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The Journal of Immunology, 2001, 167: 5697-5707.
Copyright © 2001 by The American Association of Immunologists

CD45 Modulates Galectin-1-Induced T Cell Death: Regulation by Expression of Core 2 O-Glycans1

Julie T. Nguyen*, Douglas P. Evans*, Marisa Galvan*, Karen E. Pace*, David Leitenberg{ddagger}, Thanhmy N. Bui* and Linda G. Baum2,*,{dagger}

* Department of Pathology and Laboratory Medicine and {dagger} The Jonsson Comprehensive Cancer Center, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095; and {ddagger} Department of Immunology, George Washington University, School of Medicine, Washington, DC 20037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Galectin-1 induces death of immature thymocytes and activated T cells. Galectin-1 binds to T cell-surface glycoproteins CD45, CD43, and CD7, although the precise roles of each receptor in cell death are unknown. We have determined that CD45 can positively and negatively regulate galectin-1-induced T cell death, depending on the glycosylation status of the cells. CD45+ BW5147 T cells lacking the core 2 {beta}-1,6-N-acetylglucosaminyltransferase (C2GnT) were resistant to galectin-1 death. The inhibitory effect of CD45 in C2GnT- cells appeared to require the CD45 cytoplasmic domain, because Rev1.1 cells expressing only CD45 transmembrane and extracellular domains were susceptible to galectin-1 death. Moreover, treatment with the phosphotyrosine-phosphatase inhibitor potassium bisperoxo(1,10-phenanthroline)oxovanadate(V) enhanced galectin-1 susceptibility of CD45+ T cell lines, but had no effect on the death of CD45- T cells, indicating that the CD45 inhibitory effect involved the phosphatase domain. Expression of the C2GnT in CD45+ T cell lines rendered the cells susceptible to galectin-1, while expression of the C2GnT in CD45- cells had no effect on galectin-1 susceptibility. When CD45+ T cells bound to galectin-1 on murine thymic stromal cells, only C2GnT+ T cells underwent death. On C2GnT+ cells, CD45 and galectin-1 co-localized in patches on membrane blebs while no segregation of CD45 was seen on C2GnT- T cells, suggesting that oligosaccharide-mediated clustering of CD45 facilitated galectin-1-induced cell death.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cell death is a crucial process for regulating T cell development and function. Although the mechanisms regulating different T cell death pathways are not fully understood, it is known that a variety of signals can trigger these pathways (1, 2, 3, 4, 5). Galectin-1, a dimeric lectin expressed in a variety of mammalian tissues, induces death of human and murine thymocytes and of activated human T cells through a Fas-, steroid-, and CD3- independent pathway (6, 7, 8, 9). Galectin-1 is a soluble, secreted lectin that remains associated with the cell surface or surrounding extracellular matrix by binding to saccharide ligands on several different glycoproteins (10). Dimeric galectin-1 expressed by thymic epithelial cells can mediate the carbohydrate-dependent binding of thymocytes (8). In addition, galectin-1 expressed on endothelial cells can trigger the death of adherent T cells in a carbohydrate-dependent manner (7).

Galectin-1-induced T cell death is regulated by expression of specific glycosyltransferase enzymes that create oligosaccharide ligands recognized by galectin-1, as well as specific glycoprotein receptors that bear these oligosaccharide ligands. We demonstrated that galectin-1-induced T cell death requires expression of core 2 {beta}-1,6-N-acetylglucosaminyltransferase (C2GnT)3 (11). This enzyme creates the core 2 branch on O-glycans, allowing the addition of lactosamine sequences, the preferred saccharide ligands of galectin-1. Although core 2 O-glycans have been described on both CD43 and CD45 (12, 13), two of the most abundant and highly glycosylated T cell-surface glycoproteins, it is not known which T cell-surface receptors for galectin-1 require the core 2 O-glycan modification to trigger cell death.

On human and murine T cells, while many cell-surface glycoproteins may bear lactosamine-containing oligosaccharides, a restricted set of human and murine T cell-surface glycoproteins preferentially bind galectin-1. These are CD45, CD7, and CD43 (Refs. 14 and 15 ; J. T. Nguyen, unpublished data). Deciphering the roles played by these specific glycoprotein receptors is critical to understanding how a death signal is delivered by galectin-1.

We have demonstrated that galectin-1 binding to T cells results in the segregation of CD45, CD7, and CD43 into unique membrane domains on the T cell surface, with co-localization of CD45 and externalized phosphatidylserine on membrane blebs of dying cells (14). Recent work characterizing the binding of galectin-1 to CD45 has demonstrated that multiple molecules of galectin-1 can bind to a single CD45 molecule (16). These data provide support for our model of CD45 cross-linking and segregation after galectin-1 binding (14) as an important step in the initiation of T cell death. Previous work in our laboratory and others suggested that galectin-1-induced T cell death might involve CD45, because some CD45- T cell lines were resistant to galectin-1 (7, 17, 18). However, the effect of re-expressing CD45 in these CD45- T cell lines has not been examined, so that the requirement for CD45 in galectin-1-induced cell death has never been clearly demonstrated. To determine the requirement for CD45 in T cell death mediated by galectin-1, we used a panel of CD45+ and CD45- cell lines derived from the BW5147 murine T cell lymphoma (Refs. 11 and 19, 20, 21, 22, 23 ; see Fig. 1Go). Surprisingly, we found that CD45 was not required for galectin-1 cell death. Indeed, in the absence of C2GnT expression, CD45+ cells were resistant to galectin-1 while C2GnT expression restored susceptibility of CD45+ T cells to galectin-1-induced cell death.



