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The Journal of Immunology, 2004, 173: 275-284.
Copyright © 2004 by The American Association of Immunologists

Alteration of Cell Surface Sialylation Regulates Antigen-Induced Naive CD8+ T Cell Responses1

Bhanu P. Pappu2 and Protul A. Shrikant3

Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY 14263


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The strength of interactions with APC instructs naive T cells to undergo programmed expansion and differentiation, which is largely determined by the peptide affinity and dose as well as the duration of TCR ligation. Although, most ligands mediating these interactions are terminally sialylated, the impact of the T cell sialylation status on Ag-dependent response remains poorly understood. In this study, by monitoring TCR transgenic CD8+ T cells, OT-I, we show that biochemical desialylation of naive OT-I T cells increases their sensitivity for agonist as well as partial agonist peptides. Desialylation enhances early activation and shortens the duration of TCR stimulation required for proliferation and differentiation, without increasing apoptosis. Moreover, desialylation of naive OT-I T cells augments their response to tumor-presented Ag. These results provide direct evidence for a regulatory role for sialylation in Ag-dependent CD8+ T cell responses and offer a new approach to sensitize or dampen Ag-specific CD8+ T cell responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Most glycosylated proteins and lipids are terminally modified by sialylation (1, 2). The role for sialyl-glycans in the normal functioning of the immune system is amply demonstrated by loss of regulated homing (3), aberrant activation that leads to autoimmunity (4), and loss of T cell homeostasis in animals with genetic deficiencies for various glycosyltransferase activities (5, 6, 7).

Naive T cell activation is regulated by multiple factors including the concentration and affinity of Ag, duration of Ag stimulation, and the costimulatory signals and the cytokine milieu present at the time of Ag interrogation (8, 9). In contrast, Ag-experienced T cells have less stringent requirements for Ag dose, duration, and costimulatory context for their activation (10). This condition is thought to occur due to inherent differences in their protein and gene expression that endows the Ag-experienced T cells with enhanced ability to integrate activation signals during their secondary exposure to Ag (11, 12). Conversely, the changes produced in the cell surface by activation (e.g., expression of adhesion molecules, cytokine receptors) and the reduced sialylation, as demonstrated by increased binding of the plant lectin peanut agglutinin (PNA),4 may permit distinct qualitative and/or quantitative receptor ligand interactions that sensitize Ag-experienced T cell responses (7, 11, 13, 14). The notion that the sialyl-glycans can impart regulatory potential to T cells is supported by several reports in which it has been demonstrated that genetic disruption of the normal N-glycosylation pathway promotes aberrant naive T cell activation resulting in autoimmunity, whereas overexpression of branched O-glycans decreases primary T cell responses to Ag (4, 15). This may occur due to the extent of sialyl-glycans on peripheral T cell surface that by virtue of their size and charge, may tune Ag recognition by regulating the thresholds for activation (16). Furthermore, several elegant studies have recently presented compelling evidence indicating that the differential sialylation of CD8{alpha}{beta} coreceptor modulates the TCR affinity during thymocyte selection and contributes to their dampening of the dynamic range for peptide recognition upon maturation in the thymus and egress into the periphery (17, 18, 19), and it has been reported that sialylation regulates the dimerization and activity of CD45; a phosphatase that restricts TCR signaling, clearly implicating sialyl-glycans in the regulation of Ag-specific peripheral T cell responses (20). Collectively, these findings have generated significant interest in better understanding the precise impact of T cell sialylation on Ag recognition in the hope that thresholds for Ag-dependent activation of T cells can be either augmented (tumor immunity) or attenuated (transplantation, autoimmunity) by altering sialyl-glycans on T cell surfaces.

To directly test the impact of the extent of sialylation on the Ag-specific responses of CD8+ T cells, we have biochemically reduced cell surface sialylation on naive TCR transgenic CD8+ T cells (OT-I) in vitro and studied the effects of dose, affinity, and duration of stimulation by Ag presented on either professional APCs (dendritic cells (DC)) or tumor cells. Our results show that surface sialylation attenuates the dynamic range of naive CD8+ T cells for Ag recognition and desialylation promotes their capacity to integrate the instructive signals that program their proliferation and differentiation in an Ag-specific manner.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and cells

The OT-I TCR transgenic mice (21), originally a gift from Dr. F. Carbone (Monash Medical School, Victoria, Australia) were bred to B6.PL-Thy1a/Cy mice (The Jackson Laboratory, Bar Harbor, ME) and generously provided by Dr. M. F. Mescher (University of Minnesota, Minneapolis, MN). They were backcrossed up to 10 generations for Rag2 deficiency; OT-I/Pl-Rag–/– referred throughout the report as OT-I mice. C57BL/6 mice were purchased from The Jackson Laboratory. All animals were maintained under specific pathogen-free conditions and the experiments performed were in compliance with the relevant laws and institutional guidelines under a protocol approved by the Institutional Animal Care and Use Committee of the Roswell Park Cancer Institute. Adherence-depleted lymph node cells from OT-I mice were the source of CD8+ T cells for all experiments. In some experiments, the CD8+ T cells were negatively selected using CD8 Cellect columns (Cedarlane Laboratories, Hornby, Ontario, Canada) and according to manufacturer recommendations. The purity of cells used was confirmed by flow cytometry and was typically >98% CD8 cells with <1% of CD4 T cells. JAWS II, a bone marrow-derived immature DC line, and EL-4, a thymoma, were obtained from American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 complete medium and according to the supplier’s instructions.

