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International Immunology Advance Access originally published online on November 15, 2005
International Immunology 2006 18(5):627-635; doi:10.1093/intimm/dxh344
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© The Japanese Society for Immunology. 2005. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Sialylation regulates peripheral tolerance in CD4+ T cells

Patrick J Brennan1,2,, Sandra J Saouaf1, Steve Van Dyken3, Jamey D Marth3, Bin Li1, Avinash Bhandoola1 and Mark I Greene1,2,

1 Department of Pathology and Laboratory Medicine, 252 John Morgan Building, 36th & Hamilton Walk, and
2 Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6082, USA
3 Department of Cellular and Molecular Medicine, Howard Hughes Medical Institute, University of California San Diego, La Jolla, CA 92093, USA

Correspondence to: M. I. Greene; E-mail: greene{at}reo.med.upenn.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Decreased binding by the 6C10 auto-antibody serves as a unique marker for CD4+ T cell unresponsiveness after the induction of T cell tolerance in Vß8.1 TCR transgenic mice. We further define the nature of the epitope recognized by the 6C10 antibody to be a subset of Thy-1 bearing incompletely sialylated N-linked glycans, and furthermore, we demonstrate that tolerant CD4+ T cells have an increased degree of cell-surface sialylation. To test the significance of the altered glycosylation state identified by the 6C10 auto-antibody in the tolerant CD4+ T cell population, surface sialic acid was cleaved enzymatically. Treatment of purified peripheral CD4+ T cells with Vibrio cholerae sialidase (VCS) leads to increased 6C10 binding, significantly enhances proliferation in the tolerant CD4+ population and corrects defects in phosphotyrosine signaling observed in the tolerant CD4+ T cell. Furthermore, in vivo administration of VCS enhances proliferation in both tolerant and naive CD4+ T cell subsets. These studies suggest that sialylation of glycoproteins on the surface of the CD4+ T cell contributes to the regulation of T cell responsiveness in the tolerant state.

Keywords: T cell, tolerance, sialylation, superantigen


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Mechanisms directed toward the avoidance of self-reactivity are thought to occur at multiple stages during the life of the CD4+ T cell. Of these mechanisms, none has been better studied than central tolerance, a thymic process involving a combination of positive and negative selection that leads to the generation of a diverse T cell repertoire (1). It has become increasingly clear, however, that thymic selection is not sufficient for the prevention of self-reactivity, and that peripheral mechanisms for the modulation of T cell responsiveness are also necessary. To study peripheral T cell tolerance, we have used TCR Vß8.1 transgenic mice that can be rendered tolerant of the minor lymphocyte-stimulating antigen 1a (Mls-1a) (2). CD4+ T cells from these tolerant mice are hyporesponsive to in vitro stimulation (3–5) and tolerant mice display delayed allograft rejection in vivo (6). Additionally, we have reported altered protein tyrosine phosphorylation patterns in tolerant CD4+ T cells (3). We have previously identified the 6C10 antibody as a unique surface marker of T cell responsiveness in the Mls-1a tolerance system. Induction of tolerance is associated with a rapid and prolonged down-modulation of 6C10 binding in the CD4+ T cell compartment (4). During the maintenance phase of tolerance, 6C10lo T cells are completely unresponsive, while 6C10hi T cells, at least partially, regain proliferative capacity (5). The 6C10 antibody has also been associated with developmentally defined T cell subsets and differential T cell responsiveness in the wild-type mouse (7, 8). The 6C10 recognizes a subset of Thy-1 molecules in a glycosylation-dependent manner (9), though how the specific epitope recognized by 6C10 differs from structures found on bulk Thy-1 molecules has not been defined.

The role of protein glycosylation in the immune response is under increasing scrutiny, and many well-studied surface receptors involved in the immune response are glycosylated. Recent development of mice with targeted deletions of genes that produce specific glycan linkages has provided a new insight into the importance of protein glycosylation in the immune system, identifying a critical role for protein sialylation in T cells (10–13). Here, we demonstrate that the 6C10 antibody binds to Thy-1 species with a low degree of sialylation and serves as a global marker for a subset of incompletely sialylated glycans in the CD4+ T cell population. In vitro desialylation of tolerant CD4+ T cells restores the proliferative capacity and reverses hypophosphorylation of signaling proteins in response to TCR cross-linking. In vivo desialylation corroborates these findings. Our studies indicate that increased sialylation contributes to the tolerant phenotype in CD4+ T cells tolerized to Mls-1a.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
Vß8.1 transgenic mice bred onto a CBA/Ca background have been previously described (2). CBA/J (Mls-1a, H-2k) and CBA/Ca (Mls-1b, H-2k) mice were purchased from The Jackson Laboratories. Thy-1 knockout (KO) mice on a CB17 background were generously supplied by K. Hayakawa at the Fox Chase Cancer Center, Philadelphia, PA, USA.