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  		FIGURE 1. Derivation of cell lines used in this study. All cell lines are derived from the murine BW5147 T cell line (21 ).

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and reagents

The BW5147, BW5147(Rev)1.1 (Rev1.1), and T200- cell lines were gifts of Dr. R. Hyman (21, 22). The BW(T200-)TCR cell line expressing CD3 (CD45-) and the BWTCR/CD45Null cell line (CD45R0) were gifts of Dr. K. Bottomly (19, 20). The BW5147PharR2.1 cell line was the gift of Dr. M. Pierce (23). The BW5147, Rev 1.1, and T200- cell lines were maintained in complete DMEM (Life Technologies, Rockville, MD) supplemented with 10% heat-inactivated FBS (HyClone Laboratories, Logan, UT), 2 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 1 mM MEM sodium-pyruvate solution (Sigma-Aldrich, St. Louis, MO). The CD45- cell line was maintained in complete Bruff’s medium (Click’s medium and EHAA (Irvine Scientific, Santa Ana, CA) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 10 mM HEPES, 50 µM 2-ME, and 0.5 mg/ml geneticin (G418; Sigma-Aldrich)). The CD45R0 cell line was maintained in selective complete Bruff’s medium with 0.5 mg/ml hygromycin (Roche Molecular Biochemicals, Indianapolis, IN). The BW5147, CD45-, and CD45R0/C2GnT-transfected cell lines were maintained in selective Bruff’s medium with 0.5 mg/ml hygromycin and 0.5 mg/ml zeocin (Sigma-Aldrich). The {theta}35 thymic-stromal-epithelial cell line was obtained from Dr. K. Dorshkind (24) and cultured in MEM D-valine with 10% heat-inactivated FBS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Cells were grown at 37°C at 5% CO2 for {theta}35 cells and 10% CO2 for murine T cells, in a humidified atmosphere.

Recombinant human galectin-1 was prepared as previously described (25). Before use, galectin-1 was dialyzed in 8 mM DTT in PBS (10 mM NaPO4, 140 mM NaCl, pH 7.4).

The following mAbs were used in this study: biotinylated or FITC-conjugated CD45 (clone 30-F11); rPE-conjugated CD4 (clone H129.19); FITC-conjugated CD8 (clone H11-86.1); PE-conjugated CD43, high m.w. (clone 1B11) and biotin-, FITC-, or PE-conjugated isotype-matched controls (BD PharMingen, San Diego, CA).

The following reagents were purchased from the indicated suppliers: protein-tyrosine-phosphatase inhibitor, potassium bisperoxo(1,10-phenanthroline)oxovanadate(V) (bpV(phen)) (Calbiochem, La Jolla, CA); annexin V/propidium iodide (PI) staining kit (R&D Systems, Minneapolis, MN); DTT (Fisher Scientific, Fairlawn, NJ); {beta}-lactose, 10x PBS, and BSA (Sigma-Aldrich); and Ficoll-Paque (Pharmacia, Piscataway, NJ).

Death assays

Flow cytometry data were acquired using a BD Biosciences (San Jose, CA) FACScan and analyzed using CellQuest software. In all death assays, 0.2x 106 cells were used in a final reaction volume of 200 µl, and all assays were done in triplicate. Galectin-1 death assays were performed as previously described (7, 8, 9), except that incubation times are indicated for each experiment. For inhibition assays, cells were preincubated for 3 h in the presence or absence of 40 µM bpV(phen), before a 3-h incubation with 20 µM galectin-1 or buffer control at 37°C. A range of bpV(phen) concentrations (10–50 µM) was initially assayed, and the lowest concentration at which augmentation of cell death was observed was used for subsequent experiments (data not shown). Cells were washed with PBS and either 30-F11 mAb or an isotype-matched control was added to a final concentration of 0.2 µg/sample for 30 min at 37°C. Cells were washed in PBS, resuspended in complete media, and incubated at 37°C for an additional 2 h. Before analysis, 0.1 M {beta}-lactose (final concentration) was added to dissociate galectin-1 from the cells and cells were washed with PBS. Ten-thousand events were acquired per sample for death assays of T cell lines and thymocytes.

The percent cell death was calculated by determining the percent of viable cells: [percentage of viable = (percentage of annexin V-, PI-, reagent treated)/(percentage of annexin V-, PI-, control treated) x 100 and percentage of death = 100 - percentage of viable] Results are expressed as the mean ± SD, unless otherwise noted.

For phenotypic analysis, 0.2 x 106 cells were washed in cold PBS and incubated with the relevant mAb at a final concentration of 0.2 µg/sample in PBS/1% BSA at 4°C for 45 min. Cells were washed in cold PBS and resuspended in PBS/1% BSA containing 2 µg/ml 7-aminoactinomycin D. Five-thousand cells were acquired for phenotypic analysis, nonviable cells (7-aminoactinomycin D) were excluded, and expression of the relevant Ags was determined by flow cytometry, as described above.

Isolation of galectin-1 binding proteins

Isolation of galectin-1 binding proteins from murine T cells was performed exactly as we have previously described for human T cells (14). Briefly, cell-surface glycoproteins were biotinylated, cellular plasma membrane preparations were isolated and solubilized, and the solubilized plasma membrane preparation was applied to a galectin-1 affinity column, washed, and eluted with {beta}-lactose. Glycoproteins eluted from the galectin-1 affinity column were immunoprecipitated with CD45 Ab (30-F11; BD PharMingen) or isotype control, and the precipitate was separated by SDS-PAGE and electroblotted on the nitrocellulose membrane, Hybond C-extra (Amersham, Arlington Heights, IL). The membrane was blocked with 5% skim milk in TBS (20 mM Tris-HCl, 0.5 M NaCl, pH 7.5), probed with HRP-conjugated streptavidin (Zymed Laboratories, San Francisco, CA), washed extensively with TBS, and the precipitated proteins were visualized via ECL (Amersham).

