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The Journal of Immunology, 2005, 174: 4051-4059.
Copyright © 2005 by The American Association of Immunologists

An Alternate Core 2 {beta}1,6-N-Acetylglucosaminyltransferase Selectively Contributes to P-Selectin Ligand Formation in Activated CD8 T Cells1

Jasmeen S. Merzaban*, Jonathan Zuccolo*, Stéphane Y. Corbel*, Michael J. Williams* and Hermann J. Ziltener2,*,{dagger}

* Biomedical Research Centre and {dagger} Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Core 2 {beta}1,6-N-acetylglucosaminyltransferase (C2GlcNAcT) synthesizes essential core 2 O-glycans on selectin ligands, which mediate cell-cell adhesion required for lymphocyte trafficking. Although gene-deletion studies have implicated C2GlcNAcT-I in controlling selectin ligand-mediated cell trafficking, little is known about the role of the two other core 2 isoenzymes, C2GlcNAcT-II and C2GlcNAcT-III. We show that C2GlcNAcT-I-independent P-selectin ligand formation occurs in activated C2GlcNAcT-Inull CD8 T cells. These CD8 T cells were capable of rolling under shear flow on immobilized P-selectin in a P-selectin glycoprotein ligand 1-dependent manner. RT-PCR analysis identified significant levels of C2GlcNAcT-III RNA, identifying this enzyme as a possible source of core 2 enzyme activity. Up-regulation of P-selectin ligand correlated with altered cell surface binding of the core 2-sensitive mAb 1B11, indicating that CD43 and CD45 are also physiological targets for this alternate C2GlcNAcT enzyme. Furthermore, C2GlcNAcT-I-independent P-selectin ligand induction was observed in an in vivo model. HYtg CD8 T cells from C2GlcNAcT-Inull donors transferred into male recipients expressed P-selectin ligand in response to male Ag, although at reduced levels compared with wild-type HYtg CD8 T cells. Our data demonstrate that multiple C2GlcNAcT enzymes can contribute to P-selectin ligand formation and may cooperate with C2GlcNAcT-I in the control of CD8 T cell trafficking.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Lymphocytes become activated upon contact with foreign Ags presented by APCs and subsequently travel through the blood to the site of inflammation. This trafficking of leukocytes is a complex process tightly controlled by a cascade of molecular interactions between endothelial cells and leukocytes. The selectin family of carbohydrate-binding proteins consists of three closely related cell surface molecules termed P-selectin, E-selectin, and L-selectin (reviewed in Ref. 1), which control the earliest steps of leukocyte interaction with blood vessel walls. Interactions of selectins with selectin ligands promote the capture of leukocytes from circulating blood, leading to leukocyte tethering and rolling on endothelial cells. The leukocyte then stops and emigrates through the endothelial cells in a process mediated by chemokines and integrins (2, 3, 4).

The best characterized selectin ligand is P-selectin glycoprotein ligand 1 (PSGL-1),3 a type I membrane protein with an extracellular mucin domain (5, 6, 7). Although all three selectins can interact with PSGL-1 (8, 9, 10, 11, 12), it is the primary ligand for P-selectin (13, 14, 15, 16, 17, 18, 19, 20, 21). PSGL-1 is expressed as a functional P-selectin ligand on myeloid cells and T cell subsets (22, 23, 24). The interaction of PSGL-1 with P-selectin requires expression of specific sialylated (25) and fucosylated core 2 O-glycans in addition to tyrosine sulfation (26, 27, 28, 29, 30, 31, 32) of the PSGL-1 backbone. Targeted gene deletion studies have demonstrated that both the {alpha}1-3-fucosyltransferase-VII (33, 34, 35) and the core 2 {beta}1,6-N-acetylglucosaminyltransferase (C2GlcNAcT)-I (36, 37, 38, 39) are essential for this interaction and are crucial to the recruitment process of cells.

C2GlcNAcT isoenzymes create the core 2 O-glycan branch (40) by adding GlcNAc to the Gal {beta}1-3 GalNAc core 1 structure expressed on serine or threonine residues. Capping of the core 2 branch with a sialyl LewisX moiety creates the P-selectin counter receptor. Three separate C2GlcNAcT enzymes, termed C2GlcNAcT-I, C2GlcNAcT-II, and C2GlcNAcT-III, have been described in humans (41, 42, 43, 44). C2GlcNAcT-I, the most studied of the three isoenzymes, is widely expressed, and its contribution to the biosynthesis of selectin ligands has been well established (36, 39, 45, 46). C2GlcNAcT-II is expressed in mucous epithelial cells, where it participates in mucin production (41, 42), and C2GlcNAcT-III is highly expressed in the thymus, possibly reflecting a unique role in T cell development (44). Mice with deficiencies in C2GlcNAcT-II or C2GlcNAcT-III have not yet been reported, whereas analyses of C2GlcNAcT-Inull mice have shown that it is essential for PSGL-1 modifications on neutrophils (36, 37, 39) consistent with in vitro studies (5, 26). Despite the essential role of C2GlcNAcT-I in P-selectin ligand formation, C2GlcNAcT-Inull mice possessed a relatively mild phenotype, showing only a partial reduction in selectin ligands and essentially no effect on lymphocyte homing (36), which may reflect redundancy of the C2GlcNAcT isoenzymes. Whether C2GlcNAcT-II and/or C2GlcNAcT-III contribute to selectin ligand formation and to cell trafficking has not yet been determined.

