for
each sample. After the final washing, cells were resuspended in PBA
buffer containing 1 µg/ml 7-aminoactinomycin D (Calbiochem) to
identify nonviable cells. Samples were incubated for 20 min at 4 °C
prior to acquisition on a FACScan flow cytometer (Becton Dickinson).
Sections of thymus (6 µm) on acetylated glass slides were treated with proteinase K and hybridized with the digoxigenin-labeled probes exactly as described(17) . Hybridization was detected by an anti-digoxigenin-alkaline phosphatase conjugate (Boehringer Mannheim), followed by overnight development with the chromogenic substrate nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Bio-Rad). Color development was stopped after 12 h.
The thymus contains T cell precursors at various stages of maturation. To examine changes in the sialylation of N-linked oligosaccharides during thymocyte maturation, we used two sialic acid-specific lectins that have been shown to discriminate between two linkages on N-glycans. Biotinylated MAA and SNA were bound to the surface of freshly isolated human thymocytes, and the cells were analyzed by flow cytometry. As shown in Fig. 1A, the MAA lectin bound to the entire population of thymocytes (solid line). The level of MAA binding to thymocytes was slightly higher than that seen with mature peripheral blood T cells (dotted line).
Figure 1:
Binding of sialic acid-specific
lectins differs among T cell subsets. Thymocytes (solid line)
and PBMC (dotted line) were stained with biotin-conjugated MAA
or SNA (diluted 1:100 in PBA buffer) and FITC-labeled avidin and
analyzed by flow cytometry. A, reactivity of thymocytes and
PBMC with the MAA lectin, which recognizes SA2,3Gal sequences; B, reactivity of thymocytes and PBMC with SNA, which
recognizes SA
2,6Gal sequences.
In contrast, when the binding of SNA to thymocytes was
examined, two distinct populations of cells were observed (Fig. 1B). The majority of the cells did not bind
appreciable levels of SNA, compared with background staining with
biotin-labeled BSA (biotin-labeled BSA staining not shown). However, a
small population of SNA cells were present. These
cells bound the SNA lectin at a level comparable to that observed for
peripheral blood T cells (dotted line). These data suggested
that expression of the SA
2,6Gal sequence was restricted to a
discrete subset of thymocytes.
The human thymus can be divided into
three major functional and anatomic compartments(33) . The
subcapsular cortical cells represent the most recent immigrants from
the bone marrow, and they do not express T cell receptor
(CD3). The cortical thymocytes are immature cells
undergoing T cell receptor gene rearrangement, and they express low
levels of cell-surface T cell receptor complex (CD3
). The
medullary cells are mature cells that are poised to emigrate to the
peripheral circulation, and they express high levels of T cell receptor
complex (CD3
). To relate the level of SNA binding to the
stage of thymocyte maturation, we performed dual-color flow cytometric
analysis of thymocytes using SNA and CD3 antibody. As shown in Fig. 2A, virtually all of the SNA
cells were in the CD3
population, while the majority
of the CD3
and CD3
cells bound
background levels of SNA, compared with biotin-labeled BSA indicated by
the dashed line. These results demonstrated that the mature
thymocytes were SNA
and that the level of SNA binding
to these cells did not change appreciably as the T cells emigrated to
the peripheral circulation (see Fig. 1B).
Figure 2:
Mature thymocytes bind SNA. A,
dual-color analysis of thymocytes with SNA (x axis) and
CD3-conjugated PE (y axis) by flow cytometry. Cursors (dashed lines) represent background levels of staining and
were set using biotin-labeled BSA and FITC-labeled avidin for the x axis and an isotype-matched PE-conjugated irrelevant murine
monoclonal antibody for the y axis. The CD3 cells
represent the most mature thymocyte subset and were the only population
that stained with SNA. B, treatment of thymocytes with
cytokines, which drive T cell maturation and proliferation, results in
increased binding of SNA to the cell surface. Freshly isolated
thymocytes were cultured for 12 days in medium alone or in medium
containing the indicated cytokine. Cells were stained with
biotin-conjugated SNA and FITC-labeled avidin and analyzed by flow
cytometry. The percent of SNA
cells in each sample was
as follows: control, 45%; IL-2, 77%; IL-4, 77%; and IL-7,
87%.