Antibodies and flow cytometry
Antibodies to CD4 (GK1.5), CD8 (53-6.7) and mouse IgM (II/41), as well as fluorophore–streptavidin conjugates, were from PharMingen. Biotinylated lectins ECA, SNA, MAL II and peanut agglutinin (PNA) were from Vector Laboratories. For flow cytometry, lectins SNA, MAL II and PNA were used at 1 µg ml–1, and ECA lectin was used at 5 µg ml–1. The 6C10 hybridoma (14) was a gift from K. Hayakawa. Cell viability was assessed using TO-PRO-3 (Molecular Probes). Flow cytometry was performed on a Becton/Dickinson FACSCalibur (Becton Dickinson) in the University of Pennsylvania flow cytometry core facility and analyzed with CELLQUEST software (Becton Dickinson). Flow cytometric data are shown in log 10 fluorescence.

Cell preparation
Splenic CD4+ T cells were enriched by negative selection (using antibodies to CD8{alpha}, CD11b, CD45R, DX5, Ter-119) over a magnetic column using a MACS CD4+ T cell purification kit (Miltenyi) according to the manufacturer's instructions. Purity of >97% was routinely obtained. T cell-depleted splenocytes were prepared by treatment with anti-Thy-1.2 mAb (HO-13.4) followed by Low-Tox-M rabbit complement (Cedarlane). Purity of cell populations was confirmed by flow cytometric analysis. Treatment of purified CD4+ T cells with Vibrio cholerae sialidase (VCS) (Sigma) was carried out in RPMI at 37°C for 1 h. Inactivated enzyme was prepared by heating at 95°C for 10 min.

Tolerance induction
Tolerance induction in Vß8.1 transgenic mice has been previously described (3, 4, 15). Briefly, 1.5 x 107 CBA/J T cell-depleted splenocytes (Mls-1a) were injected intravenously via the tail vein into female 6- to 12-week-old Vß8.1 transgenic mice (Mls-1b). Tolerance was assessed 14 days after injection. For in vivo administration of sialidase, 0.5 units of VCS (Sigma) was injected intravenously on day 13 of tolerance induction.

Proliferation assays
Cell proliferation was assessed in vitro using both [3H]thymidine ([3H]TdR) incorporation and carboxy-succinimidyl-fluorescein-ester labeling. Briefly, 5 x 104 purified CD4+ T cells were incubated for 72 h with 5 x 105 irradiated (2000 R), T cell-depleted CBA/J splenocytes, unless otherwise indicated. Assays were carried out in RPMI supplemented with 10% FCS, L-glutamine, sodium pyruvate and ß-mercaptoethanol. For [3H]TdR incorporation studies, the culture was pulsed with 1 µCi per well [3H]TdR for the last 8 h. For CFSE dilution studies, labeling was performed using a protocol provided by Wells (16). Briefly, 5 x 106 cells ml–1 were incubated for 3 min with CFSE at a concentration of 2 µM in PBS. The labeling reaction was quenched with 20% FCS and the cells were washed. After a 72-h culture period with antigen-presenting cells (APCs), the mixed culture was stained with anti-CD4 (GK1.5) and TO-PRO-3 prior to flow cytometric analysis.

Electrophoresis and western blotting
CD4+ T cell populations were prepared as described above. Unsorted single-cell thymocyte suspensions were obtained from freshly isolated tissue. For Thy-1 and 6C10 analysis, surface biotinylation was performed after centrifugation over a cushion of Lympholyte-M (Cedarlane). Briefly, 2.5 x 107 ml–1 cells were incubated with 1 mg ml–1 Sulfo-NHS-LC biotin (Pierce) at 4°C in PBS pH 8.0 with agitation, followed by extensive washing to remove excess biotin. Cells were lysed for 30 min on ice in 150 mM NaCl, 50 nM Tris pH 8.0, 2% NP-40, 0.1% saponin, supplemented with phenylmethylsulfonylfluoride (PMSF) (Calbiochem) and aprotinin (Sigma). This detergent combination has been shown to completely solubilize Thy-1 (17). Lysate was pre-cleared with either protein-A–sepharose (Sigma) or anti-mouse-IgM–sepharose (Zymed). For immunoprecipitations, monoclonal anti-Thy-1 (YBM 29.2.1, a gift from S. Cobbold) was pre-linked to protein-A–sepharose, while 6C10 was pre-linked to anti-mouse-IgM–sepharose. Pre-cleared lysates were incubated overnight with the appropriate pre-linked sepharose beads. Twelve hours later, the immunoprecipitate was washed four times with lysis buffer before dissociation of the immunoprecipitated material from sepharose beads.