Reagents

For all in vitro cell culture, RPMI 1640 complete medium containing 200 µg/ml penicillin, 200 µg/ml streptomycin, 2 mM L-glutamine, 50 mM 2-ME, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 10 mM HEPES buffer (all from Mediatech, Herndon, VA) and 10% FBS (Life Technologies, Invitrogen, Carlsbad, CA) was used. Cell cultures were maintained in a humidified incubator at 37°C with 5% CO2. The peptides SIINFEKL (OVAp), SIIGFEKL (G4), and Kb-control peptide (THYLFRNL) have been previously described (21, 22). The OVAp and Kb-control peptide were synthesized at peptide synthesis facility of Medical College of Georgia (Augusta, GA). The G4 peptide was kindly provided by Dr. S. Jameson (University of Minnesota, Minneapolis, MN). The following fluorochrome-conjugated or unconjugated reagents were used for flow cytometric staining: mAbs against CD69, CD8{alpha}, Fc block (anti-CD16/32), IFN-{gamma}, and Streptavidin-PE were purchased from BD PharMingen (San Diego, CA); PE-conjugated annexin V was purchased from Caltag Laboratories (Burlingame, CA); biotinylated-PNA was purchased from Vector Laboratories (Burlingame, CA). For plate-bound stimulation of T cells, anti-CD3{epsilon} Ab (clone 2C11) and murine B7.1-Fc chimeric fusion protein were purchased from BD PharMingen and R&D Systems (Minneapolis, MN), respectively.

Flow cytometry

Cells from culture were reacted with Fc block before staining for specific cell markers. For monitoring sialylation changes, cells were stained with biotin-conjugated lectins followed by streptavidin-fluorochrome conjugates. After incubating with Abs/lectins, cells were washed, fixed, and analyzed by flow cytometry. For intracellular IFN-{gamma} staining, the cells were incubated with brefeldin-A during last 4 h of stimulation then stained, fixed, permeabilized with 0.5% saponin and reacted with mAb for IFN-{gamma}. All flow cytometric analyses were performed on a FACScan flow cytometer (BD Biosciences, San Jose, CA). Samples were gated for live cells that were CD8+, and 10,000 gated events were typically acquired and analyzed using CellQuest software (BD Biosciences).

In vivo immunization

Naive OT-I TCR transgenic mice were immunized by i.p. injection of chicken OVA323–339 peptide (OVAp) in LPS (Sigma-Aldrich, St. Louis, MO) at 5 µg and 25 µg per mouse, respectively. Control mice were injected with equivalent volumes of LPS mixed with PBS.

In vitro OT-I stimulation

Purified OT-I T cells from naive OT-I mice were incubated with irradiated bone-marrow derived DCs or EL-4 thymoma cells or mitomycin C-treated JAWS II cells (as DCs) with indicated concentration of antigenic peptide for 2 h at 37°C and washed thrice with complete medium to remove unbound peptide. To monitor T cell proliferation, CFSE (Molecular Probes, Eugene, OR) labeling of naive OT-I cells was performed as previously described (23). Briefly, 107 OT-I cells were incubated with CFSE in 1 ml PBS at a final concentration of 1 µM for 2 min at room temperature. Labeling was quenched by adding excess of RPMI 1640 complete medium and cells were washed twice with culture medium. CFSE-labeled OT-I lymph node cells (105/well) were incubated with Ag-pulsed APCs (2 x 104/well) in 96-well flat-bottom plates (Discovery Labware; BD Biosciences). To evaluate the impact of duration of T cell stimulation; 96-well flat-bottom Maxisorp plates (Falcon Labware; BD Biosciences) were coated with anti-CD3 Ab (1 µg/ml) and murine B7.1-Fc chimeric fusion protein (50 ng/ml) of in PBS overnight at 4°C. Plates were washed three times with PBS and 2 x 105 OT-I cells were added per each well. Plates were spun immediately to allow the synchronized interaction of OT-I cells with the Abs. Cells were harvested at indicated times and stained for flow cytometric analysis. Dilution of CFSE fluorescence was analyzed using FACScan flow cytometer and CellQuest software (BD Biosciences). To stimulate OT-I cells with the G4 peptide, syngeneic spleenocytes pulsed with indicated concentration of peptide for 1 h and OT-I cells were cocultured for defined periods. Cells were harvested at indicated time points, stained, and evaluated by flow cytometry.

Biochemical desialylation

For desialylation of naive T cell surface, OT-I cells (106 cells/ml) were incubated in PBS containing 1 mM CaCl2 and 50 mU of recombinant Vibrio cholerae neuraminidase (Calbiochem, La Jolla, CA) for 1 h at 37°C. Some OT-I cells were incubated under similar conditions without neuraminidase and used as intact cells. After neuraminidase treatment the cells were washed three times with complete medium. The efficiency of neuraminidase treatment was routinely assessed by the extent of staining with fluorescein-conjugated PNA and flow cytometry on CD8+ gated populations.