To further
examine the relationship between SNA binding and thymocyte maturation,
we evaluated the ability of specific cytokines, which have been shown
to induce maturation or proliferation of thymocytes in vitro,
to alter the binding of SNA to the cell surface. Treatment of
thymocytes with IL-2(31) , IL-4(34) , and IL-7 (30) results in an increase in the number of CD3 cells in culture, which also express CD45RA, another marker of
thymocyte maturation. IL-2 induces proliferation of only the mature
CD3
cells; IL-4 induces expression of
maturation-associated cell-surface markers with minimal proliferation;
and IL-7 induces both proliferation and maturation of thymocytes. To
assess the effects of these cytokines on SNA binding, thymocytes were
cultured with or without cytokine for 12 days and analyzed for the
ability to bind SNA. Treatment with all three cytokines resulted in an
increase in the percent of SNA
cells (Fig. 2B). These data demonstrate that acquisition of
the ability to bind SNA is a marker of thymocyte maturation and may be
regulated by specific cytokines during T cell development.
Prior
work from our laboratory demonstrated that the level of mRNA encoding
the Gal1,3GalNAc
2,3-sialyltransferase and the core 2
GlcNAc-transferase correlated precisely with the expression of the
relevant oligosaccharide structure on the thymocyte cell
surface(16, 17) . To evaluate the relationship between
binding of MAA and SNA and expression of mRNA encoding the relevant
sialyltransferases, we performed in situ hybridization
experiments with sections of human thymus (Fig. 3). As shown in Fig. 3(B and C), hybridization with an
antisense RNA probe for ST3N produced uniform staining of all regions
of the thymus. The control section, hybridized with a sense orientation
probe, demonstrated negligible background staining (Fig. 3A). The uniform expression throughout the thymus
of mRNA encoding ST3N correlated precisely with the pan reactivity of
thymocytes with MAA (Fig. 1A).
Figure 3:
Expression of mRNA encoding ST3N and ST6N
in human thymus. Hybridization of digoxigenin-labeled RNA probes was
detected by anti-digoxigenin antibody conjugated to alkaline
phosphatase. A, control sense orientation probe for ST3N
(magnification 40); B and C, antisense probe
for ST3N (magnification
40 and 100, respectively); D,
control sense orientation probe for ST6N (magnification
40); E and F, antisense probe for ST6N (magnification
40 and 100, respectively). Uniform staining of all regions of
the thymic lobules is seen with both antisense probes. c,
cortex; m, medulla.
mRNA encoding ST6N was also present in all regions of the thymus (Fig. 3, E and F), although SNA reactivity was restricted to mature thymocytes. No difference in the level of staining with the antisense probe could be discerned among the three anatomic compartments of the thymus. Control sections, hybridized with a sense orientation probe, showed negligible background reactivity (Fig. 3D). In contrast with the correlation between ST3N mRNA expression and MAA reactivity, the detection of mRNA encoding the ST6N enzyme did not correlate with expression of the relevant oligosaccharide sequence on the thymocyte cell surface.
Like ST6N mRNA, the ST6N protein was observed in both cortical and medullary thymocytes, as detected by immunohistochemical staining of thymus sections with a polyclonal antibody to ST6N(28) . Fig. 4demonstrates focal cytoplasmic staining of thymocytes in the cortical (panel A) and medullary (panel B) regions. Although the amount of transferase present in the cells cannot be quantified, these results demonstrate that ST6N is translated in both of these anatomic regions, although only medullary cells bind SNA.
Figure 4:
Immunohistochemical detection of ST6N in
human thymocytes. Immunoreactive material was detected in focal
perinuclear patches in both cortical (A) and medullary (B) thymocytes (magnification 400). Scattered stromal
cells in both regions also showed reactivity with the antibody. Control
sections, incubated with a preimmune rabbit serum, showed no reactivity (C). In situ hybridization with the antisense (D) and sense (E) orientation probes for ST6N is
shown at the same magnification.
Given the lack of anatomic
correlation between ST6N expression and the ability of thymocytes to
bind SNA, we examined separated populations of immature and mature
thymocytes for SNA binding and ST6N expression. To avoid cellular
activation that is triggered by CD3 binding, we enriched for immature
thymocytes by removing the CD5 mature cells and
enriched for mature thymocytes by removing immature CD1
cells. In Fig. 5, the CD1-depleted (CD1
)
thymocytes are enriched for CD3
cells, with an increased
number of cells that are CD45RA
, compared with the
starting material. The CD5-depleted cells (CD5
) are
primarily CD3
and CD3
. In addition, very
few CD45RA
cells are present in the CD5-depleted
subset.
Figure 5:
Isolation of immature and mature thymocyte
subsets. Freshly isolated thymocytes were incubated with either CD5 or
CD1 antibody, and the cells that bound antibody were removed by
magnetic bead separation. The remaining cells were assayed for
expression of CD3 and CD45RA, a marker of mature thymocytes. The
CD5-depleted (CD5) subset is enriched for
CD3
and CD3
cells, while the
CD1-depleted (CD1
) subset is enriched for CD3
cells (upper panels). Dual-color analysis (lower
panels) demonstrates that the mature CD1-depleted cells express
significantly higher levels of CD45RA than the immature CD5-depleted
cells.