For SDS-PAGE analysis, the immunoprecipitate was dissociated from beads by boiling in Laemmli buffer in the presence of ß-mercaptoethanol for 10 min followed by centrifugation. Samples were run on 12.5% Tris–glycine gel. For isoelectric focusing (IEF) analysis, samples were dissociated in 8.5 M urea, 2% NP-40, 10% Pharmalyte 3-10 (Sigma) for 1 h at room temperature followed by centrifugation.

IEF was carried out in a vertical minigel apparatus (Invitrogen) on an 8.5 M urea, 5% polyacrylamide gel containing 5% Pharmalyte 3-10 and 2% NP-40 using stepped voltages of 100 mV (30 min), 150 mV (1 h), 200 mV (3 h), 0.1 N NaOH (upper chamber), 0.6% phosphoric acid (lower chamber). IEF gels were prepared for western blotting by washing five times for 10 min in 10x gel volume per wash in 5 mM Tris pH 8.0, 50% methanol, 1% SDS (18).

Western blotting on nitrocellulose membrane (Bio-Rad) was performed using standard methods. For biotinylated samples, membranes were blocked in 5% non-fat milk/PBS/0.5% Tween 20 (Bio-Rad), washed, blotted using streptavidin–HRP (Amersham) in 5% BSA/PBS/0.5% Tween 20 and visualized with ECL-Plus (Amersham).

For phosphotyrosine studies, purified CD4+ T cells were incubated with anti-CD3{varepsilon} (145-2C11, purified over protein G, from hybridoma culture supernatant) followed by cross-linking with polyclonal anti-hamster IgG (ICN). Cells were lysed in TNE buffer (150 mM NaCl, 50 mM Tris pH 8.0, 1% NP-40, 2 mM EDTA, supplemented with PMSF, aprotinin and Na3VO4). Anti-phosphotyrosine mAb 4G10 (Upstate biotechnology) was added to the lysate for 12 h followed by immunoprecipitation with protein-A–sepharose and SDS-PAGE on a 10% Tris–glycine gel. PY-20–HRP (Transduction Laboratoriess) was used for western blotting. Anti-Cbl-b (G1) and anti-phospho-ERK1/2 (sc-16982) were from Santa Cruz. Anti-SLP-76 was a gift from G. Koretzky, University of Pennsylvania.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
The 6C10 auto-antibody in the CD4+ T cell compartment
As we and others have previously shown, peripheral CD4+ T cells display a heterogeneous 6C10-binding profile, with 60–70% of peripheral T cells being 6C10hi, 30–40% being 6C10lo in the mature CBA/Ca mouse (Fig. 1A; 4, 7, 8). We have reported that 6C10 serves as a marker of the proliferative capacity following the induction of T cell tolerance to Mls-1a superantigen in Vß8.1 TCR transgenic mice (4). In this system, long-lived tolerance to Mls-1a observed in CD4+ T cells is accompanied by a reduction in 6C10 binding. Four days after injection of tolerogenic, Mls-1a-bearing, T cell-depleted splenocytes, 6C10 staining was almost completely absent from the CD4+ population (Fig. 1B). As we have previously shown (4), after the initial decrease in 6C10 staining, the proportion of 6C10hi T cells slowly increases but does not return to pre-tolerance levels. At 14 days, approximately half of CD4+ T cells are again 6C10hi (Fig. 1C). In vitro stimulation of CD4+ T cells also leads to rapid down-modulation of 6C10 binding (data not shown).


Figure 1
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  		Fig. 1. Functional modulation of 6C10 binding. 6C10 staining decreases in CD4+ T cells following in vivo or in vitro stimulation. (A–C) Tolerance was induced in naive Vß8.1 transgenic mice by injecting T cell-depleted Mls-1a splenocytes. The 6C10-staining profiles at day 4 (B) and day 14 (C). Shaded tracing represents uninjected. Representative data from CD4+ gated flow cytometry plots are shown.