Statistical analysis

Statistical analysis and p values are calculated using paired Student’s t test, and p < 0.05 is considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cell surface sialylation undergoes dynamic changes during CD8 T cell response

Previously it has been reported that CD8+ T cell surface undergoes desialylation during thymic development as well as peripheral response to viral challenge (13, 14). To address whether immunization with Ag in an adjuvant setting would result in reduced sialylation upon activation, naive OT-I mice were immunized i.v. with OVAp and LPS or left unimmunized (LPS alone). The lymph node cells harvested on day 4 postimmunization were analyzed by flow cytometry and demonstrated an activated phenotype as indicated by increased CD44 expression (Fig. 1A). The activated OT-I T cells had reduced cell surface sialylation as demonstrated by increased PNA lectin binding; the population CD44high and PNAhigh increased from 4.4% to 38% (Fig. 1A, right panel, upper right quadrant). Cells harvested from unimmunized mice showed low to intermediate CD44 expression and maintained a PNAlow phenotype (Fig. 1A, left panel, upper right quadrant). These observations demonstrate that naive CD8+ T cells undergo desialylation during activation and support the previously reported findings on reduced cell surface sialylation detected on CD8+ T cells responding to viral infection in vivo.



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  		FIGURE 1. Activation of naive OT-I T cells with Ag leads to desialylation. A, In vivo. Lymph node and spleen cells harvested from PBS (nonimmunized; left panel) or LPS plus SIINFEKL (OVAp; 10 mg) (immunized) (right panel) injected OT-I mice on day 4 were gated for live CD8+ T cells and analyzed for CD44 and PNA staining by flow cytometry. The expression of CD44 (x-axis) and PNA (y-axis) on CD8+ T cells is shown. The numbers in the upper right quadrant indicate the percentage of CD8+ T cells that are CD44high and PNAhigh. A representative of two independent experiments is shown. B, In vitro. CFSE-labeled naive OT-I cells were stimulated with DCs pulsed with 10 nM OVAp for 48 h. The gated CD8+ T cells were evaluated for PNA staining and CFSE dilution. The data are representative of three independent experiments. C, In vitro. The PNA binding profile of naive OT-I cells (filled histogram), OT-I T cells after 48 h of Ag activation (solid line) and biochemically desialylated naive OT-I cells (dotted line) are comparatively represented in an overlay histogram. The data are representative of three independent experiments.

 
Although, recently several papers have addressed the role for sialylation in thymic development, the impact of sialylation changes on Ag-specific responses mounted by mature peripheral T cells remains poorly understood. To specifically characterize the impact of desialylation on mature CD8+ T cells response to Ag, we have used an in vitro model system that permits study of 1) the change sialylation on Ag-specific CD8+ T cells (OT-I) without altering their gene expression or activation status, 2) the comparative ability of desialylated vs intact naive T cells to respond to varying antigenic stimulatory conditions, and 3) to elucidate the impact of desialylation on distinct phases of Ag-induced CD8+ T cell responses.

To validate the use of this in vitro system to specifically discern the impact of sialylation on Ag responses of CD8+ T cells, we used naive OT-I cells that were either treated with V. cholerae neuraminidase to remove most cell surface sialic acids (referred to as "desialylated" OT-I cells) or left unmanipulated (referred as "intact" cells), and then labeled them with CFSE to follow the extent and rate of their cell division. The CFSE-labeled intact OT-I T cells were reacted with APCs pulsed with 10 nM SIINFEKL for 48 h. The live-gated CD8+ cells were evaluated for PNA binding and CFSE dilution by flow cytometry. Ag stimulation of intact OT-I cells resulted in proliferation that was accompanied by desialylation, as noted by an increase in PNA binding along with CFSE dilution (Fig. 1B). Typically, PNA binding was increased by ~60-fold in OT-I T cells that had divided once and the binding reached maximum levels in cells that had undergone three to four divisions, whereas the OT-I T cells that had not divided remained PNAlow (Fig. 1B). The PNA binding on naive T cells reached maximum levels after 1 h of neuraminidase treatment as evidenced by increased PNA binding (Fig. 1C). Biochemical desialylation was achieved without aberrant T cell activation and alterations in cell surface activation marker expression, as evidenced by significant increase in PNA binding (Fig. 1C) and reduction in SNA and MAL-II staining lectins, which require sialic acid for binding (data now shown), but maintenance of low to moderate levels of CD44 expression. The biochemical alteration in cell surface sialylation achieved with neuraminidase treatment although more dramatic, is comparable to that observed by Ag activation (Fig. 1C). These results clearly demonstrate that comparable changes in cell surface sialylation occur in our in vitro model system as observed in vivo, and the results validate the use of this model and biochemical desialylation for understanding the impact of surface sialylation on Ag recognition, activation, and development of naive CD8+ T cell responses.