Transfection

Murine C2GnT cDNA (the kind gift of Dr. M. Fukuda, The Burnham Institute, La Jolla, CA) was subcloned into the PstI and XbaI site of the pcDNA3.1–Zeo+ vector (Invitrogen, Carlsbad, CA) as previously described (26). CD45- and CD45R0 cells were transfected with the vector containing the C2GnT cDNA or with mock vector via standard electroporation methods. Ten micrograms of linearized vector containing C2GnT insert or vector alone were added to 1x 106 cells in electroporation cuvettes, incubated on ice for 15 min, and pulsed for 1 s with a voltage/capacitance setting of 330 V/1000 mf using an Invitrogen Electroporator II. Selective Bruff’s medium was added and cells were cultured for 2 days. Cells were split 1:10 into selective Bruff’s with zeocin added to a final concentration of 0.5 mg/ml to select for C2GnT transfectants. Transfected cells were washed with selective medium every 3 days. Clones were isolated by limiting dilution, and C2GnT expression was confirmed by flow cytometric analysis with the 1B11 mAb that recognizes core 2 O-glycans on CD43.

Cell-cell conjugate assays and confocal microscopy

{theta}35 cells were plated on coverslips in 12-well plates. At 80–90% confluence, 106 T cells were added to the coverslips and allowed to bind for 1–8 h. Unbound cells were removed by washing in PBS. For cell death assays, coverslips were incubated with annexin V/biotin (1 µg/ml) in binding buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 2.5 mM CaCl2, and 1 mM MgCl2) for 20 min at 4°C. The coverslips were washed and incubated with 2% paraformaldehyde for 30 min at 4°C. After washing with PBS, coverslips were incubated with 0.2 M glycine in PBS for 10 min at 4°C, rinsed with PBS and blocked in 10% goat serum in PBS for 45 min. After washing with PBS, bound annexin V was detected with anti-biotin FITC (1/25 in PBS with 2% goat serum) for 90 min at 4°C. After washing, coverslips were mounted onto slides with 25 µl Prolong Anti-fade mounting medium (Molecular Probes, Eugene, OR) and the slides were visualized on a Fluoview laser scanning confocal microscope (Olympus, Melville, NY), using the x60 and x100 objectives.

For localization of galectin-1 and CD45 on the T cell-{theta}35 cell conjugates, T cells bound to {theta}35 cells on coverslips were fixed with 2% paraformaldehyde, quenched with 0.2 M glycine, rinsed, and blocked with 10% goat serum. The fixed cells were incubated with either galectin-1 antiserum or preimmune rabbit serum (1/1000), and with biotinylated CD45 (30-F11) Ab or IgG2b isotype control (10 µg/ml), all diluted in PBS with 2% goat serum for 90 min at 25°C. After washing, the bound Ab was detected with Texas Red-conjugated goat anti-rabbit antiserum (1/1200) and streptavidin-FITC (5 µg/ml) for 90 min at 25°C. After washing, coverslips were mounted to slides and examined by confocal microscopy as described above.

To detect FITC- and Texas Red-labeled Ags, samples were excited at 488 and 568 nm with argon and krypton lasers, respectively, and the light emitted between 525–540 nm was recorded for FITC and above 630 nm for Texas Red. Images were collected at 0.5 micron optical slices, and ~20 horizontal (X-Y) confocal sections were obtained for each sample. Dual emission fluorescent images were collected in separate channels. The images were processed using the Fluoview image analysis software (version 2.1.39; Olympus). Areas of red and green overlapping fluorescence were represented with a yellow signal.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD45 negatively modulates galectin-1-induced T cell apoptosis

To examine the role of CD45 in galectin-1-induced cell death, we used a panel of murine T cell lines derived from the parental line BW5147 (see Fig. 1Go for cell lines used in this study). The BW5147, PhaR2.1, T200-, CD45-, and CD45R0 cell lines have all been well-characterized in previous studies (19, 20, 21, 22, 23). The parental BW5147 cell line is CD45+CD3-. The T200- and CD45- derivatives of the parental line lack CD45 and the CD45R0 cell line was generated by transfection of CD45- cells with cDNA encoding the CD45R0 isoform (19, 20, 21, 22).

We first compared the parental cell line, BW5147, with the T200- cell line that lacks CD45. As shown in Fig. 2GoA, the parental BW5147 cell line was minimally susceptible to galectin-1. In contrast, the T200- cells were susceptible to galectin-1-induced death. Cell death of the T200- cells was shown by change in forward vs side scatter, as well as by an increase in annexin V/PI staining (Fig. 2GoB). Galectin-1 also induced death of the CD45- cell line, a derivative of the T200- cell line that expresses CD3 (19). Importantly, these results clearly demonstrated that CD45 expression was not absolutely required for galectin-1-induced T cell death, as both the T200- and CD45- cell lines were susceptible to galectin-1.