 
The 6C10 antibody binds to Thy-1 with a low degree of sialylation
The 6C10 antibody is known to recognize Thy-1 in a glycosylation-dependent manner (9). Mouse Thy-1 has been well characterized biochemically, and contains three N-linked glycosylation sites. In lymphocytes, two sites are heterogeneously glycosylated, containing a mixture of bi- and tri-antennary complex-type glycans, while the third, homogenous glycosylation site is of the high-mannose type (19, 20). To further characterize the epitope recognized by 6C10, we undertook biochemical analysis of the protein product immunoprecipitated by this antibody.

Differential glycosylation of Thy-1 is known to lead to variability in charge as well as in the apparent molecular weight (MW) (20). The Thy-1 species immunoprecipitated by 6C10 runs at an apparent MW of 30 kDa by SDS-PAGE, and unlike the bulk Thy-1 species, is restricted to a single band (Fig. 2A). In order to assess whether the Thy-1 species immunoprecipitated by 6C10 also displays charge restrictions, we developed a technique to perform one-dimensional, denaturing IEF followed by western blotting. As has been previously reported (20–22), Thy-1 molecules of thymic origin exist as more basic species than the majority of peripheral Thy-1 molecules (Fig. 2B, lanes 1, 2). The 6C10 immunoprecipitate resolves as a single band by IEF, and has a pI in the thymic range (Fig. 2B, lane 3). A weak band co-migrating with the 6C10 band is also seen in the immunoprecipitate from peripheral CD4+ T cells (Fig. 2B, middle lane). Because the charge of a given Thy-1 molecule is determined by the degree of terminal sialic acid modification (20), these data indicate that 6C10 binds Thy-1 species with a relatively low number of sialic acid moieties.


Figure 2
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  		Fig. 2. Biochemical analysis of 6C10 immunoprecipitate. Immunoprecipitated material from surface-biotinylated CBA/Ca thymocytes or purified CD4+ splenocytes was subjected to SDS-PAGE (A) or denaturing IEF (B) followed by western blotting. Cell lysate from freshly isolated thymocytes or CD4+ splenocytes, as indicated, was immunoprecipitated with anti-Thy-1 mAb YBM 29.2.1 pre-coupled to protein-A–sepharose. The 6C10 immunoprecipitations were from thymocyte lysate after pre-coupling 6C10 antibody to anti-IgM–sepharose beads.

 
Enzymatic desialylation increases 6C10 binding on peripheral CD4+ T cells
As the 6C10 antibody binds to Thy-1 molecules that have a low level of sialic acid modification, we hypothesized that the enzymatic removal of sialic acid moieties would reveal the epitope recognized by 6C10, increasing the level of 6C10 binding in flow cytometric analysis. VCS was used to cleave sialic acid, as this enzyme exhibits partial desialylation activity under physiologic conditions suitable for mammalian cells (23).

Treatment of purified CD4+ T cells in vitro for 1 h with VCS leads to enhanced 6C10 binding immediately following treatment (Fig. 3A). There is an increase in both the proportion of the 6C10hi fraction as well as in the mean 6C10 fluorescence. A more pronounced shift from 6C10lo to 6C10hi populations is observed after VCS treatment of CD4+ T cells from the neonatal mouse (Fig. 3B), in which the starting CD4+ population is skewed toward the 6C10lo fraction.


Figure 3
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  		Fig. 3. Partial enzymatic desialylation increases 6C10 binding to peripheral CD4+ T cells. The 6C10 binding is increased after VCS treatment. Freshly isolated CD4+ T cells were incubated for 1 h with 40 mU ml–1 VCS at 37°C followed by cell-surface staining with the 6C10 antibody and flow cytometric analysis. VCS-treated cells are indicated by black tracings, untreated by shaded tracings. CD4+ T cells from (A) 8 weeks CBA/Ca, (B) 5 days CBA/Ca mice, (C) Vß8.1 transgenic mice tolerized to Mls-1a 7 days earlier and (D) CBA/Ca mice stimulated in vitro with solid-phase anti-CD3{varepsilon} for 24 h. (E) VCS treatment had no effect on 6C10 staining of CD4+ T cells from Thy-1 –/– mice. (F) A Thy-1 +/+ background-matched wild-type CB17 control.