Desialylation increases Ag sensitivity of naive OT-I T cells

To initially characterize the effect of reduced sialylation on Ag recognition and early activation, intact and desialylated OT-I cells were reacted for 6 h with APCs pulsed with varying doses of OVAp, harvested and evaluated by flow cytometry for their surface expression of early activation markers CD69 and CD25 (IL-2R {alpha}-chain). The percentage of intact OT-I expressing CD69 shows Ag dose-dependent increases, which are enhanced in desialylated OT-I cells (Fig. 2A; p < 0.05). Furthermore, the relative increase in the mean fluorescence intensity of desialylated OT-I T cells is also augmented over intact cells (Fig. 2B). The expression of CD25, part of receptor complex that designates IL-2 cytokine responsiveness, is also augmented by desialylation (Fig. 2C). Taken together, these observations demonstrate that biochemical desialylation renders naive CD8+ T cells sensitive to weak antigenic stimulation.



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  		FIGURE 2. Biochemical desialylation enhances early activation of naive OT-I T cells. Naive OT-I cells stimulated with DCs pulsed with various concentrations of OVAp were harvested 6 h postculture and stained for CD8, CD69, and/or CD25. The CD8+ gated cells were analyzed for CD69 and CD25 expression by flow cytometry. The percentage of intact OT-I cells ({square}) or desialylated OT-I cells ({blacksquare}) expressing (A) CD69 and (B) the mean fluorescence intensity (MFI) of the positive population is plotted. The percentage of CD8+ T cells positive for CD25 expression is plotted (C). The data represent the values for four independent experiments and the error bars represent the SEM. The p values are calculated using the two-tailed Student t test.

 
The proliferation of T cells is a qualitative outcome of productive TCR engagement that allows sufficient activation (8). It is increasingly appreciated that the early interactions between naive T cells and the APCs instructs the naive T cells for proliferation and differentiation programs (24, 25). To determine the impact of desialylation on the commitment of naive CD8+ T cells for proliferation, we reacted intact or desialylated OT-I T cells labeled with CFSE with varying doses of Ag and evaluated the dilution of CFSE dye by flow cytometry at 48 h. As shown in Fig. 3A, at 1 µM peptide concentration, most intact and desialylated OT-I cells (99%) were activated and underwent multiple cell divisions. However, at lower Ag concentrations, substantially more desialylated OT-I cells underwent CFSE dilution, as evidenced by smaller percentage of OT-I T cells that retained high intensity of CFSE dye in comparison to intact cells (Fig. 3, 100 pM and 1.0 pM SIINFEKL). Strikingly, at 1.0 pM peptide concentration, CFSE dilution was not detected in intact OT-I cells, whereas ~30% of desialylated OT-I cells had undergone cell division and diluted their CFSE dye. These results clearly indicate that desialylation promotes the commitment of naive CD8+ T cells for proliferation at low doses of Ag, perhaps by lowering their threshold for activation. The desialylation mediated increase in early activation (Fig. 2) and proliferation (Fig. 3A) were Ag-specific as no CFSE dilution in desialylated OT-I cells was noted with high doses of an irrelevant peptide with similar H-2Kb binding affinity (Fig. 3, 100 µM THYLFRNL).



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  		FIGURE 3. Desialylation enhances proliferation and differentiation of naive CD8+ T cells. CFSE-labeled naive OT-I cells stimulated with DCs pulsed with varying concentrations of OVAp in vitro for 48 h were stained and analyzed by flow cytometry. A, The CD8+ gated cells were evaluated for CFSE intensity. The numbers above the respective histograms represent the percentage of CD8+ cells that did not undergo cell division. Data are representative of five independent experiments. B, Desialylated and intact naive OT-I cells stimulated with DCs pulsed with 1.0 nM OVAp for 48 h were harvested, stained, and evaluated for CD8 and intracytoplasmic expression of IFN-{gamma}. The overlay histogram represents relative IFN-{gamma} expression of intact OT-I (solid line) and desialylated OT-I (dashed line) cells that were CD8+. The percentage and mean fluorescence intensity of CD8+ T cells positive for IFN-{gamma} expression is indicated at the top. The filled histogram is the Ab isotype control for desialylated cells, and data are representative of three independent experiments.

 
Sustained T cell activation and proliferation lead to subsequent differentiation into effector cells. The ability to produce IFN-{gamma} is a useful attribute for CD8+ effector T cells as they are shown to be important for control of tumors and intracellular infections (26, 27). When stimulated with APCs pulsed with 10 nM SIINFEKL peptide, only 34% of the intact OT-I cells that underwent cell division expressed IFN-{gamma}, as evaluated by intracytoplasmic staining and flow cytometry. Almost 80% of the desialylated OT-I cells expressed IFN-{gamma} under the same conditions of stimulation (Fig. 3B). Moreover, desialylation also increased the amount of IFN-{gamma} produced as demonstrated by increase in the IFN-{gamma} mean fluorescence intensity of desialylated OT-I T cells (Fig. 3B). These results demonstrate that desialylation also enhances differentiation of Ag-activated CD8+ T cells into IFN-{gamma}-producing effectors.