In the present study, we have sought to determine in vitro and in vivo cell activation conditions that stimulate P-selectin binding to PSGL-1 in the absence of C2GlcNAcT-I. We found that activated C2GlcNAcT-Inull CD8 T cells can form functional PSGL-1 recognized by P-selectin, whereas activated C2GlcNAcT-Inull CD4 T cells do not, implicating alternate C2GlcNAcTs in the biosynthesis of functional P-selectin ligand.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Mice aged 6–10 wk were used for analyses. C57BL/6 and PSGL-1null mice, originally from The Jackson Laboratory, were bred at the Biomedical Research Centre (University of British Columbia). C2GlcNAcT-Inull mice (36) were kindly supplied by Dr. J. Marth (Howard Hughes Medical Institute, University of California at San Diego, La Jolla, CA) and were backcrossed seven generations with C57BL/6 mice. The C2GlcNAcT-Inull mice were mated with CD43null mice (47, 48) to produce mice that lack both C2GlcNAcT-I and CD43. C2GlcNAcT-Inull mice were also mated with PSGL-1null mice to produce mice that lack both C2GlcNAcT-I and PSGL-1. HY TCR-transgenic mice on the C57BL/6 background (49) were crossed with C2GlcNAcT-Inull mice to yield mice with a HY+C2GlcNAcT-Inull genotype.

Media

Cell culture suspensions were prepared in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 8% FCS, 5 x 10–5 M 2-ME, 100 U/ml penicillin, 100 U/ml streptomycin (StemCell Technologies), and 2 mM glutamine (Sigma-Aldrich).

Abs and flow cytometry

Anti-CD43 mAb, 1B11-PE (09695A), was obtained from BD Pharmingen. Conditioned media from the anti-CD43 hybridoma line 1B11 (50) and from the anti-pan-CD43 hybridoma line S11 (kindly supplied by Dr. J. Kemp, Department of Pathology, University of Iowa, Iowa City, IA) (51) were used for Western blotting experiments. P-selectin-hIgG fusion protein (28111A; BD Pharmingen) and E-selectin-hIgG fusion protein (575-ES; R&D Systems) were detected with biotinylated anti-human IgG (109-065-098; Jackson ImmunoResearch Laboratories) and CyChrome-conjugated streptavidin (554062; BD Pharmingen) or just with anti-human IgG-PE (109-116-098; Jackson ImmunoResearch Laboratories). Rabbit polyclonal anti-peptide Ab H18 was raised against a peptide corresponding to the C terminus of CD43 and affinity purified on immobilized peptide (50). CD8{alpha}-allophycocyanin (553035; BD Pharmingen) and CD4-FITC (01064D; BD Pharmingen) were used for FACS staining. Neutralizing anti-PSGL-1 mAb, 2PH-1 (52), was kindly provided by Dr. D. Vestweber (University of Münster, Münster, Germany). For FACS staining, cells were suspended in DMEM (Invitrogen Life Technologies) supplemented with 8% FCS and incubated with Abs for 15–30 min on ice in 96-well round-bottom plates (163320; Nunc). Cells were washed twice and analyzed on a FACSCalibur flow cytometer (BD Biosciences). For negative controls, cells were either incubated with the neutralizing anti-PSGL-1 Ab 2PH-1 before P-selectin-hIg staining, or EDTA (5 mM) was added during staining with P-selectin-hIgG or E-selectin-hIgG to prevent binding. T3.70-biotin mAb specific for the HY-transgenic TCR-{alpha}-chain was biotinylated in our laboratory.

Western blotting

Cells were washed with PBS and lysed at 108 cells/ml on ice in buffer containing 20 mM Tris (pH 7.5), 0.15 M NaCl, 0.5% Triton X-100, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 174 µg/ml PMSF, and 5 µg/ml pepstatin. Lysates were rotated for 1 h at 4°C before centrifugation at 14,000 rpm for 5 min in an Eppendorf centrifuge 5402. Supernatants were then recovered for SDS-PAGE or used for immunoprecipitations. Abs were bound to protein G-coupled Sepharose beads (Pharmacia). After washing beads free of unbound Ab with PBS, cell lysates were mixed with beads and incubated at 4°C for 1–2 h. Immunoprecipitates were washed in TBS containing 0.1% Tween 20 (Fisher Scientific), and an immunoprecipitated protein was extracted from beads with Laemmli loading buffer. 2-ME was added to reduce the Ab where necessary, and samples were then loaded for SDS-PAGE with a 4% stacking gel and 8% resolving gel. Resolved protein was transferred to PROTRAN-BA85 nitrocellulose membrane (Schleicher & Schuell Microscience) that was subsequently blocked with 5% BSA. Blots were probed with Abs diluted in TBS containing 0.5% BSA and 0.5% Tween for 60 min. Blots were washed in TBS with 0.1% Tween and were detected with goat anti-mouse Ig-HRP or goat anti-rabbit HRP where appropriate (Invitrogen Life Technologies), washed, and developed with ECL reagent (Amersham Biosciences) for autoradiography with Biomax film (Eastman Kodak), according to the manufacturer’s instructions. Molecular mass standards in the high range were used (Bio-Rad).

Lymphocyte cultures

Splenocytes were cultured in culture plates (08-757-13 and 08-757-13A; BD Biosciences) at 106 cells/ml in RPMI 1640 medium, 8% FCS, and stimulated with 4 µg/ml Con A (C-0412; Sigma-Aldrich) and IL-2 for 48 h at 37°C in 5% CO2. Cells were then harvested, washed, counted, and replated at various secondary cell culture densities. Cells were cultured at various densities with 2% IL-2 supernatant obtained as conditioned medium from the myeloma X.653 transfected with the cDNA for murine IL-2 (F. Melchers, Basel Institute of Immunology, Basel, Switzerland).

Core 2 and core 4 enzymatic assays

Cells were washed in PBS and lysed in 150 mM NaCl and 0.25% Triton X-100 with protease inhibitors (10 µg/ml soybean trypsin inhibitor, 40 µg/ml phenylmethyl-sulfonylfluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 5 µg/ml pepstatin) at 4°C. Transferase assays were performed in triplicate according to established protocols (43, 53). The reaction mixtures for the C2GlcNAcT assay contained 50 mM MES (pH 7.0), 0.5 µCi of uridine diphosphate-N-acetyl-D-glucosamine (glucosamine-6-[3H](N)) (NEN), 1 mM uridine 5'-diphospho-N-acetylglucosamine (Sigma-Aldrich), 0.1 M GlcNAc, 1 mM acceptor, and 25 µl of cell lysate in a total volume of 50 µl. The acceptor substrates were Gal {beta}1-3GalNAc{alpha}-pNp and GlcNAc{beta}1-3GalNAc{alpha}-pNp (Toronto Research Chemicals) for the core 2 and core 4 enzyme activity assays, respectively. Negative control samples were run for each assay with all the reactants in the absence of acceptor substrates to determine the background signal for normalization purposes. The mixtures were incubated for 3 h at 37°C, processed by C18 Sep-Pak (Waters) column chromatography, and eluates were counted in a scintillation counter.