 
The effect of VCS treatment on 6C10 staining is apparent not only on resting CD4+ T cells but also on T cells after the functional down-modulation of 6C10 binding. The decreased 6C10 staining observed following in vivo (Fig. 3C) or in vitro (Fig. 3D) stimulation is almost completely reversed following VCS treatment. As 6C10 binds to a Thy-1 glycoform, CD4+ T cells from the Thy-1 –/– mouse are not bound by 6C10 (Fig. 3E). VCS treatment of CD4+ T cells from Thy-1 –/– had no effect on 6C10 binding, demonstrating that the effect of VCS on 6C10 binding is Thy-1 dependent (Fig. 3E). In all experiments, recovery of T cells following VCS treatment was identical to recovery following mock treatment.

The 6C10 is a marker of global differential sialylation in CD4+ T cells
Biochemical data described above suggest that the 6C10 antibody recognizes a form of Thy-1 that is restricted in both MW and charge. Since the variability observed in Thy-1 species is due to differential glycosylation, we hypothesized that 6C10 binding may serve as a marker for the global glycosylation state of the CD4+ T cell. To test this hypothesis, plant lectins, having well-characterized binding specificities, were used for flow cytometric analysis. Because N-glycans on Thy-1 are known to be sialylated, the binding of lectins SNA, which binds {alpha}-2,6-linked sialic acid, and MAL II, which binds {alpha}-2,3-linked sialic acid (24, 25) was studied. In addition, we tested the binding of PNA, which binds exposed galactosyl (ß-1,3) N-acetylgalactosamine (26) found on mammalian core 1 O-linked glycans, and ECA, which binds exposed galactosyl (ß-1,4) N-acetylglucosamine (27) found on mammalian N-linked glycans and core 2 O-linked glycans. The binding of PNA and ECA lectins is blocked by sialylation of their respective O- and N-linked glycan substrates (28–30).

In CD4+ T cells isolated from the naive Vß8.1 transgenic mouse, 6C10 and ECA binding showed a direct relationship, with 6C10hi CD4+ T cells binding ECA to a greater extent than the 6C10lo CD4+ population (Fig. 4A). This relationship between 6C10 and ECA binding was also observed in wild-type CBA/Ca CD4+ T cells (data not shown). Thus, 6C10 binding identifies CD4+ T cells with higher surface density of incompletely exposed galactosyl (ß-1,4) N-acetylgluosamine moieties recognized by ECA lectin. No relationship between 6C10 and SNA, MAL II or PNA was evident (Fig. 4D–F) in the CD4+ T cell population. The 6C10hi CD4+ and 6C10loCD4+ T cell subsets have similar overall surface levels of sialic acid, as evidenced by similar SNA- and MAL II-binding profiles (Fig. 4D and F). The PNA-binding profile indicates that incompletely sialylated core 1 O-linked glycan density is not related to 6C10 binding in the CD4+ population (Fig. 4E).


Figure 4
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  		Fig. 4. 6C10 and surface glycoprotein sialylation. 6C10 is a marker for galactosyl (ß-1,4) N-acetylglucosamine, a substrate for sialylation on N-linked glycans recognized by ECA lectin. (A–C) CD4+ gated splenocytes from naive (A) and tolerant (B) Vß8.1 transgenic mice, with single-color ECA-binding profiles (C) of naive (shaded) and Mls-1a-tolerant (overlay) CD4+ T cells. (D–F) CD4+ gated splenocytes naive Vß8.1 transgenic mice, with 6C10 binding shown on the horizontal axis, binding of each lectin indicated shown on the vertical axis. (G–I) Lectin-staining profiles on splenic CD4+ T cells from naive (shaded tracing) and Mls-1a-tolerant (overlay) Vß8.1 transgenic mice.

 
As T cell populations rendered tolerant to Mls-1a display decreased 6C10 binding, we asked whether such tolerant cells also display decreased ECA binding. A small, reproducible decrease in ECA binding was observed in the tolerant CD4+ T cell population, just as 6C10 was seen to decrease (Figs 1C and 4B). SNA, PNA, and MAL II lectin-binding profiles were similar in tolerant and naive CD4+ T cells, suggesting no apparent difference in surface levels of overall protein sialic acid content or incompletely sialylated core 1 O-linked glycans (Fig. 4G–I) in these two populations. The 6C10, then, is a global marker of incompletely sialylated ECA-reactive glycans in both the naive and tolerant CD4+ T cell.