Rapid kinetics of activation by weak agonist peptides upon desialylation

The peptide SIIGFEKL or G4, acts as an altered peptide ligand (weak agonist) for the TCR transgenic OT-I T cells. G4 peptide induces kinetically delayed activation of naive OT-I cells when compared with agonist peptide (OVAp) and takes 1000- to 10,000-fold excess to produce comparable levels of activation (22). Based on the previously discussed results, we reasoned that desialylation would augment weak agonist G4-induced activation of OT-I cells. To test this possibility, we reacted intact and desialylated naive OT-I cells with syngeneic APCs pulsed with varying concentrations of the G4 peptide and evaluated its ability to result in early activation and proliferation in a dose-dependent manner. We consistently observed 30–36% of intact OT-I cells that expressed CD69 after 6 h of stimulation with the highest dose (100 µM) of G4 peptide, which was increased to 84% by 24 h (Fig. 4, A and B); a finding that is entirely consistent with previously published reports (22). However, as predicted by our previous observations, nearly 85% of the desialylated naive OT-I cells expressed CD69 at 6 h poststimulation. More dramatically, 70% of desialylated OT-I cells expressed CD69 6 h at 100-fold lower concentration of G4 peptide (1 µM), whereas only 17% of the intact OT-I cells were CD69+ (Fig. 4A). Although, it must be noted that by 24 h both intact and desialylated OT-I cells show comparable percentages of OT-I cells expressing CD69 at all doses of G4 peptide tested (Fig. 4B). These results demonstrate that desialylation results in quicker integration of activation signals provided by weak agonist stimulation, which over time are rendered equivalent. Furthermore, G4 peptide stimulation also increased proliferation of desialylated OT-I cells at 48 h (Fig. 4C). At lower concentrations of G4 peptide (100 nM), few desialylated OT-I cells undergo one to two rounds of cell division, whereas intact OT-I cells remain unresponsive (Fig. 4C, bottom right panel). These data illustrate the ability of desialylation to not only increase sensitivity to low concentration of agonist peptide stimulation, but also increase sensitivity to stimulation by weak agonist peptides by producing more rapid and robust responses in naive CD8+ T cells.



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  		FIGURE 4. Desialylation augments early activation and proliferation of OT-I cells stimulated by altered peptide ligands (weak agonists). CFSE-labeled naive OT-I cells stimulated with varying concentrations of SIIGFEKL (G4) peptide presented by syngeneic spleenocytes were harvested at either 6 or 24 h poststimulation and stained for CD8 and CD69 expression. The intact ({square}) and desialylated ({blacksquare}) cells were analyzed by flow cytometry, and the percentage of CD8+ T cells positive for CD69 expression at 6 h (A) and 24 h (B) are plotted in relation to varying peptide concentrations. CFSE-labeled OT-I T cells were stimulated with spleenocytes pulsed with G4 peptide at indicated concentrations and cells harvested at 48 h were stained for CD8 and analyzed by flow cytometry (C). The overlay histogram shows relative CFSE intensity of CD8 gated intact (filled) and desialylated (solid line) OT-I cells. Data are representative of two independent experiments.

 
Desialylated OT-I cells require shorter duration of stimulation to commit for proliferation

The naive T cells have more stringent requirements for Ag stimulation than Ag-experienced T cells (28, 29, 30). The duration of Ag stimulation is one of the several parameters that differentiates the activation requirements for naive and effector T cells (31). In light of the recent reports indicating that only 2 h of Ag stimulation was sufficient to commit naive CD8+ T cells for programmed proliferation and differentiation (24, 25), and our observations demonstrating increased ability of naive CD8+ T cells to integrate activation signals for activation and differentiation upon desialylation (Figs. 2 and 3), we hypothesized that biochemical desialylation may reduce the duration of Ag stimulation required to commit naive T cells for proliferation. To directly test this prediction, we chose to use plate-bound Ab-mediated stimulation as it allows us to precisely control the duration of stimulation received by naive OT-I cells. The CFSE-labeled OT-I cells were stimulated with plate-bound anti-CD3 Ab and recombinant chimeric B7.1 protein, and at indicated periods after stimulation the cells were transferred and recultured in fresh plates without any further stimulation. At 48 h after the initial stimulation, cells were harvested, stained, and evaluated by flow cytometry. As shown in Fig. 5, naive OT-I cells undergo proliferation after only 4 h of stimulation, whereas desialylated OT-I cells proliferate after as little as 2 h of stimulation (35% of the desialylated cells diluted CFSE by 48 h), a time point in which no proliferation was noted in intact OT-I cells. Furthermore, increased percentage of desialylated cells underwent three to four rounds of proliferation when compared with intact cells even after longer periods of Ag stimulation (Fig. 5; 8 h).



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  		FIGURE 5. Desialylation shortens the duration of stimulation required to commit OT-I cells for proliferation. Purified CFSE-labeled intact or desialylated OT-I T cells reacted with plate-bound anti-CD3{epsilon} and chimeric B7.1-Fc protein. At times indicated, the cells were transferred to fresh plates without any stimulation and harvested at 48 h. The gated CD8+ T cells were analyzed for CFSE dilution by flow cytometry. The plots represent the CFSE intensity and the numbers indicate the percentage of CD8+ T cells that have undergone more than one cell division. Data are representative of three independent experiments.