Real-time RT-PCR

Activated splenocytes underwent positive selection to enrich for CD8 T cells before RNA extraction using the AutoMACS cell separation system (Miltenyi Biotec). Activated C2GlcNAcT-Inull and C57BL/6 T cell cultures were stained for CD8 using CD8-PE (BD Pharmingen), washed, and resuspended in 80 µl of buffer (0.5% BSA and 5 mM EDTA in PBS) per 1 x 107 total cells. Twenty microliters of MACS anti-PE microbeads (Miltenyi Biotec) were added per 1 x 107 total cells, and the suspension was incubated for 15 min at 4°C, washed, and finally resuspended in 500 µl of buffer per 1 x 108 total cells before sorting on the AutoMACS. RNA was extracted from CD8 T cells (>85% purity) using TRIzol Reagent (Invitrogen Life Technologies), according to the manufacturer’s instructions. Reverse transcription was performed with ~1 µg of DNase-treated total RNA using Superscript (Invitrogen Life Technologies) in a 20-µl volume. The reverse transcription reaction was then diluted 20-fold. Fifteen microliters of a LightCycler Reaction Mastermix containing 9 µl of water, 1 µl of forward primer (0.5 µM), 1 µl of reverse primer (0.5 µM), and 4 µl of LightCycler FastStart DNA Master Plus SYBR Green I (Roche Diagnostic Systems) and 5 µl of cDNA, added as a PCR template, were mixed and transferred into the LightCycler glass capillaries. The LightCycler experimental run protocol used was as follows: denaturation program (95°C for 5 min), amplification, quantification program repeated 40 times (95°C for 10 s, 55°C for 10 s, and 72°C for 30 s with a single fluorescent measurement), melting curve program (60°C to 95°C with a heating rate of 0.2°C/s and a continuous fluorescent measurement), and finally a cooling step to 40°C. Transcription levels of different mRNAs were determined by preparing a standard curve for each of the genes of interest and using hypoxanthine phosphoribosyltransferase (HPRT) as an internal reference gene to normalize the results. PCR primers for the core 2 isoenzymes were purchase from Qiagen, optimized to an annealing temperature of 55°C and were as follows: C2GlcNAcT-I (EMBL accession no. D87332), forward primer, 5'-GAAGGACCTGTACAGAATGAATG-3', and reverse primer, 5'-ACTTGTTGCTTGAGGGGAAAGAA-3'; C2GlcNAcT-II (NCBI accession no. NM028087), forward primer, 5'-CTTGCTTCAGAGCCCCGTGCC-3', and reverse primer, 5'-GTTGCCGGGCTTTTGAGTTACTG-3'; and C2GlcNAcT-III (Ensembl accession no. ENSMUST00000049586), forward primer, 5'-CCTCCTCAAGTCTTCCGTTCAG-3', and reverse primer, 5'-GGAGACGTTCGTCTTTACTGG-3'. PCR primers for HPRT (EMBL accession no. BC004686) were exon 7 forward primer, 5'-CTCGAAGTGTTGGATACAGG-3', and HPRT exon 9 reverse primer, 5'-TGGCCTATAGGCTCATAGTG-3' (Invitrogen Life Technologies), and were specific for separate exons to be able to detect potential contaminating genomic DNA. Genomic contamination was tested by performing HPRT-specific PCR on DNase-treated RNA samples for presence of a 1100-bp product that would have indicated that genomic DNA had resisted the DNase treatment. Duplicate samples that had no cDNA added were included with each batch of PCR to check for external contamination. Data analysis was performed with the relative expression software tool using the HPRT as the internal reference gene (54). This software package compares the levels of transcript present in different tissue samples and performs a pairwise-fixed reallocation randomization test to determine the statistical significance of the changes in transcript level. In addition, the PCR product was checked for size by gel electrophoresis on a 1.5% agarose Tris Borate EDTA gel and finally sequenced to verify the identity of the PCR products.

Rolling assays

Activated CD8 T cell interactions with immobilized P-selectin and E-selectin under physiologic flow conditions were assessed using an in vitro flow chamber, according to established protocols (55, 56). Petri dishes (catalog no. 430588; 35-mm Corning Cell Culture Dishes) were coated with P-selectin-hIg or E-selectin hIgG (5 µg/ml) in PBS. After overnight incubation at 4°C, the coated spot was rinsed four times with PBS and blocked with 1 ml of 1% BSA in HBSS for 1 h at 37°C. The substrate-coated petri dishes were mounted in a parallel plate flow chamber. For most experiments, HBSS was drawn through the chamber at a shear stress of 2.0 dyne/cm2 (0.55 ml/min) with a syringe pump (Harvard Apparatus). Activated CD8 T cells (106/ml) were suspended in DMEM with or without EDTA (5 mM) and then perfused through the chamber for a 6-min period. Cell rolling was observed using an inverted phase contrast microscope (Zeiss) and was videotaped using an Exwave HAD color video camera (model SSC-DC50A; Sony) with a Super VHS ET video recorder (model HR-S9911U; JVC) and an attached time-date ID generator (CREST Electronics) at x10 objective. Multiple random fields were recorded for at least 20 s at the end of the 4-min perfusion period. The total number of rolling cells within each 0.16-mm2 field was determined by analyzing the videotapes, with a minimum of five fields analyzed for each experiment. Stable adhesion was defined as attachment without movement for a minimum of 10 s. Assays were run at least five times for each cell type studied.

Statistical analysis

Student’s t test was used to determine the significance of differences between sample means. For flow chamber assays, the unpaired Student’s t test was used (Microsoft Excel). Values of p < 0.05 were considered significant. Data are presented as mean ± SEM.