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  		FIGURE 2. CD45 expression inhibits galectin-1-induced cell death. A, The parental BW5147 and three derivative cell lines were treated with 20 µM galectin-1 or buffer control and the percent cell death was evaluated as described in Materials and Methods. T200- and CD45- cell lines do not express CD45. CD45R0 cells were generated by expression of the CD45R0 isoform in CD45- cells (see Fig. 1Go for phenotypes of all cell lines used). The level of CD45 expression for each cell line, measured as peak fluorescence channel, is indicated under the graph. B, Scatter plots of BW5147 and T200- cells treated with galectin-1 or buffer control (CT) to demonstrate the parameters of cell death. T200- cells treated with galectin-1 demonstrate significant cell loss, detected by decreased forward scatter and increased side scatter, but there is minimal loss of BW5147 cells treated with galectin-1. Similarly, T200- cells treated with galectin-1 demonstrate a significant increase in the fraction of annexin V+/PI+ cells, compared with galectin-1-treated BW5147 cells.

 
Because the BW5147 cell line was resistant to galectin-1, compared with the T200- and CD45- cell lines, this suggested that CD45 expression in this cell line actually inhibited galectin-1-induced cell death. To address this, we examined the CD45R0 cell line, previously made by Bottomly and coworkers (19, 20), that was created by expressing the CD45R0 isoform in CD45- cells (see Fig. 1Go). The CD45R0 cells demonstrated reduced susceptibility to galectin-1, compared with the CD45- cells (Fig. 2GoA). The level of CD45 expression on the CD45R0 cell line was lower than on the parental BW5147 cells (peak fluorescence channel 255 vs 1500, shown under the graph); however, in the CD45R0 cell line, this level of CD45 expression correlated with decreased susceptibility to galectin-1.

Inhibition of death involves the cytoplasmic domain of CD45

Two possible mechanisms could account for the inhibitory affect of CD45 on galectin-1-induced cell death. First, the inhibitory effect could involve the cytoplasmic protein tyrosine-phosphatase domains of CD45; galectin-1 binding has been shown to modulate CD45 tyrosine-phosphatase activity (17, 18). Second, galectin-1 binds to saccharide ligands on the extracellular domain of CD45 (14), so that the CD45 extracellular domain could compete with other proapoptotic receptors, such as CD7 (15), for galectin-1. To address this, we examined the Rev1.1 cell line, a spontaneous revertant of the T200- cell line that expresses only the transmembrane and extracellular domains of CD45, but lacks the two cytoplasmic tyrosine-phosphatase domains (22). We confirmed that galectin-1 bound to the extracellular domain of the Rev1.1 cells, by isolating galectin-1-binding proteins from the Rev1.1 cells (Fig. 3GoC), as well as from the parental BW5147 cell line and the derivative PhaR2.1 cell line that is susceptible to galectin-1 (11). As expected, CD45 from the Rev1.1 cells had an Mr of ~135 kDa, due to loss of the cytoplasmic domain.



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  		FIGURE 3. The inhibitory effect of CD45 requires the cytoplasmic domains. A, The Rev1.1 cell line, derived from the T200- cell line, expresses only the transmembrane and extracellular domains of CD45, and is susceptible to galectin-1-induced death. BW5147, T200-, and Rev1.1 cells were treated with galectin-1 or buffer control and cell death was determined as described in Materials and Methods. The level of CD45 expression for each cell line, measured as peak fluorescence channel, is indicated under the graph. B, The tyrosine-phosphatase inhibitor bpV(phen) (40 µM) enhanced susceptibility of the BW5147 cells to galectin-1. No bpV(phen) enhancement was seen with the T200- cell line that does not express CD45. C, CD45 from BW5147, PhaR2.1, and Rev1.1 cells bind to galectin-1. Cell-surface proteins were biotinylated and galectin-1-binding glycoproteins were isolated on a galectin-1 affinity matrix. Eluate from the galectin-1 column was immunoprecipitated with CD45 or isotype control and immunoblotted, and the blot was probed with streptavidin-HRP. There was no appreciable difference in the Mr of CD45 from BW5147 and PhaR2.1 cells (cells that differ in expression of the C2GnT). CD45 from Rev1.1 cells migrated faster, due to the loss of the two cytoplasmic domains. As expected, no CD45 was detected among the galectin-1-binding proteins from the T200- cells.

 
As shown in Fig. 3GoA, the Rev1.1 cells demonstrated increased susceptibility to galectin-1 compared with the BW5147 cells, although the level of Rev 1.1 cell death was slightly less than that of the T200- cells. Of note, the level of CD45 on the Rev1.1 cells was significantly higher than the level of CD45 on the CD45R0 cells (Fig. 2Go). Thus, although galectin-1 can bind to the extracellular domain of CD45 on the Rev1.1 cells, the extracellular domain of CD45 did not appear to be solely responsible for the inhibitory effect on cell death, by acting as a "sink" for galectin-1 binding. Rather, the susceptibility of Rev1.1 cells to galectin-1 suggested that the inhibitory effect of CD45 involved the cytoplasmic tyrosine-phosphatase domains that are absent in Rev1.1 cells.

CD45 phosphatase domains inhibit cell death

If the phosphatase domains of CD45 participated in the galectin-1 resistance of BW5147 cells, we reasoned that the addition of a phosphatase inhibitor to BW5147 cells would restore sensitivity to galectin-1. We treated cells with bpV(phen), a tyrosine-phosphatase inhibitor (27), before the addition of galectin-1. As shown in Fig. 3GoB, treatment of BW5147 cells with 40 µM bpV(phen) resulted in a 4-fold increase in the percent of apoptotic cells in the galectin-1 treated samples, compared with control BW5147 cells not treated with bpV(phen). This concentration of bpV(phen) had no effect on the viability of the BW5147 cells in the absence of galectin-1 (data not shown). Although bpV(phen) is not a specific inhibitor of the CD45 tyrosine phosphatase, the augmentation of galectin-1 sensitivity in BW5147 cells likely involves CD45 inhibition, because we did not observe enhancement of galectin-1-induced apoptosis by bpV(phen) in the T200- cell line that lacks CD45 (Fig. 3GoB). Thus, the data in Fig. 3Go, A and B, indicated that the CD45 cytoplasmic tyrosine-phosphatase domains participated in the resistance to galectin-1 in BW5147 and CD45R0 cells.