To identify SNA-reactive glycoproteins on mature and
immature thymocytes, we performed lectin blotting on extracts of CD1-
and CD5-depleted cells. As shown in Fig. 6, SNA bound to a
prominent band of M = 200,000 in the total
thymocyte extract and in the mature CD1-depleted thymocytes, while this
band was significantly reduced in the immature CD5-depleted cells.
Treatment of the blot with A. ureafaciens sialidase diminished
SNA reactivity of this band, indicating that SNA binding of this
glycoprotein was sialic acid-dependent. The reactivity of this
glycoprotein correlated with the pattern of SNA binding to thymocytes (Fig. 2), suggesting that this molecule was responsible for the
selective binding of SNA by mature thymocytes. Probing of the blot with
antiserum to ST6N demonstrated immunoreactive material in all three
lanes, although the intensity of staining in the CD1- and CD5-depleted
lanes was not identical.
Figure 6:
SNA
binding and ST6N expression in isolated thymocyte subsets. Equal
numbers of immature (CD5) and mature
(CD1
) thymocyte pools as well as total thymocytes (T) were separated by SDS-polyacrylamide gel electrophoresis
and analyzed by Western and lectin blotting. The blot was probed with
SNA, with monoclonal antibodies to CD45 and CD45R0, and with
affinity-purified antiserum to ST6N (
2,6ST) and reprobed
with SNA after treatment with A. ureafaciens sialidase. The
SNA-reactive band that is sialidase-sensitive is indicated by an arrowhead, and the CD45 isoforms are indicated by arrows.
The relative mobility of the SNA-reactive
band in extracts of mature CD1-depleted thymocytes suggested that this
glycoprotein was an isoform of CD45. CD45, which constitutes 10%
of the total cell-surface glycoproteins on T cells, is highly
sialylated(35) . In addition, Hart and co-workers (7) have demonstrated that mature thymocytes have fully
sialylated CD45, while immature thymocytes can be sialylated by
exogenous ST6N enzyme. To determine whether the SNA-reactive band was
CD45, we probed the blot with antibodies to CD45. As shown in Fig. 6, CD45 antibody bound primarily to a band of M
= 200,000 in the extracts of total
thymocytes and mature CD1-depleted cells, with only faint reactivity
seen in the lane containing the immature CD5-depleted cell extract.
This band migrated slightly above the band detected by an antibody to
the 180-kDa CD45R0 isoform, which was present in approximately equal
amounts in both the mature and immature populations. These data
indicated that SNA was binding to a maturation-specific isoform of
CD45, most likely CD45RA(36) . We were unable to detect any
reactivity on this blot with the CD45RA-specific antibody 4KB5 (data
not shown); however, Fig. 5demonstrates that CD45RA was present
primarily in the mature CD1-depleted cells. These results strongly
suggest that CD45RA is a glycoprotein counter-receptor for SNA on
mature thymocytes.
We have examined the pattern of expression in the human
thymus of four glycosyltransferase enzymes, the Gal1, 3GalNAc
2,3-sialyltransferase(17) , the core 2 Gal
1,3GalNAc
1,6GlcNAc-transferase(16) , and ST3N and ST6N (Fig. 1Fig. 2Fig. 3). Comparison of the pattern of
mRNA expression and the relevant cell-surface glycotype is shown in Table 1. Two conclusions can be drawn from these data. First, the
tight control of cell-surface glycosylation in the thymus suggests that
specific oligosaccharide sequences participate in distinct functions
during thymocyte development. It is striking that, for the four
transferases examined, four different patterns of expression were
observed. Second, the level of expression of mRNA encoding a particular
enzyme does not always correlate with the amount of the relevant
oligosaccharide sequence on the cell surface. For the Gal
1,3GalNAc
2,3-sialyltransferase, the core 2 Gal
1,3GalNAc
1,6GlcNAc-transferase, and ST3N, lectin and monoclonal antibody
binding studies demonstrated that specific oligosaccharide sequences
were always detected in thymocyte subsets that expressed the cognate
transferase mRNA. However, although mRNA encoding ST6N was detected
throughout the thymic lobule (Fig. 3), only mature cells bound
SNA (Fig. 2A). We detected SNA binding to thymocytes by
flow cytometry since histochemical staining of the thymus with SNA
revealed staining of stromal structures as well as thymocytes, so that
the reactivity of thymocyte subsets was difficult to ascertain by
lectin histochemistry (data not shown).
The reactivity of CD3 medullary thymocytes with SNA resembles that found with CD22, a B
lymphocyte lectin that recognizes
2,6-linked sialic acid (37) and that can bind to
2,6-linked sialic acid on
CD45(23) . The reactivity of CD22 with murine medullary
thymocytes suggested that expression of the ST6N enzyme might be
limited to mature thymocytes in medulla(19, 20) .