Reversal of CD4+ T cell tolerance following VCS treatment
As a marker, 6C10 has been associated with functionally distinct CD4+ T cell subsets in both the naive and the tolerant animal. Our ECA-binding results indicate that 6C10 binding serves as a marker for incompletely sialylated exposed galactosyl (ß-1,4) N-acetylglucosamine moieties. Tolerant CD4+ T cells, then, bear a subset of glycoproteins which are more completely sialylated relative to naive CD4+ T cells. In order to test the significance attributable to the differential sialylation state identified by 6C10 binding in the tolerant CD4+ population, sialidase was used to enzymatically reduce surface sialic acid modification. Naive and tolerant Vß8.1 transgenic animals were treated with VCS in vivo, 24 h prior to CD4+ T cell recovery and analysis. VCS-treated CD4+ T cells from both naive and tolerant animals showed similar proliferation to re-stimulation with Mls-1a APCs, and this level of proliferation was increased with respect to the proliferation of CD4+ T cells from untreated naive animals (Fig. 5A).


Figure 5
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  		Fig. 5. Partial enzymatic desialylation reverses CD4+ T cell tolerance. The proliferation of tolerant cells before ({square}) and after ({blacksquare}) pre-treatment with VCS. The proliferation of naive T cells before ({circ}) and after (•) VCS pre-treatment. (A) Naive and tolerant Mls-1a Vß8.1 transgenic mice were treated in vivo with 0.5 units VCS 24 h prior to CD4+ T cell purification. Proliferation was assayed by [3H]thymidine incorporation to the indicated ratio of Mls-1a APCs. (B) Purified CD4+ T cells from naive or tolerant Vß8.1 transgenic mice were treated in vitro with 40 mU ml–1 VCS, and proliferation was assayed as in (A). (C) Proliferation after in vitro incubation of naive or tolerant CD4+ T cells with heat-inactivated enzyme ({diamondsuit}, {blacktriangleup}). (D) Proliferation of tolerant CD4+ T cells following VCS treatment with increasing dose of VCS. (E) The effect of in vitro VCS treatment of CD4+ T cells on proliferation to Mls-1a APCs as measured by CFSE staining. CFSE dilution at 72 h in viable CD4+ gated populations is shown.

 
Since in vivo sialidase treatment may alter T cell function indirectly by affecting APCs as well as T cells, we addressed the direct effects of sialic acid cleavage on the T cell in vitro. Purified naive and tolerant CD4+ T cell populations were treated in vitro with VCS and their proliferative capacity was subsequently assayed. The proliferation of tolerant CD4+ T cells was significantly enhanced by VCS treatment as measured by [3H]TdR incorporation (Fig. 5B) as well as by CFSE dilution (Fig. 5E), an effect that exhibits dose dependence (Fig. 5D). At the highest dose of VCS used (40 mU ml–1), proliferation of VCS-treated tolerant cells was similar to that of mock-treated naive CD4+ T cells, indicating that cleavage of sialic acids is sufficient to restore the proliferation of tolerant CD4+ T cells to a pre-tolerance level. The proliferation of naive CD4+ T cells was also enhanced by VCS treatment, albeit to a lesser extent (Fig. 5B and E). Treatment of either naive or tolerant CD4+ T cells with VCS did not stimulate proliferation in response to syngeneic APCs (data not shown). Pre-treatment with heat-inactivated enzyme had no effect on proliferation of either naive or tolerant CD4+ T cells (Fig. 5C).

Alteration of phosphotyrosine signaling in VCS-treated CD4+ T cells
Defective phosphotyrosine signaling is a hallmark of tolerance in the Mls-1a system. We previously reported differential phosphorylation patterns of cellular substrates in Mls-1a-tolerant CD4+ T cells. Differentially phosphorylated proteins in the ~40 and ~75 kDa range have been observed but were not specifically identified (3). We have now identified these proteins as ERK and SLP-76 (Fig. 6A). In addition, a third clearly differentially phosphorylated band at 120 kDa has been identified as Cbl-b (Fig. 6A). Phosphorylation of Cbl-b is apparent in CD4+ T cells upon CD3{varepsilon} cross-linking, and its phosphorylation is reduced in tolerant CD4+ T cells. Since we have shown a functional effect of VCS treatment on CD4+ T cells, we investigated the biochemical effects of VCS treatment on tyrosine phosphorylation of cellular substrates. Interestingly, VCS treatment of tolerant CD4+ T cells reversed the observed defect in tyrosine phosphorylation of cellular substrates (Fig. 6B). Sialidase treatment leads to the full reversal of phosphorylation defects observed for ERK and SLP-76, while leading to partial reversal of Cbl-b hypophosphorylation.