Glycosyltransferase expression regulates CD45 inhibition of cell death

We have demonstrated that expression of the C2GnT is required for galectin-1-induced cell death (11). The C2GnT creates branched O-glycans bearing the lactosamine ligand preferentially recognized by galectin-1 (26, 28, 29). Expression of the C2GnT in galectin-1-resistant BW5147 cells made these cells very sensitive to galectin-1 (11). Moreover, a derivative of the BW5147 cell line, PhaR2.1, that expresses both CD45 and the C2GnT, was very susceptible to galectin-1 (11). Because the BW5147, T200-, CD45-, and CD45R0 cell lines do not express the C2GnT (see Fig. 1Go), we asked whether C2GnT expression would overcome the galectin-1 resistance of the CD45+ BW5147 and CD45R0 cell lines (see Fig. 2Go, A and B).

We expressed the C2GnT in the CD45R0 and CD45- cell lines. Expression of core 2 O-glycans on cell-surface glycoproteins of C2GnT-transfected cells was confirmed by staining with the 1B11 mAb, that detects core 2 O-glycans on the CD43 polypeptide (Fig. 4Go). As shown in Figs. 2Go and 5Go, the C2GnT- CD45R0 cells are minimally susceptible to galectin-1. However, expression of the C2GnT in CD45R0 cells dramatically increased the susceptibility of the cells to galectin-1 (Fig. 5Go). As a control, we also examined the galectin-1 susceptibility of the CD45- cell line transfected with the C2GnT. We observed no enhancement in the galectin-1 susceptibility of the CD45- cells expressing the C2GnT, compared with CD45- cells transfected with vector alone. These data suggest that, in the absence of core 2 O-glycans, CD45 expression interferes with the proapoptotic galectin-1 signal delivered through other cell-surface receptors. However, the presence of core 2 O-glycans appears to specifically modulate the inhibitory effect of CD45.



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  		FIGURE 4. Expression of the C2GnT. The CD45R0 cell line was transfected with cDNA-encoding C2GnT (CD45R0/C2GnT) or with vector alone (CD45R0/mock). Expression of core 2 O-glycans was detected with the 1B11 Ab. CD45R0/C2GnT cells showed increased expression of the 1B11 epitope, indicating C2GnT activity compared with CD45R0 mock cells. The identical protocol was used for the CD45- mock and CD45-/C2GnT cell lines (not shown).

 


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  		FIGURE 5. Cells that express the C2GnT are susceptible to galectin-1-induced death. CD45R0/C2GnT and CD45R0 mock cell lines, and CD45- C2GnT and CD45- mock cell lines, were treated with galectin-1 and cell death was measured as described in Materials and Methods. The CD45R0/C2GnT cells showed a dramatic enhancement in galectin-1-induced cell death compared with CD45R0 mock cells. No enhancement in galectin-1 susceptibility was observed for the CD45- cells expressing the C2GnT, compared with mock-transfected controls.

 
C2GnT expression is required for death of T cells on thymic stromal cells

In the experiments described above, T cell death was triggered by the addition of soluble recombinant galectin-1. Although galectin-1 is secreted from cells that express it, galectin-1 is found primarily on the cell surface or associated extracellular matrix (10, 30, 31, 32). Thus, in vivo, T cells are most likely to encounter cell- or tissue-associated galectin-1. It was therefore critical to determine whether the features required for cell death induced by soluble galectin-1 were the same features required for death when T cells bound to galectin-1 on stromal cells.

To examine this, we used murine {theta}35 cells, derived from murine thymic stroma (24). These cells abundantly express galectin-1 (Fig. 6Go) which remains associated with the cell surface or with the extracellular matrix material produced by these cells (D. P. Evans, M. Donnell, and L. G. Baum, unpublished data). T cells were added to confluent monolayers of {theta}35 cells and allowed to bind for 1 h. After binding, the unbound T cells were removed by washing and biotin-conjugated annexin V was added to label externalized phosphatidylserine on dying T cells. After fixation, bound annexin V was detected with anti-biotin FITC. The total number of bound T cells on the {theta}35 monolayers was determined by counting 4–6 high power fields by phase microscopy. The number of annexin V+ cells on the same fields was determined by confocal microscopy to detect fluorescence. One hour of interaction with the {theta}35 cells was sufficient to achieve the level of T cell death shown in Fig. 7Go. Prolonged incubation, up to 8 h, did not result in a significant increase in the percent of annexin V+ T cells, compared with that observed at 1 h (data not shown).



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  		FIGURE 6. {theta}35 cells express cell-surface galectin-1. Monolayers of {theta}35 thymic stromal cells on coverslips were fixed and stained with (A) preimmune serum or (B) anti-galectin-1 antiserum. Bound Ab was detected with Texas Red-conjugated goat anti-rabbit serum and cells were visualized by confocal microscopy. A x60 image is shown.