In vivo HY stimulation

Thymocytes from TCR-transgenic HY female mice (HYtg wild-type, HYtgC2GlcNAcT-Inull, or HYtgCD43nullC2GlcNAcT-Inull) (3 x 107 cells) were treated with 5 µM CFSE (Molecular Probes) and adoptively transferred into normal male or female C57BL/6 recipient mice. After 45 or 72 h, spleen cells were harvested and stained for CD8, T3.70, 1B11, and P-selectin-hIg. The presence of TCR-transgenic cells was assessed by flow cytometric analysis based on CFSE and T3.70 signals.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
P-selectin binds PSGL-1 on Con A-activated T cells from C2GlcNAcT-Inull mice

Mice lacking C2GlcNAcT-I were shown previously to exhibit a defect in leukocyte recruitment to sites of inflammation due to a deficiency in P-selectin ligand formation in neutrophils (36) and in activated CD4 T cells (37). In an attempt to search for functional PSGL-1 expression and hence P-selectin binding in the absence of C2GlcNAcT-I on T cells, different in vitro culture stimulation conditions were tested. Previous studies in our laboratory using wild-type C57BL/6 mice had shown that cell culture densities ranging from 0.05 x 106 to 0.25 x 106 cells/ml significantly affected the level of P-selectin ligand formation in activated splenocytes, with cells kept at the highest densities having the highest P-selectin binding (57). Splenocytes from C2GlcNAcT-Inull mice were activated with Con A for 48 h and then cultured in IL-2 for an additional 48 h under varying high-density culture conditions, ranging from 0.5 x 106 to 2 x 106 cells/ml. Our experiments revealed that cells cultured under these very high densities exhibited significant P-selectin binding (Fig. 1A). Time course studies demonstrated that there was a tight temporal control of P-selectin ligand formation with significant P-selectin binding on days 3 and 4 and loss of binding by day 5 (Fig. 1B). P-selectin binding was shown to be specific for PSGL-1 because binding could be inhibited with the neutralizing {alpha}-PSGL-1 Ab 2PH-1 (Fig. 1B) or by treatment with EDTA (Fig. 1B). Furthermore, there was no detectable P-selectin ligand on PSGL-1null splenocytes activated under high-density conditions (Fig. 1E). Expression of PSGL-1 was comparable on activated cells from C57BL/6 and C2GlcNAcT-Inull mice at all cell culture densities tested (Fig. 1C).



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  		FIGURE 1. P-selectin ligand formation in Con A-activated C2GlcNAcT-Inull splenocytes is cell culture density dependent. A, P-selectin ligand expression (thick line) on mitogen-stimulated C2GlcNAcT-Inull spleen cells maintained at culture densities of 0.5 x 106, 1.0 x 106, and 2.0 x 106 cells/ml. B, P-selectin ligand expression on mitogen-stimulated C2GlcNAcT-Inull spleen cells maintained for 3, 4, and 5 days (2.0 x 106 cells/ml, thick line); P-selectin-hIgG binding is blocked by the anti-PSGL-1 mAb 2PH-1 (thin line). C, PSGL-1 expression on day 4-activated C57BL/6 and C2GlcNAcT-Inull splenocytes cultured at 0.5 x 106 cells/ml (dashed line), 1.0 x 106 cells/ml (thin line), or 2.0 x 106 cells/ml (thick line). D, E-selectin-hIgG binding to day 4-activated splenocytes cultured at either 0.25 x 106 cells/ml (thin line) or 2.0 x 106 cells/ml (thick line) is shown for C57BL/6 and in C2GlcNAcT-Inull cells. E, P-selectin-hIgG and E-selectin-hIgG binding on activated PSGL-1null splenocytes cultured at either 0.25 x 106 cells/ml (thin line) or 2.0 x 106 cells/ml (thick line). A total of 5 mM EDTA (dashed line) was added during incubation with P-selectin-hIgG and E-selectin-hIgG as a negative control in all experiments.

 
E-selectin can bind to cells in a PSGL-1- and C2GlcNAcT-I-dependent fashion (12, 26, 36, 37, 39), as well as in a PSGL-1-independent fashion via alternate E-selectin ligands (13, 58, 59, 60). Consistent with this, we observed high levels of E-selectin binding on activated wild-type cells, while binding on activated C2GlcNAcT-Inull (Fig. 1D) cells was reduced significantly. In contrast to P-selectin ligand expression, we found that expression of E-selectin ligand on C2GlcNAcT-Inull cells was not influenced by cell culture density (Fig. 1D). The level of E-selectin binding on activated cells from PSGL-1null and C2GlcNAcT-Inull mice was also comparable (Fig. 1, D and E), indicating that E-selectin binding observed in the absence of C2GlcNAcT-I is PSGL-1 independent. These data indicate that P-selectin binds activated C2GlcNAcT-Inull cells in a PSGL-1-dependent manner, whereas E-selectin binds in a PSGL-1-independent manner.

C2GlcNAcT-I and C2GlcNAcT-III RNA is expressed in activated splenocytes

Core 2 O-glycan branching is required for binding of P-selectin to PSGL-1 (26, 36, 46); thus, activity of an alternate C2GlcNAcT isoenzyme is the likely cause for P-selectin ligand formation observed in C2GlcNAcT-Inull splenocytes. In the human system, two additional enzymes capable of catalyzing core 2 O-glycan structures have been identified, expressed, and characterized (41, 42, 44). A database search identified the murine orthologs of C2GlcNAcT-II and C2GlcNAcT-III shown in Fig. 2. There is significant amino acid sequence homology among the three mouse C2GlcNAcT isoenzymes especially evident in the C-terminal catalytic domain of these enzymes.



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  		FIGURE 2. Murine C2GlcNAcT-I, C2GlcNAcT-II, and C2GlcNAcT-III show high degrees of homology. Multiple amino acid sequence analysis (ClustalW) of the murine C2GlcNAcT is shown. Dark gray regions depict identity and light gray regions depict similarity.