 
Based on these results, we conclude that desialylated naive OT-I cells require shorter duration of stimulation to integrate signals required to commit naive T cell for proliferation. Importantly, even when both intact and desialylated OT-I T cells (activated for >6 h) showed similar numbers of T cell participating in the Ag response, the desialylated OT-I cells underwent more rigorous cell divisions as demonstrated by larger proportion of cells with reduced CFSE. These results confirm our prediction and reaffirm the notion that the sialylation status affects the capacity of the naive T cells to kinetically integrate activation signals that instruct the cell for proliferation and differentiation.

Biochemical desialylation does not alter apoptosis of naive and Ag-activated T cells

The sialic acids are negatively charged and form ubiquitous terminal modifications on cell surface glycans. Pan desialylation by neuraminidase treatment could result in aberrant aggregation of cell surface receptors, siglec interactions and/or ganglioside interactions that are implicated in inducing cell death. Moreover, recently the sialylation status of Fas has been implicated in regulating Fas-induced apoptosis in T cells (5, 32). Thus to determine that desialylation does not promote activation that results in cell death, we evaluated induction of apoptosis in the intact and desialylated OT-I cells by monitoring surface phosphotidylserine expression by annexin V labeling after 48 h stimulation with APCs pulsed with either Kb-control peptide (Fig. 6, no Ag) or agonist peptide (Fig. 6, OVAp). The annexin V labeling detected on intact and desialylated OT-I cells was virtually identical as shown in Fig. 6, suggesting that although desialylation promoted naive OT-I T cell activation, proliferation, and differentiation, it did not enhance the induction of apoptosis. Thus increasing the sensitivity of naive T cells for subthreshold Ag activation by desialylation does not lead to exuberant T cell responses that result in increased attrition, but rather promotes greater sensitivity for Ag and subtle enhancement of subsequent responses.



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  		FIGURE 6. Apoptosis is not influenced by desialylation of OT-I cells. Naive intact or desialylated OT-I cells stimulated with DCs pulsed with 10 µM OVAp or control peptide for 24 h were harvested, stained for CD8 and annexin V, and then analyzed by flow cytometry. The histograms of CD8+ gated cells stained with annexin V are plotted. The overlay histograms represent the relative annexin V binding for intact (dotted line) and desialylated (solid line) OT-I cells. Data shown are representative of three independent experiments.

 
Desialylated cells show increased responsiveness to stimulation by tumors as APCs

Most tumors are poor stimulators of T cell responses as they are derived from nonprofessional APCs lacking appropriate costimulatory ligands and perhaps low levels of MHC expression on their cell surface (33, 34). Although, it is argued that most tumor Ag-specific T cell responses are primed via cross presentation, there are reports suggesting the role for direct Ag presentation by tumor cells in shaping the onset of antitumor T cell responses in vivo that eventually produces either tolerance or direct killing of tumors (35, 36). Hence, we next inquired whether the desialylated state of naive OT-I T cells could promote their recognition of Ag presented by tumor cells. Intact and desialylated OT-I T cells labeled with CFSE were reacted with EL-4 thymoma cells pulsed with 10 nM OVAp and the proliferation was evaluated by monitoring the dilution of CFSE at 48 h post culture. Only 38% of intact OT-I T cell underwent cell divisions, whereas 64% of desialylated OT-I T cells had proliferated in response to peptide presented by EL-4 thymoma (Fig. 7, top panel). Significantly, 72 h after stimulation, most desialylated OT-I cells had undergone CFSE dilution (86%), but only 68% of the intact OT-I cells had undergone cell division (Fig. 7, bottom panel). These results clearly demonstrate that desialylated naive T cells are more sensitive to nonprofessional Ag presentation than intact cells.



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  		FIGURE 7. Desialylated OT-I T cells proliferate better to tumor-induced Ag presentation. CFSE-labeled intact and desialylated OT-I cells stimulated with syngeneic EL-4 thymoma pulsed with 10 nM OVAp were gated for CD8+ and analyzed at 48 or 72 h for CFSE intensity. The percentage of CD8+ T cells that have undergone at least one cell division is indicated inside the respective histograms, whereas the percentage outside indicate the OT-I T cells that remain undivided. Data are representative of four independent experiments.