Figure 6
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  		Fig. 6. Partial enzymatic desialylation reverses phosphotyrosine signaling defect in tolerant CD4+ T cells. (A) Purified splenic CD4+ T cells were stimulated by cross-linking CD3{varepsilon}Rs for 5 min at 37°C. Phosphotyrosine-containing proteins were immunoprecipitated using mAb 4G10 from CD3{varepsilon} cross-linked and control lysates and resolved by SDS-PAGE followed by anti-phosphotyrosine immunoblotting. Naive and tolerant populations were compared with and without VCS pre-treatment. Three major differentially phosphorylated bands at 42, 76, and 120 kDa are identified by anti-phosphotyrosine immunoprecipitation followed by western blotting using antibodies to phospho-ERK1/2, SLP-76 and Cbl-b, respectively. (B) Naive and Mls-1a-tolerized CD4+ T cells either pre-treated or mock treated with VCS followed by CD3{varepsilon} cross-linking, immunoprecipitation and immunoblotting (as above) to measure tyrosine phosphorylation of cellular substrates.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The 6C10 antibody serves as an interesting and unique marker in CD4+ T cells, as both functionally and developmentally significant T cell populations are identified by their ability to bind this antibody. We hypothesized that 6C10 binding may serve as a marker for the glycosylation state of a subset of cell-surface proteins, and moreover, that the glycosylation state of these proteins could have functional consequences for the tolerant CD4+ T cell. In order to test this hypothesis, we set out to further define the nature of the determinant recognized by the 6C10 auto-antibody and to better understand the functional T cell subsets identified by this antibody in CD4+ T cell tolerance.

The Hayakawa laboratory has previously demonstrated that 6C10 fails to bind murine Thy-1 in the complete absence of glycosylation (9), and we sought, herein, to further investigate the contribution of Thy-1 glycosylation to the binding of this antibody. The majority of peripheral Thy-1 molecules in the lymphoid compartment of the mouse are reported to contain four to six sites of potential sialic acid modification, and the number of sialic acid moieties determines the charge of each Thy-1 molecule (19–22). Figure 2 demonstrates that the 6C10 antibody immunoprecipitates a Thy-1 species restricted in both MW and charge, and furthermore, that the relative charge of the species immunoprecipitated co-migrates in the range of thymic Thy-1, suggesting a low degree of sialylation. A low-density band in the thymic Thy-1 pI range was also seen in Thy-1 immunoprecipitate from peripheral CD4+ T cells, demonstrating the presence of a rare Thy-1 species in the periphery with a low degree of sialylation, and this band co-migrates with 6C10 immunoprecipitate. Our data suggest that 6C10 binds to this incompletely sialylated form of Thy-1 in the periphery. Further supporting our model, partial enzymatic desialylation significantly increased the binding of the 6C10 antibody in peripheral CD4+ T cells, whether the starting cell population demonstrated a naive 6C10-binding profile or down-modulated 6C10 binding (Fig. 3). Importantly, VCS treatment of CD4+ T cells from Thy-1 KO mice does not lead to 6C10 binding, demonstrating that increased 6C10 binding in wild-type mice following VCS treatment is specific to the Thy-1 molecule (Fig. 3E and F). Collectively, these data suggest that the 6C10-binding epitope is present in the 6C10lo populations studied, but that this epitope is masked by the degree of sialylation on Thy-1 species.

We show that 6C10 binding correlates with ECA binding on CD4+ T cells (Fig. 4), demonstrating that 6C10 binding serves as a marker of the surface density of exposed galactosyl (ß-1,4) N-acetylglucosamine, and hence the degree of terminal sialylation of this glycan. ECA and 6C10 do not bind the same epitope in competition experiments (unpublished results), so it is unlikely that the 6C10 auto-antibody binds directly to galactosyl (ß-1,4) N-acetylglucosamine, but rather that the ability of the 6C10 antibody to bind is dependent on the sialylation state of Thy-1. Glycan haptens sufficient to block the binding of the ECA lectin do not alter 6C10 binding (unpublished results), also suggesting that 6C10 does not directly bind galactosyl residues on Thy-1. The failure of saccharide haptens to alter 6C10 binding also argues against the possibility that 6C10 binding to Thy-1 is dependent on a complex between an unidentified galactosyl glycan-binding lectin and Thy-1. Taken together, these studies suggest that 6C10 binds only to Thy-1 with a low degree of sialic acid modification, and that this binding serves as a marker for the sialylation state of a subset of surface proteins. Our lectin-binding studies demonstrate differential binding of both 6C10 and ECA between naive and tolerant T cells (Fig. 4A–C), suggesting that the sialylation state of these two cell populations differs not only on Thy-1 but also on an unidentified subset of glycoproteins bearing N-linked glycans.