 
Real-time RT-PCR was conducted to identify and quantify levels of C2GlcNAcT isoenzyme RNA in activated wild-type and C2GlcNAcT-Inull splenocytes. Our analysis revealed, as expected, significant levels of C2GlcNAcT-I RNA in activated T cells from wild-type control mice, whereas no signal was detected in C2GlcNAcT-Inull T cells (Fig. 3A). C2GlcNAcT-II RNA was absent in all splenocyte samples tested, consistent with data for human C2GlcNAcT-II described to be present in mucosal tissue and absent in spleen (41). The only core 2 isoenzyme detected in activated C2GlcNAcT-Inull T cells was C2GlcNAcT-III (Fig. 3A). RNA for this enzyme was detected at comparable levels in activated T cells from both wild-type and C2GlcNAcT-Inull mice (Fig. 3B), implicating C2GlcNAcT-III as the most likely source of residual core 2 activity in C2GlcNAcT-Inull T cells.



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  		FIGURE 3. RT-PCR demonstrates expression of C2GlcNAcT-I and C2GlcNAcT-III RNA in activated CD8 T cells. A, RT-PCR was conducted for C2GlcNAcT-I (lane 1), C2GlcNAcT-II (lane 2), and C2GlcNAcT-III (lane 3) from RNA isolated on day 3 from CD8 T cell-enriched C2GlcNAcT-Inull or C57BL/6 high-density (HD; 2.0 x 106 cells/ml) and low-density (LD; 0.25 x 106 cells/ml) cultures. RT-PCR products were separated on an agarose gel; DNA ladder is standard (L). No cDNA controls were performed as described in Materials and Methods. B, Comparisons of relative C2GlcNAcT-III RNA expression levels were determined by real-time RT-PCR. Cells from HD and LD splenocyte cultures are compared among and between C2GlcNAcT-Inull (C2) and wild-type C57BL/6 (WT).

 
Increased core 2 O-glycan formation on CD43 and CD45RB correlates with increased P-selectin ligand formation

To confirm that core 2 enzyme activity correlates with P-selectin ligand formation, we used several approaches. Core 2 enzyme activity was measured by a standard enzyme assay using lysates from activated C2GlcNAcT-Inull cells. Activated splenocytes cultured at high density had significantly more core 2 activity than corresponding cells cultured at low density for both wild-type and C2GlcNAcT-Inull cells. As expected, C2GlcNAcT-Inull cells expressed lower core 2 activity than wild-type cells (Fig. 4A).



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  		FIGURE 4. High-density (HD) cultured C2GlcNAcT-Inull Con A blasts express significant core 2 but not core 4 activity. Lysates from HD (2.0 x 106 cells/ml) and low-density (LD; 0.25 x 106 cells/ml) cultured C57BL/6 or C2GlcNAcT-Inull splenocytes were tested for core 2 enzyme activity (A). Lysates from HD cultures were also tested for core 4 enzyme activity (B). Intestinal tissue lysate from a C57BL/6 mouse was used as a positive control for the core 4 assay. Values were normalized against background levels obtained from assays done on samples in the absence of the acceptor substrates as outlined in Materials and Methods. CD43 precipitated from day 4-activated C2GlcNAcT-Inull splenocytes grown at 0.5 x 106, 1.0 x 106, or 2.0 x 106 cells/ml was immunoblotted with a pan-CD43 mAb, S11, and the core 2-dependent mAb, 1B11. Activated splenocytes and naive lymph node cells (LNC) from wild-type C57BL/6 (B6) were run in parallel as controls (C).

 
C2GlcNAcT-II has been shown to have core 2 as well as core 4 O-glycan branching activity (41, 42), whereas C2GlcNAcT-III is specific for core 2 O-glycan branching (44). Additionally, to exclude the possibility that C2GlcNAcT-II is responsible for the residual core 2 activity, lysates were tested for core 4 enzyme activity using GlcNAc{beta}1-3GalNAc{alpha}-pNp as an acceptor substrate. No core 4 activity was found in these lysates, whereas, as expected, lysates of intestinal tissue displayed significant core 4 activity (Fig. 4B). Thus, lack of core 4 activity in splenocyte lysates supports the RNA results above that the residual core 2 enzyme activity in C2GlcNAcT-Inull CD8 splenocytes is not due to the C2GlcNAcT-II isoenzyme.

CD43 is a well-established substrate of C2GlcNAcT-I (61, 62) and ectopic expression of the two human core 2 isoenzymes, C2GlcNAcT-II and C2GlcNAcT-III, has been shown to increase CD43 O-glycan branching (41, 44), while this has not yet been shown for the murine orthologs. To determine whether the residual core 2 activity observed in C2GlcNAcT-Inull cells targets physiological substrates other than PSGL-1, we evaluated properties of CD43 with regard to changes in its electrophoretic mobility and to reactivity with the core 2 glycosylation-sensitive anti-CD43 mAb 1B11 (50, 63, 64). CD43 immunoprecipitated from cultures of activated wild-type and C2GlcNAcT-Inull splenocytes with the pan-CD43-specific Ab H18 can be detected with the pan-CD43 mAb S11 in lysates of unstimulated wild-type and C2GlcNAcT-Inull lymph node cells, whereas mAb 1B11 reactive core 2-branched CD43 is virtually absent (Fig. 4C). As expected, mAb 1B11-reactive 130-kDa CD43 was strongly detected in activated wild-type cells. CD43 precipitated from activated C2GlcNAcT-Inull cells cultured at 0.5 x 106 cells/ml displayed a relatively modest 1B11 signal, whereas a significant increase in the 1B11-CD43 signal was observed with CD43 precipitated from cultures kept at densities of 1.0 x 106 and 2.0 x 106 cells/ml.

mAb 1B11 has dual specificity for core 2 O-glycan-dependent epitopes on both CD43 and CD45RB. Core 2 O-glycans are required for 1B11 recognition of the CD43 epitope, whereas 1B11 recognition of a CD45RB epitope, exclusively expressed on CD8 T cells, indicates lack of core 2 O-glycans (64). On CD43+ CD8 T cells, 1B11 reactivity with CD45RB is obscured by reactivity with CD43. Three-color analysis of activated CD43+C57BL/6 and CD43+/C2GlcNAcT-Inull splenocytes shows that P-selectin-positive cells have an increased 1B11 signal (Fig. 5, A and B). On CD43null CD8 T cells, 1B11 reactivity is exclusively CD45RB dependent, and three-color analysis of activated CD43null/C2GlcNAcT-Inull splenocytes shows that P-selectin-positive cells have a decreased 1B11-CD45RB signal (Fig. 5C) (i.e., an increased C2GlcNAcT enzyme activity). This data collectively suggests that the residual core 2 activity detected in these C2GlcNAcT-Inull splenocytes targets CD43 and CD45 in addition to PSGL-1.