 
Collectively, we submit that surface sialylation on naive CD8+ T cells regulates their ability to respond to Ag and alterations in the sialylation status of T cells can be used to finely modulate their recognition and response to Ag. For the results presented, we have obtained comparable results when negatively enriched CD8+ (OT-I) cells (<2% CD4+ve) were used and when desialylation was achieved by using recombinant pan-neuraminidase from other sources. Moreover, heating neuraminidase for 1 h at 60°C completely abrogated the desialylation capability and the subsequent enhanced naive T cell responses.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The surface of naive T cells is highly decorated with sialic acids, the reduction of which has been used to effectively differentiate between naive and effector-memory T cells responding to a viral challenge (13). Several studies have demonstrated that 1) N-glycosylation regulates activation thresholds of T cells and aberrant N-glycosylation results in autoimmunity (4), and 2) sialylation modulates CD45 dimerization, which is required to dampen primary T cell activation via the TCR (20). An elegant recent study by Starr et al. (37) showed that sialylation of O-glycans modulates sensitivity of the TCR signaling in developing thymocytes. However, most of these studies have provided minimal insights into the impact of surface sialylation on Ag driven peripheral naive T cell responses. Our data in Fig. 1, A and B, demonstrates that naive OT-I T cells are highly sialylated and rapidly lose their sialylation after Ag activation and proliferation, this corroborates well with the reported observations by Harrington et al. (14) in a viral infection model. Importantly, our results using the in vitro model (Fig. 1B) recapitulate the in vivo desialylation of T cells after Ag activation, and go on to show that desialylation does not aberrantly cause the activation of naive T cells without Ag stimulation (Fig. 3A, THYLFRNL). Furthermore, biochemical desialylation leads to reduction in sialyl-glycans but not other alteration that typically follow T cell activation. Thus the use of this in vitro system to delineate the impact of surface sialylation on different phases of Ag-induced CD8+ T cell response is validated. The data indicates that desialylation increases the percentage of naive OT-I cells induced to express early activation markers CD69 and CD25, especially at lower Ag doses (Fig. 2, A and C), and the level of CD69 expression is enhanced in desialylated compared with intact OT-I cells (Fig. 2B). Moreover, the observation that desialylated OT-I T cells also undergo more rounds of cell division and have fewer cells retain their original levels of CFSE (Fig. 3A) provides evidence indicating that the enhanced sensitivity to Ag produced by biochemical desialylation translates into increased proliferation. Thus suggesting that the high sialylation status of peripheral naive CD8+ T cells contributes to their lower Ag sensitivity as recently reported for the fine tuning of TCR sensitivity during thymic maturation (37).

Stimulation of naive T cells by altered peptide ligands often elicits only a subset of activation events in a delayed manner that results in abortive responses. The partial activation is thought to be due to the inability of the weak Ag affinity to stimulate the TCR and overcome activation thresholds that can integrate downstream signals that drive full activation (22, 38, 39). As shown in Fig. 4, desialylated OT-I cells responded more readily than intact OT-I cells to G4 peptide stimulation. Moreover, at low concentrations of G4 peptide, 30% of desialylated OT-I cells were CD69+, whereas only 8% of intact cells express CD69. More importantly, only desialylated OT-I cells showed proliferation in response to 100 nM G4 stimulation at 48 h (Fig. 4C, bottom right). Recently, it has been reported that reduced sialyl-glycans achieved by either neuraminidase treatment or targeted disruption of ST3Gal-I activity results in CD8+ T cells that have increased basal levels of conjugate formation, higher CD69 expression, and integrin activation, when stimulated by the G4 peptide (37). Our data confirm these findings and provide compelling evidence to suggest that by reducing the T cell surface sialylation status we can regulate the capacity of naive T cells to respond to weak Ag stimulation.

Productive activation of naive T cells is thought to require sustained triggering of the TCR determined by the duration of Ag signaling (8, 31). Our data indicate that desialylated OT-I cells require shorter duration of stimulation to commit for proliferation, in comparison to intact OT-I cells (Fig. 5). This suggests that desialylation lowers the activation thresholds in naive T cells that are more readily overcome by shorter duration of stimulation, which may also explain how a larger proportion of desialylated OT-I cells participate in the reaction at a fixed Ag dose within a given period of Ag exposure. Although, it can be argued that the enhanced responsiveness is due to increased nonspecific binding ("stickiness") of desialylated OT-I cells, our unpublished observations and the recent report demonstrating equivalent conjugate formation by intact and desialylated thymocytes and OT-I cells dispel this notion (37). We conclude that the high sialylation status attenuates their sensitivity to Ag by regulating the activation thresholds in naive CD8+ T cells. Furthermore, as the biochemical desialylation did not produce nonspecific activation of naive OT-I T cells (Fig. 3), and no impact of desialylation was noted when the naive OT-I T cells were subjected to activation by super physiologic stimuli (PMA+ionomycin; data not shown), it is suggested that the biochemical desialylation regimen can only marginally affect activation processes and once the threshold of activation is achieved it cannot further augment T cell responsiveness. This observation is further confirmed by our preliminary findings demonstrating that although desialylation of naive T cells does produce increased sensitivity for Ag recognition, the enhancement is considerably weaker when compared with previously activated T cells.

Surface sialic acids are shown to influence tumor cell recognition in mixed lymphocyte reactions (40, 41). The CD8+ T cells mediate several important effector functions by exerting control on intracellular infections and cancer. It is well accepted that IFN-{gamma} is a principal mediator of CD8+ T cell effector functions (26, 27). Hence factors that influence the naive CD8+ T cell differentiation into IFN-{gamma}-producing cells will have profound impact on immunity against infections and tumors. Our data indicate that desialylation of naive T cells enhances the naive T cell differentiation into IFN-{gamma}-producing effector cells, both quantitatively and qualitatively (Fig. 3B). This finding provides yet another rationale for developing strategies to alter cell surface sialyl-glycans as a novel means to enhance effector CD8+ responses against tumor and infected cells.