 


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  		FIGURE 7. Galectin-1 expressed on {theta}35 cells kills T cells expressing the C2GnT. The indicated T cell lines were added to monolayers of {theta}35 thymic stromal cells on coverslips. The BW5147, BW5147/mock, and CD45R0 cells do not express the C2GnT. The PhaR2.1, BW5147/C2GnT, and CD45R0/C2GnT cells express the C2GnT. After 1 h of binding, the cells were labeled with annexin V biotin. After fixation, bound annexin V was detected with streptavidin FITC, and the cells were examined at x60 by confocal microscopy. The total number of bound T cells was determined by counting five microscopic fields by light microscopy. Apoptotic cells were detected in each field by the binding of annexin V and the percent of bound T cells that were annexin V+ was determined. The percent of annexin V+ cells for each of the different cell lines was normalized to that observed for the PhaR2.1 cells in each experiment; absolute numbers of annexin V+/PhaR2.1 cells ranged between 50–70%. The mean ± SD for duplicate experiments is shown, with p values for the indicated comparisons: *, p = 0.004, **, p = 0.006, ***, p = 0.006.

 
After 1 h of binding, 50–70% of the adherent PhaR2.1 cells that express both CD45 and the C2GnT bound annexin V. Annexin V binding of all cell lines was normalized to that observed for the PhaR2.1 cells in each experiment (Fig. 7Go). The BW5147 cells demonstrated significantly less annexin V binding, compared with the PhaR2.1 cells. However, expression of the C2GnT in the BW5147 cells made these cells highly susceptible to cell death on the {theta}35 cells. The percent of BW5147/C2GnT cells that bound annexin V was comparable to the level we observed for the PhaR2.1 cells (91 and 92% of the level of death observed for the PhaR2.1 cells, respectively). The CD45R0 cells demonstrated a modest level of cell death on {theta}35 cells, comparable to that seen with the parental BW5147 cells. However, expression of the C2GnT in the CD45R0 cells (CD45R0/C2GnT) also resulted in increased death on the {theta}35 cells. Thus, the cells that expressed the C2GnT, i.e., PhaR2.1, BW5147/C2GnT and CD45R0/C2GnT, all demonstrated increased susceptibility to death on thymic stromal cells, compared with the C2GnT- BW5147 and CD45R0 cells. These results indicated that optimal T cell death induced by galectin-1 expressed on thymic stromal cells required that T cells expressing CD45 also express the C2GnT.

C2GnT expression results in CD45 clustering on the T cell surface

Our laboratory has previously demonstrated that the binding of soluble recombinant galectin-1 to human T cells and thymocytes results in the clustering of CD45 and segregation of CD45 with externalized phosphatidylserine on membrane blebs of dying cells (14). We wished to determine whether this same event occurred when T cells bound to native galectin-1 expressed on stromal cells, because the use of cells as a source of galectin-1 is closer to an in vivo system. Also, we examined whether the clustering of CD45 on the T cell surface required expression of the C2GnT. The BW5147, PhaR2.1, BW5147/mock, and BW5147/C2GnT T cell lines were added to monolayers of {theta}35 cells, as described above. (CD45 localization on CD45R0 and CD45R0/C2GnT cells could not be analyzed by this method, due to the low level of CD45 expression relative to wild-type BW5147 cells.) After 1 h of binding, nonadherent T cells were removed by washing and the adherent cells were fixed in paraformaldehyde. Galectin-1 (red) and CD45 (green) were detected by immunofluorescent Ab labeling and the samples were analyzed by confocal microscopy. As shown in Fig. 8Go, adherent BW5147 cells were primarily round cells, with CD45 distributed uniformly over the cell surface. On these cells, there was no significant colocalization of CD45 and galectin-1, demonstrated by the absence of yellow color.



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  		FIGURE 8. Apoptotic T cells expressing the C2GnT demonstrate segregation of CD45 and colocalization of CD45 with galectin-1. BW5147, PhaR2.1, BW5147/mock, and BW5147/C2GnT T cells bound to {theta}35 thymic stromal cells were labeled with Ab to CD45 (green) and galectin-1 (red). On BW5147 and BW5147/mock cells bound to {theta}35 thymic stromal cells, there is uniform distribution of CD45 with little or no colocalization of CD45 and galectin-1. These cells display round morphology with no membrane blebs. PhaR2.1 and BW5147/C2GnT cells bound to {theta}35 thymic stromal cells demonstrate irregular cellular contours, with prominent membrane blebs typical of dying cells. On these cells, CD45 is clustered in discrete patches primarily on membrane blebs, with colocalization of galectin-1 (yellow) in some areas. In some sections (right column), a portion of the underlying {theta}35 stromal cell is seen, with abundant cell-surface expression of galectin-1. Three representative 0.5 µm slices of each cell type are shown, taken from a total of seven experiments.

 
In contrast, adherent PhaR2.1 cells had irregular plasma membrane contours with membrane blebs protruding from the cell, morphologic features that are characteristic of cell death. On the PhaR2.1 cells, CD45 was unevenly distributed on the cell surface, and was localized primarily on the membrane blebs. On these cells, CD45 colocalized with galectin-1 (as demonstrated by the yellow color) indicating close approximation of the two fluorochromes.

In contrast to the round morphology observed for BW5147 cells, BW5147/C2GnT cells also displayed irregular membrane contours when bound to the {theta}35 cells. On BW5147/C2GnT cells, we also observed clustering of CD45 to membrane protrusions, as seen with PhaR2.1 cells. BW5147/mock cells demonstrated the round morphology and uniform distribution of CD45 that we had observed for parental BW5147 cells. These data demonstrate that expression of the C2GnT was sufficient to allow the clustering of CD45 on the T cell surface following binding to galectin-1. Thus, the presence of core 2 O-glycans on CD45 may allow galectin-1 to physically segregate CD45 and counteract the negative effect of CD45 on galectin-1-induced T cell death.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
CD45 has been proposed to both augment and inhibit many T cell functions (33, 34), including cell death. Ab-mediated cross-linking of CD45-induced apoptosis of both human T cells and murine thymocytes (35, 36). In addition, overexpression of CD45 in HY-TCR-transgenic mice increased the efficiency of TCR-mediated apoptosis (37). In other systems, CD45 has been shown to suppress T cell apoptosis triggered by cellular de-adhesion, CTL clones, or activation with PHA and IL-2 (38, 39, 40). Apoptosis during T cell development also appears to be suppressed by CD45. Intestinal intraepithelial lymphocytes from CD45-deficient mice demonstrated increased susceptibility to spontaneous apoptosis (41) and impaired thymocyte development in CD45-deficient mice has been proposed to result from increased thymocyte death "by neglect" (42, 43).