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  		FIGURE 5. CD43 and CD45 on C2GlcNAcT-Inull CD8 T cells are modified by an alternate C2GlcNAcT enzyme. Spleen cells prepared from C57BL/6 (A), C2GlcNAcT-Inull (B), and CD43null/C2GlcNAcT-Inull (C) mice were activated as outlined in Materials and Methods and harvested on day 4. A three-color flow cytometric analysis was performed by staining for CD8, P-selectin-hIg, and 1B11 to determine both P-selectin ligand expression and the glycosylation status of CD43 (1B11-CD43) and CD45 (1B11-CD45) on CD8 T cells.

 
P-selectin ligand formation in Con A-activated C2GlcNAcT-Inull cells is restricted to CD8 T cells

Previous data had indicated that C2GlcNAcT-I expression is essential for generating ligands for P-selectin in primary CD4+ T cells. Snapp et al. (37) measured rolling of activated CD4+ T cells from C2GlcNAcT-Inull and wild-type mice on P-selectin. No rolling was observed with C2GlcNAcT-Inull CD4 T cells. Given that Con A-mitogenic stimulation of lymphocytes results in cultures that predominantly contain CD8+ T cells, we examined whether P-selectin ligand formation was restricted to CD8+ T cells. To compare P-selectin ligand formation on CD4+ against CD8+ cells in our cultures, we stained for both markers along with P-selectin. As shown in Fig. 6A, CD8+ T cells displayed significant P-selectin binding, whereas CD4+ T cells showed only weak P-selectin staining. 1B11-CD43 staining, an indicator of core 2 enzyme activity, was consistent with the P-selectin ligand expression pattern. Activated C2GlcNAcT-Inull CD4 T cells show only a modest 1B11 signal and correspondingly have low C2GlcNAcT activity, whereas activated C2GlcNAcT-Inull CD8 T cells show significant 1B11-CD43 reactivity, indicating considerable core 2 activity (Fig. 6B).



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  		FIGURE 6. P-selectin ligands are predominantly induced in C2GlcNAcT-InullCD8+ T cells. A, FACS analysis was performed on activated C2GlcNAcT-Inull spleen cells to determine P-selectin ligand (thick line) on CD8 and CD4 T cell subsets. Binding of P-selectin-hIg was inhibited with either EDTA (dotted line) or anti-PSGL-1 Ab 2PH-1 (thin line). B, 1B11 binding of CD43 (1B11-CD43) was determined on CD8 and CD4 subsets from C2GlcNAcT-Inull (shaded graph)- and wild-type (thick line)-activated spleen cells. Isotype control staining for 1B11 is shown in the dashed line.

 
Activated C2GlcNAcT-Inull cells roll on immobilized P-selectin in a PSGL-1-dependent manner

We tested the ability of activated C2GlcNAcT-Inull CD8 T cells to roll on P-selectin using an attachment and rolling assay under defined shear flow in vitro and found a direct correlation between the level of P-selectin binding and the capacity of cells to roll on P-selectin. Activated C2GlcNAcT-Inull T cells, from cultures grown at high density, attach and roll on immobilized P-selectin-hIg at 2.0 dyne/cm2, whereas attachment and rolling observed for cells activated under lower density conditions were reduced significantly (Fig. 7A). The number of rolling C2GlcNAcT-Inull cells was approximately half the number of rolling wild-type C57BL/6 cells. Addition of EDTA to this system completely abolished all rolling, reflecting the bivalent cation dependency of the rolling process (data not shown). As expected, activated T cells from PSGL-1null mice showed no rolling on immobilized P-selectin, demonstrating its dependence on PSGL-1. As an additional control, cells from mice with genetic deletions in both PSGL-1 and C2GlcNAcT-I were tested to eliminate the possibility that alternate P-selectin ligands are up-regulated in response to the C2GlcNAcT-I deletion. Cells from these double-deficient mice also showed no rolling on immobilized P-selectin. These data emphasize that activated T cells can roll in a PSGL-1-dependent manner on immobilized P-selectin in the absence of C2GlcNAcT-1.



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  		FIGURE 7. Activated C2GlcNAcT-Inull T cells roll on immobilized P-selectin and E-selectin. A, Splenocytes from C57BL/6, C2GlcNAcT-1null, PSGL-1null, and C2GlcNAcT-1null/PSGL-1null mice activated at low density (0.25 x 106 cells/ml; {square}) or high density (2.0 x 106 cells/ml; {blacksquare}) were perfused through an in vitro flow chamber over immobilized P-selectin at a shear force of 2 dyne/cm2. Data are expressed as the mean number of rolling cells/mm2 ± SEM. B, Splenocytes from C57BL/6, C2GlcNAcT-1null, PSGL-1null, and C2GlcNAcT-1null/PSGL-1null mice activated at low density ({square}) or high density ({blacksquare}) were perfused through an in vitro flow chamber over immobilized E-selectin at a shear force of 2 dyne/cm2. Data are expressed as the mean number of rolling cells/mm2 ± SEM. Differences between results for C2GlcNAcT-Inull cells cultured at low density vs high density, as well as wild-type cells cultured at low density vs high density, were found to be significant (*, p < 0.05) for P-selectin experiments.