To investigate the functional significance of the sialylation state of the tolerant CD4+ T cell, enzymatic desialylation was utilized to alter the glycosylation state of this cell. Interestingly, we were able to demonstrate the reversal of tolerance after enzymatic desialylation, in vivo and in vitro (Fig. 5). Although the effect of in vitro sialidase treatment on the proliferation of tolerant CD4+ T cells is greater than that observed in the naive T cell population, the proliferative capacity of naive T cells is also enhanced by desialylation, suggesting that sialylation may also have a negative regulatory effect on the naive CD4+ T cell population. Consistent with our data, moderate enhancement of naive CD8+ T cell responsiveness has been reported following enzymatic desialylation in a TCR transgenic system (31).

The observed decrease in phosphorylation of Cbl-b, ERK1/2 and SLP-76 in tolerized CD4+ T cells in response to TCR cross-linking is qualitatively reversed by VCS pre-treatment (Fig. 6B), showing that cleavage of sialic acid from surface proteins has an immediate effect that is T cell intrinsic in nature. Recent studies have pointed to the role of ubiquitination in limiting T cell responsiveness, identifying Cbl-b as one of the three E3 ubiquitin ligases contributing to the unresponsive state (32). Although these studies point to increases in Cbl-b levels in anergic cells, our observations reveal a more marked alteration in Cbl-b phosphorylation levels in peripheral induced tolerant CD4+ T cells, namely, hypophosphorylation of Cbl-b (Fig. 6A). Cbl-b has also been implicated in vav-dependent re-organization of the immunologic synapse (33), which is particularly interesting as we have also identified differential synapse formation and reduced conjugate formation in Mls-1a-tolerant CD4+ T cells (unpublished results; 15). Interestingly, enzymatic desialylation has been shown to enhance immunologic synapse formation in response to low-affinity TCR ligand in CD8+ T cells (34).

Though the mechanism by which sialylation may alter signaling at the cell surface is not clear, defective receptor multimerization and clustering is an attractive hypothesis. Taking a structural approach, Rudd and colleagues model the role of glycosylation in TCR aggregation, concluding that glycosylation is likely to play an important role in regulated receptor signaling (35). Similarly, receptor clustering has been proposed to underlie the enhanced responsiveness observed in CD4+ T cells from the Mgat5 –/– mouse, deficient in specific glycan branching (36), and CD45 dimerization on T cells has been shown to be negatively regulated by sialylation (37, 38). Furthermore, sialylation of the CD8{alpha}ß co-receptor, a modification known to alter CD8 lineage lymphocyte sensitivity during thymic development, is thought to limit signaling through the inhibition of co-receptor chain interactions (39, 40). Consistent with the aforementioned studies, we favor a model of tolerance in our system whereby tolerant T cells are more completely sialylated, preventing productive receptor aggregation and subsequent downstream signaling.

Our work indicates that sialylation of cell-surface glycoproteins is a critical regulatory mechanism associated with T cell tolerance, and raises the possibility that sialylation may be a more general mechanism by which the responsiveness of the peripheral CD4+ T cell is regulated. We are in the process of identifying specific signaling pathways that are altered by receptor sialylation as well as the genes that regulate the observed sialylation patterns seen in this in vivo system. That therapeutics which alter sialylation could have an immunomodulatory role is an exciting extension of this work.


   Acknowledgements
 
The authors thank Kyoko Hayakawa for helpful discussions and the Abramson Cancer Center as well as the University of Pennsylvania Flow Cytometry and Cell Sorting Facilities for technical support. This work is supported by the National Institutes of Health (AI043620) and the Abramson Family Cancer Research Institute.


   Abbreviations
 
APC, antigen-presenting cell
CFSE, carboxy-succinimidyl-fluorescein-ester
IEF, isoelectric focusing
KO, knockout
Mls-1a, minor lymphocyte-stimulating antigen 1a
MW, molecular weight
PMSF, phenylmethylsulfonylfluoride
PNA, peanut agglutinin
VCS, Vibrio cholerae sialidase
[3H]TdR, [3H]thymidine

   Notes
 
Transmitting editor: T. Hamaoka

Received 18 August 2005, accepted 30 September 2005.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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