However, previous studies have not demonstrated that CD45 directly blocks a specific proapoptotic signal. The present data indicate that CD45 can be a negative regulator of the galectin-1 death pathway. The parental BW5147 cells were resistant to galectin-1, while the T200- cells that have lost CD45 expression were susceptible to galectin-1. The T200- cell line was selected after chemical mutagenesis of the parental BW5147 cells (21), raising the formal possibility that other mutations may confer susceptibility to galectin-1 in T200- cells. However, re-expression of the CD45R0 isoform in a T200--derived cell line correlated with resistance to galectin-1 cell death (Fig. 2Go), implicating CD45 as the negative regulator. Although we did not examine mock-transfected cell lines along with the CD45R0 cells (Fig. 2Go), the finding that expression of the C2GnT in CD45R0 cells restored galectin-1 sensitivity indicates that the CD45R0 cells had an otherwise intact galectin-1 death pathway.

It is clear that CD45 expression on T cells does not absolutely inhibit galectin-1-induced cell death (7, 8). Several human and murine cell lines that express CD45 are susceptible to galectin-1, as are human and murine thymocytes and activated human T cells. We propose that differences in CD45 glycosylation regulate susceptibility of T cells to galectin-1.

We reported that expression of the C2GnT is required for galectin-1 death of T cells (11). In the present work, C2GnT expression was sufficient to increase galectin-1 susceptibility of cell lines expressing CD45 (Fig. 5Go). In addition, all of the CD45+ human and murine cell lines that we have found to be susceptible to galectin-1 express the C2GnT (Ref. 32 ; J. T. Nguyen and L. G. Baum, unpublished data). In vivo, the T cell subsets that are susceptible to galectin-1, i.e., double positive thymocytes and activated peripheral T cells, also express the C2GnT (11, 32, 44, 45, 46, 47), indicating that susceptibility to galectin-1 is controlled by regulated glycosyltransferase expression during specific stages in T cell maturation. Moreover, we have previously shown that there is no clustering of CD45 after galectin-1 binding on mature human thymocytes that do not express the C2GnT (14, 32).

Increased expression of core 2 O-glycans on T cell-surface glycoproteins has been described in diseases such as Wiskott-Aldrich syndrome and HIV infection (48, 49). In vivo, aberrant T cell expression of core 2 O-glycans and increased susceptibility to galectin-1 may contribute to T cell depletion seen in these diseases. Intriguingly, Marth and coworkers (50) have found that increased expression of core 2 O-glycans on T cells from ST3Gal I-/- mice resulted in virtual absence of peripheral CD8+ T cells due to increased apoptosis.

Several T cell-surface glycoproteins may be potential substrates for the C2GnT, including CD43 and CD45 (12, 13, 51). The present study indicates that galectin-1-induced T cell death requires the presence of core 2 O-glycans on CD45. It is important to note that C2GnT expression is not absolutely required for binding of soluble CD45 to galectin-1, as CD45 from the BW5147 cells that do not express the C2GnT bound to a galectin-1 affinity matrix (Fig. 3GoC). In this case, galectin-1 may bind to other CD45 glycans that bear the lactosamine sequences (13, 34, 52, 53). However, the interaction of {theta}35 stromal cell galectin-1 with CD45 on T cells appeared markedly reduced on BW5147 cells lacking the C2GnT (Fig. 8Go). In contrast, colocalization of galectin-1 and CD45 was clearly evident on PhaR2.1 and BW5147/C2GnT cells expressing the C2GnT (Fig. 8Go). Thus, interaction of galectin-1 with T cell-surface receptors at the cell-cell interface is likely a complex process regulated by glycosylation, accessibility of saccharide ligands, protein-protein interactions, and other factors (53, 54).

Although the C2GnT can potentially modify a number of T cell-surface glycoproteins, addition of core 2 O-glycans to CD45 appears to be specifically required for galectin-1-induced death. Because the T200- and CD45- cell lines, which express neither CD45 nor the C2GnT, are susceptible to galectin-1, expression of the C2GnT is not absolutely essential for galectin-1 death. In addition, expression of the C2GnT in CD45- cells did not enhance susceptibility to galectin-1 (Fig. 5Go), indicating that addition of core 2 O-glycans to other galectin-1 receptors such as CD7 or CD43 did not affect susceptibility to galectin-1. Of note, the increased T cell death associated with increased core 2 O-glycan expression observed by Marth and coworkers (50) also did not appear to involve CD43. In this study, we have examined only the CD45R0 isoform. However, because different CD45 isoforms have been shown to regulate different aspects of T cell function (19, 20, 55), future studies will determine which CD45 isoforms can be modified by the C2GnT, and which CD45 isoforms bear core 2 O-glycans in vivo. In addition, our study suggests that it may be necessary to assess the functions of CD45 isoforms in cell lines that express a physiologically relevant complement of glycosyltransferases.