 
Activated C2GlcNAcT-Inull cells roll on immobilized E-selectin in a PSGL-1-independent, core 2-independent manner

Activated C2GlcNAcT-Inull splenocytes were also tested for rolling on E-selectin using an attachment and rolling assay under defined shear flow in vitro. Activated C2GlcNAcT-Inull T cells from high- and low-density cultures displayed identical rolling on immobilized E-selectin-hIg at 2.0 dyne/cm2 (Fig. 7B). This rolling interaction was independent of both PSGL-1 and C2GlcNAcT-I because cells from mice double deficient in C2GlcNAcT-I and PSGL-1 adhered and rolled equally on immobilized E-selectin.

In vivo TCR signaling induces P-selectin ligand formation in C2GlcNAcT-Inull CD8 T cells

To determine whether P-selectin ligand formation in C2GlcNAcT-Inull CD8 T cells occurs under in vivo stimulation conditions, we compared CD8 T cell responses from C2GlcNAcT-Inull and wild-type control mice transgenic for the {alpha}{beta}TCR specific for male Ags (HY) presented by the class I MHC molecule H-2Db (19). CFSE-labeled HYtg CD8 T cells were transferred from female donors into male recipients, resulting in TCR activation of the transferred CD8 T cells due to stimulation by endemic male Ag. We analyzed CD8 T cell responses 3 days after transfer. HYtg wild-type and HYtgC2GlcNAcT-Inull CD8 T cells were compared for P-selectin ligand formation and for their respective proliferation rates by FACS. As illustrated in Fig. 8A, there was a strong increase in P-selectin ligand formation in wild-type HYtg CD8 T cells transferred into male recipients, whereas the same cells transferred into female control recipients were unresponsive. There was also a notable level of P-selectin binding on HYtgC2GlcNAcT-Inull CD8 T cells transferred into male recipients, as compared with control female recipients; nevertheless, this increase was less pronounced than that observed for HYtg wild-type CD8 T cells. To assess the level of core 2 enzyme activity in transferred HYtg CD8 T cells, we measured the 1B11-CD45 status on CD8 T cells from female HYtgCD43null/C2GlcNAcT-Inull mice (Fig. 8B). There was, as expected, a comparable P-selectin ligand induction on these cells as observed with HYtgC2GlcNAcT-Inull CD8 T cells. Analysis of the 1B11-CD45RB epitope on the CD43null background shows a decreased 1B11 signal and hence increased C2GlcNAcT activity on cells transferred into male recipients, confirming that core 2 enzyme activity is also induced in the HYtgCD43null/C2GlcNAcT-Inull cells. CFSE dilution assessed 45 h after cell transfer showed that the proliferative response of C2GlcNAcT-Inull and wild-type HYtg CD8 T cells was equal, indicating that lack of C2GlcNAcT-I did not affect the proliferative T cell response (Fig. 8C).



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  		FIGURE 8. In vivo activated C2GlcNAcT-Inull CD8 T cells can form functional P-selectin ligand. CFSE-labeled thymocytes from TCR-tg HY (HYtg) female mice (wild type (WT) or C2GlcNAcT-Inull) were adoptively transferred into male (shaded graph) or female (black line) C57BL/6 recipients. Seventy-two hours later, spleens were harvested and stained for CD8, T3.70, and P-selectin-hIgG (A). As a control, EDTA was used to abrogate binding of P-selectin (dotted line). B, Thymocytes from HYtgCD43null/C2GlcNAcT-Inull mice were adoptively transferred into male (shaded graph) or female (black line) C57BL/6 recipients 72 h before flow cytometric analysis of harvested spleens with CD8, T3.70, P-selectin, and 1B11. C, The proliferative response was monitored by analyzing the CFSE dilution of injected HYtgWT or HYtgC2GlcNAcT-Inull cells 45 h after injection into male (shaded graph) and female (dashed line) recipients.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The key finding reported in this article is that PSGL-1 can serve as a functional P-selectin ligand in the absence of the C2GlcNAcT-I enzyme and that the biosynthesis of P-selectin ligand in CD8+ T cells is controlled by at least two different C2GlcNAcT isoenzymes.

Previous studies of C2GlcNAcT-Inull mice (36, 37, 39) have revealed that the lack of this enzyme results in varying deficiencies in the formation of functional ligands recognized by each of the three selectins. Lack of C2GlcNAcT-I caused only a partial loss of E-selectin ligands on neutrophils (36, 37, 39) and of L-selectin ligands on endothelial cells (45). In contrast, studies that were focused mainly on neutrophils and CD4+ T cells showed that P-selectin-dependent cell adhesion was almost exclusively dependent on this enzyme (36, 37, 39). Our analyses of these mice now show that in activated CD8 T cells, P-selectin ligand can be expressed independent of C2GlcNAcT-I. Use of anti-PSGL-1 Ab and of PSGL-1null mouse-derived cells has identified the C2GlcNAcT-I-independent P-selectin ligand as PSGL-1, excluding the possibility that P-selectin recognizes an alternate ligand.

Induction of P-selectin ligand on CD8 T cells was not easily noticeable because T cell culture conditions that are normally used for splenocyte activation do not result in P-selectin ligand formation (57). Only when 5- to 10-fold higher cell culture densities were applied was there a reproducible induction of P-selectin ligand. Furthermore, expression of P-selectin ligand appears to be under tight temporal control; cells analyzed at days 3 and 4 after mitogen stimulation showed significant binding, whereas cells outside this temporal window did not bind P-selectin. Because such extreme in vitro culture conditions were required to induce functional P-selectin ligand, we looked for more physiological conditions. For this, we analyzed and compared the in vivo responses of HYtg CD8 T cells from C2GlcNAcT-Inull and wild-type female donors that were transferred into male recipients. Both types of HYtg CD8 T cells responded with a strong proliferative response, and there was a significant induction of P-selectin ligand in HYtgC2GlcNAcT-Inull CD8 T cells, albeit at a lower level than observed for HYtg wild-type CD8 T cells. Thus, our in vivo data demonstrate that physiological stimulation of CD8 T cells is sufficient to induce P-selectin ligand formation in the absence of C2GlcNAcT-I.