The attrition in Ag-activated T cell responses determines the extent of memory generation that affords durable immunity (42). Desialylation may alter the interactions of cell surface receptors both in cis as well as in trans such that receptor aggregation, altered interactions between siglecs and gangliosides may result in cell death. Recently, sialylation status of the Fas protein has been implicated in modulating Fas-induced caspase-mediated apoptosis in T cells (32). As shown in Fig. 6, both intact and desialylated OT-I cells showed identical annexin V labeling under naive or Ag activation conditions, indicating that desialylation does not promote the induction of apoptosis. This outcome can be reconciled with those reported in specific sialylation-deficient mice by arguing that perhaps pan biochemical desialylation brings about net changes in T cell surface that subtly enhance sensitivity of Ag responses without causing overt reactivity resulting in increased apoptosis as indicated by our results (Figs. 2–6) or that perhaps the pan desialylation that is achieved with neuraminidase treatment tilts the balance toward increased sensitivity, but targeted disruption of sialylation pathways results in alterations that specifically regulate distinct aspects of T cell survival and death. Nevertheless, the understanding that desialylation does not promote exuberant T cell activation that leads to increased apoptosis is particularly satisfying in terms of its utility as an immunotherapeutic approach.

Tumors often express low levels of specific MHC-peptide complexes and lack costimulatory molecules, hence are considered to be poor stimulators of naive T cell responses and often leads to tolerance in responding T cells (34, 35, 43). Tumor cells expressing GM-CSF and costimulatory molecules have been shown to be immunogenic and induce protective immunity in many model systems (44, 45). Our finding that desialylated OT-I cells respond better than intact OT-I cells to tumor-induced activation (Fig. 7), puts forth the concept that naive CD8+ T cells can be made more sensitive to tumor-Ag recognition and thus overcome their state of tolerance due to either by ignorance or anergy by simple pan desialylation.

Our results clearly indicate that desialylation alters the early recognition events leading to naive T cell activation, proliferation and differentiation. Although, the precise basis for the regulatory role for sialyl-glycans remains uncharacterized, several clues do exist. As formation of the immunological synapse is a early event that sets the tone for subsequent T cell responses and requires concerted interactions by sialic acid-decorated cell surface molecules (16), it entirely possible that upon desialylation we may have changed the ability of cell surface glycoconjugates to segregate into lipid rafts and form the immunological synapse. In support of this finding, the recent report by Starr et al. (37) suggests that biochemical desialylation or sialyl-transferase defects may augment CD8+ T cell sensitivity by enhancing immunological synapse formation to cognate Ag. Another possible mechanism that contributes to our observations is based on the understanding that siglecs (sialic-acid binding Ig-like lectins) can bind specifically to sialic-acid containing ligands (46) and impose activating and inhibitory influences on lymphocytes (6, 47). It can be envisioned that by neuraminidase treatment, unmasked siglecs are predominantly of the activating type and thus we observe enhanced OT-I responsiveness to Ag, or on the contrary too it can be argued that the loss in siglec binding to sialic acids leads to reduced Src homology protein-1 recruitment that dampens the negative feedback provided by siglecs for the regulation of the TCR signaling threshold (48, 49). It is evident that further studies are clearly warranted to specifically identify the glycoconjugates participating in the observed effects and then determine the mode of action. Nevertheless, our demonstration of the impact of surface sialylation on Ag dependent CD8+ T cell responses, particularly the enhanced ability to integrate weak signals for proliferation, and differentiation links sialylation status with the increased sensitivity for Ag demonstrated by effector and memory T cells and provides impetus for developing better understanding for its molecular basis. However, it is rather simplistic to consider that differential sialylation status in itself may be sufficient for all qualitative differences in effector-memory T cell responses. It is more likely that sialylation changes may synergize with the changes in the expression of signaling proteins and genes to enhance the Ag specific responsiveness. To the best of our knowledge this is the first report demonstrating the impact of cell surface sialylation on Ag driven CD8+ T cell responses. These studies provide a compelling rationale for further exploration of the potential use of this novel regulatory pathway to achieve benefits against tumors, autoimmunity, infectious agents, and transplantation-associated graft-versus-host disease.


   Acknowledgments
 
We thank Dr. S. Jameson for providing the G4 peptide, Drs. A. Hurwitz, A. Frey, S. Rath, and V. Bal for critical comments and review of the manuscript.


   Footnotes
 
1 This work was supported by the Roswell Park Alliance Foundation and an Institutional Award from the American Cancer Society. P.A.S was a special fellow of the Leukemia and Lymphoma Society of America. Back

2 Current address: Department of Microbiology and Immunology, State University of New York, Buffalo, NY 14214. Back

3 Address correspondence and reprint requests to Dr. Protul A. Shrikant, Assistant Member, Department of Immunology, Roswell Park Cancer Institute, 322 Comprehensive Cancer Center, Elm and Carlton Streets, Buffalo, NY 14263. E-mail address: Protul.Shrikant{at}roswellpark.org Back

4 Abbreviations used in this paper: PNA, peanut lectin agglutinin; DC, dendritic cell. Back

Received for publication February 11, 2004. Accepted for publication April 26, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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