However, in situ hybridization, immunohistochemical analyses,
and Western blotting (Fig. 3, Fig. 4, and Fig. 6,
respectively) demonstrated that the ST6N mRNA and protein were present
in both cortical (SNA
) and medullary
(SNA
) thymocytes, suggesting that expression of
SA
2,6Gal sequences on T cell glycoproteins may be regulated at
additional points downstream of enzyme transcription and translation.
This same lack of correlation between expression of the ST6N enzyme and
SNA reactivity in various cell types has been recently described in
human and rat tissues(38) , indicating that the thymus is not
unique in this respect.
A number of factors may contribute to
differential glycosylation of cell-surface glycoproteins in different
cellular subsets. One possibility is that either the donor or acceptor
substrate is limiting. In the thymus, it is unlikely that the donor
substrate for the ST6N enzyme, CMP-SA, is limiting since subcapsular
and cortical thymocytes synthesize the SA2,3Gal sequence detected
by MAA. However, specific glycoprotein acceptor substrates
preferentially utilized by ST6N may be expressed only in the mature
thymocyte population. A number of studies have demonstrated
preferential glycosylation of potential acceptor substrates by
different
transferases(11, 13, 39, 40) . In T
cells, Kobata and co-workers (41) found differences in the
pattern of sialylation of CD45 and CD11a/CD18; N-glycans on
CD45 had exclusively SA
2,6Gal sequences (although isoforms were
not separated in this study), while N-glycans of CD11a/CD18
had SA
2,3Gal sequences. Our results suggest that CD45RA is a
preferred acceptor substrate for ST6N in the thymus and that the
increased binding of SNA to mature thymocytes results from the dramatic
increase in CD45RA expression that accompanies thymocyte
maturation(36) .
Another factor that could contribute to differential glycosylation among various cellular subsets is differential expression of enzyme isoforms, as has been shown for ST6N in B lymphoblastoid cell lines (21) . In our in situ hybridization experiments, we used full-length RNA probes to detect mRNA expression in thymus sections (Fig. 3). Thus, we cannot determine whether specific splice variants of the ST6N mRNA were synthesized in the different thymic regions. Future experiments will address whether T cell subsets, like B cell subsets, express different ST6N isoforms.
This study reinforces the observation that differences in glycosyltransferase expression and glycoprotein glycosylation may be found among subsets of cells within a single organ as well as between organs(16, 17) . Prior studies that examined expression of sialyltransferase mRNA in the thymus (3) or cell-surface glycosylation of thymocytes (7, 42, 43) may not have accounted for the relative contribution of different subsets of thymocytes to the total pool of material analyzed. Since the three major subsets of cells found within the thymus have significant phenotypic and functional differences(33) , dissection of these subsets may be important when considering the role of specific oligosaccharides during T cell development.
The coordinated expression of glycosyltransferases and
their cognate oligosaccharide sequences on the T cell surface may be
critical for proper thymocyte maturation. We have recently found that
galectin-1, expressed on thymic epithelial cells, mediated binding of
immature T cells to the thymic stroma(16) . Galectin-1 induces
apoptosis of immature thymocytes, ()and galectin-1 binding
and apoptosis are blocked by antibodies to
CD45(16, 44) . Galectin-1 binding to lactosamine, the
preferred oligosaccharide ligand, is negatively modulated by the
presence of SA
2,6Gal sequences on the termini of the
oligosaccharide chains(22) . We found that galectin-1
preferentially bound to CD3
and CD3
thymocytes(16) , the subsets that did not bind SNA (Fig. 2A and Table 1). Thus, the ability of
thymocyte subsets to bind galectin-1 and the susceptibility of these
cells to galectin-1-induced apoptosis may be modulated by the
expression of specific oligosaccharide sequences on the cell surface.
The regulation of glycoprotein glycosylation has recently been the subject of intense investigation. Determining the pattern of expression of specific oligosaccharide sequences by normal and neoplastic tissues is relevant to the study of carbohydrate-mediated adhesion in inflammation and metastasis(25) . In addition, manipulation of glycosylation in tissue culture systems is of interest in the production of recombinant glycoproteins, where glycosylation may affect the activity, stability, or immunogenicity of the material(45, 46, 47, 48) . While expression of cDNA encoding a glycosyltransferase in specific cell lines may result in the production of the predicted product(12, 15, 49) , a number of factors, such as enzyme subcellular localization, competition among endogenous and exogenous enzymes for the same acceptors, and availability of acceptor substrates(13, 39, 50, 51, 52) , may affect the pattern of oligosaccharide structures synthesized by the cell.