The murine genome encodes for three C2GlcNAcT isoenzymes that share considerable homology as has been described earlier for the human system (41, 42, 44). Given that P-selectin binding is believed to be wholly dependent on core 2 O-glycan formation (10, 65), activity of one of the two other core 2 isoenzymes is the most likely cause for the observed P-selectin ligand formation. Based on the RT-PCR data, C2GlcNAcT-II can be excluded as the source of the core 2 activity, whereas presence of RNA for the C2GlcNAcT-I and C2GlcNAcT-III isoenzymes in activated splenocytes point to C2GlcNAcT-III as the alternate core 2 isoenzyme able to support P-selectin ligand formation. These findings are consistent with the tissue distribution that was established for the human core 2 isoenzymes: RNA for hC2GlcNAcT-II was found to be associated with digestive tract but not lymphoid tissues (41, 42), and RNA for hC2GlcNAcT-III was found predominantly in thymus (44).

C2GlcNAcT-III RNA levels determined by real-time RT-PCR in C2GlcNAcT-Inull splenocytes were found to be comparable to RNA levels detected in wild-type cells, indicating that the induction of the C2GlcNAcT-III isoenzyme in C2GlcNAcT-Inull cells is not a compensatory consequence of the loss of C2GlcNAcT-I. Thus, it is likely that at least in activated CD8 T cells both enzymes may contribute to O-glycan branch formation. Interestingly, real-time RT-PCR analysis indicated that the RNA levels for both C2GlcNAcT-I and C2GlcNAcT-III did not change significantly between the high-density and low-density cultures analyzed here. However, C2GlcNAcT activity assays of lysates from high- and low-density cultures do show a significant difference in activity, although at the RNA level, similar amounts of transcript were found. This may suggest that other glycosyltransferases required for P-selectin ligand formation are the limiting factor or, alternatively, that core 2 enzyme activity may be subject to translational or posttranslational control under these cell culture conditions.

Rolling assays in a parallel flow chamber confirmed that the P-selectin ligand expressed on C2GlcNAcT-Inull CD8 T cells was functional. In contrast to P-selectin binding and P-selectin-dependent rolling, neither E-selectin binding nor E-selectin-dependent rolling was altered between high- vs low-density stimulated C2GlcNAcT-Inull splenocytes, indicating that this alternate core 2 enzyme may not support E-selectin ligand formation in contrast to C2GlcNAcT-I (Fig. 1D). Our findings also support data indicating that E-selectin ligands other than PSGL-1 can mediate E-selectin-dependent rolling (13, 66) and that these ligands do not depend on C2GlcNAcTs.

CD43 has long been recognized as a major substrate for C2GlcNAcT-I (53, 67), and ectopic expression of the human core 2 enzymes in Chinese hamster ovary cells revealed that CD43 can serve as a substrate for all three enzymes (41, 44). We confirm and extend these findings to the murine cells analyzed here. Earlier we demonstrated that expression of C2GlcNAcT-I in murine T cells results in increased O-glycan branching on several cell surface molecules, including CD43 and CD45 (62, 64). Our data here demonstrate that the alternate C2GlcNAcT enzyme induced in activated CD8 T cells can, in addition to PSGL-1, also use the leukocyte mucins CD43 and CD45 as acceptor glycoproteins. Thus, it has overlapping substrate specificity with C2GlcNAcT-I that likely extends to other glycoproteins.

Analysis of T cell subsets revealed that C2GlcNAcT-I-independent P-selectin ligand formation was observed primarily in Con A-stimulated CD8 T cells maintained under the high-density culture conditions, whereas CD4 T cells showed only marginal binding of P-selectin. Whether this signal is relevant functionally and whether P-selectin ligand can be induced more efficiently in C2GlcNAcT-Inull CD4 T cells are important issues that have not been resolved in the current study.

The reason why there is coinduction of two core 2 glycosyltransferases with identical or similar substrate specificities in CD8 T cells is not obvious. Possibly, these two core 2 enzymes differ in some fine specificity that is not yet known. For instance, the lack of effect of this alternate core 2 enzyme on E-selectin ligand formation observed in our system may be indicative for such a difference in specificity. Moreover, it is possible that induction of multiple C2GlcNAcT isoenzymes may be subject to differential regulation in specific subsets of T cells and that there may be CD8- and/or CD4-specific signaling pathways that result in enzyme induction, which remain to be discovered.

In summary, our data show that a second C2GlcNAcT enzyme, identified based on RNA expression as C2GlcNAcT-III, can contribute to P-selectin ligand formation and affect the rolling of activated CD8 T cells. However, C2GlcNAcT-I appears to remain the key enzyme that contributes to P-selectin ligand formation in activated CD8 T cells. Nevertheless, C2GlcNAcT-I and C2GlcNAcT-III may cooperate in the control of P-selectin ligand formation in CD8 T cells. Analysis of mice with genetic inactivation of the C2GlcNAcT-III enzyme will be required to determine the true respective contributions of these O-glycan branching enzymes in the control of CD8 T cell trafficking.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Acknowledgments
 
We thank J. Marth for the C2GlcNAcT-Inull mice and D. Vestweber for provision of the anti-PSGL-1 Ab 2PH1, D. Carlow and A. Lew for helpful discussions and critical evaluation of the manuscript, A. Johnson for assistance with flow cytometry, and P. Kubes, S. Kerfoot, and E. Drew for assistance with the parallel plate rolling assays.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Canadian Institutes for Health Research (CIHR) Operating Grant MOP-53162. J.S.M. was the recipient of a CIHR Transplantation Fellowship. Back

2 Address correspondence and reprint requests to Dr. Hermann J. Ziltener, Biomedical Research Centre, 2222 Health Sciences Mall, Vancouver, British Columbia, V6T 1Z3 Canada. E-mail address: hermann{at}brc.ubc.ca Back

3 Abbreviations used in this paper: PSGL-1, P-selectin glycoprotein ligand-1; C2GlcNAcT, core 2 {beta}1,6-N-acetylglucosaminyltransferase; HPRT, hypoxanthine phosphoribosyltransferase. Back

Received for publication October 4, 2004. Accepted for publication January 19, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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