We propose that developmentally regulated expression of the C2GnT, e.g., in immature thymocytes or activated effector T cells, creates galectin-1 oligosaccharide ligands on CD45 that permit galectin-1 binding (14; Fig. 8Go), and subsequent triggering of the death pathway. Galectin-1-induced cell death apparently requires both a proapoptotic signal as well as removal of the CD45 anti-apoptotic signal, because the T200- and CD45- cell lines do not spontaneously undergo apoptosis in culture, but are specifically triggered to die after binding galectin-1. Tyrosine phosphorylation accompanies galectin-1-induced death (9), and inhibition of protein tyrosine-phosphatase activity augmented galectin-1-induced death of T cell lines (Fig. 3Go) and murine thymocytes (data not shown). Thus, the CD45 phosphatase may oppose the action of tyrosine kinases activated by galectin-1 binding (9, 34).

Fig. 9Go depicts a model of cell-surface glycoprotein interactions following galectin-1 binding, modified from our previous work demonstrating the segregation of CD45 following galectin-1 binding (14). In this model, segregation of CD45 would physically remove the CD45 phosphatase domains, an effect that may be mimicked by phosphatase inhibitors. Alternatively, cross-linking of CD45 by galectin-1 may directly inhibit phosphatase activity, as has been shown by Walzel et al. (17). Clustering of CD45 may facilitate autoinhibition of CD45-phosphatase activity via interaction of the inhibitory wedge and catalytic domains, as proposed by Weiss and colleagues (56). In addition, the second phosphatase domain of CD45 binds the cytoskeletal linker molecule fodrin (57); movement of the cytoplasmic domain of CD45 may disrupt cytoskeletal interactions and thus enhance cell death (58). Miceli and colleagues (59) have proposed that CD45 clustering by galectin-1 contributes to the modulation of TCR signal transduction that they have observed, although the role of core 2 O-glycans in this process remains to be addressed.



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  		FIGURE 9. Model of galectin-1 binding to T cell glycoprotein receptors in the presence and absence of C2GnT expression. A, In the absence of C2GnT expression, galectin-1 binds to other glycoproteins bearing lactosamine sequences recognized by galectin-1. However, this is not sufficient for clustering of CD45. B, The addition of core 2 O-glycans to CD45 facilitates galectin-1 binding and clustering of CD45. N-linked, core 1 O-linked, and core 2 O-linked glycans and galectin-1 are represented as described in the key. PP, phosphatase domains of CD45.

 
Although CD45 cross-linking alone can trigger death of T cell lines and thymocytes, it does not appear that galectin-1-induced death of cells expressing CD45 and the C2GnT occurs solely via CD45 cross-linking. PhaR2.1 cells treated with both galectin-1 and CD45 cross-linking demonstrated a level of cell death that was approximately the sum of the level of death seen with galectin-1 or CD45 cross-linking alone (J. T. Nguyen and L. G. Baum, unpublished data). Moreover, the T cell line HUT78, that expresses both CD45 and the C2GnT, but lacks the proapoptotic galectin-1 counterreceptor CD7, is resistant to galectin-1 death (15). In addition, CD45 cross-linking does not result in DNA cleavage in dying cells (34), while DNA cleavage has been demonstrated during galectin-1-induced cell death (7, 8, 60). Thus, the galectin-1 death pathway does not appear to be identical to the death pathway initiated by CD45 cross-linking.

Our initial report of galectin-1-induced cell death demonstrated that a CD45- cell line, HPB.45.0, was resistant togalectin-1, suggesting that CD45 was essential for galectin-1-induced cell death (7). Walzel et al. (17) reported similar results with a CD45- mutant of the Jurkat cell line. However, we have now demonstrated that CD45- cells are susceptible to galectin-1. Galectin-1-resistant CD45-deficient cell lines derived from galectin-1-susceptible parental HPB.ALL and Jurkat cell lines may have other defects in glycosylation (61, 62) or at downstream points in the galectin-1 signaling pathway that remain to be characterized. It is likely that there are multiple levels at which galectin-1 susceptibility is controlled, including regulated expression of glycosyltransferases, expression and association of glycoprotein counterreceptors, and integration of downstream components of the death pathway.


   Acknowledgments
 
We thank Robert Hyman, Kim Bottomly, Michael Pierce, and Ken Dorshkind for providing cell lines, Minoru Fukuda and Shigeru Tsuboi for murine C2GnT cDNA, Ayyappan Rajasekaran for assistance with confocal microscopy, and Janis V. Giorgi and the staff of the Flow Cytometry Core Facility at the Jonsson Comprehensive Cancer Center at the University of California (Los Angeles, CA). We also thank Moira E. Donnell and Kent Kwan for invaluable technical assistance, and Leland D. Powell, Marcus Horwitz, and C. Fred Brewer for helpful discussions and critical reading of the manuscript.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant AI40118, American Cancer Society Grant RPG 9704901IM, a Glycoscience Research Award from Neose Technologies (to L.G.B.), and National Institutes of Health Grant CA16042 to the Jonsson Comprehensive Cancer Center. M.G. was supported in part by the National Institutes of Health Training Grant CA09120-21. Back

2 Address correspondence and reprint requests to Dr. Linda G. Baum, Department of Pathology and Laboratory Medicine, University of California, Los Angeles, School of Medicine, 10833 Le Conte Avenue, Los Angeles, CA 90095-1732. E-mail address: lbaum{at}mednet.ucla.edu Back

3 Abbreviations used in this paper: C2GnT, core 2 {beta}-1,6-N-acetylglucosaminyltransferase; bpV(phen), potassium bisperoxo(1,10-phenanthroline)oxovanadate(V); PI, propidium iodide. Back

Received for publication June 8, 2001. Accepted for publication September 6, 2001.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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