Core 2-containing O-glycans on CD43 are preferentially expressed in the memory subset of human CD4 T cells
Ryuta Mukasa,
Toshio Homma,
Takashi Ohtsuki,
Osamu Hosono,
Akiko Souta,
Toshio Kitamura1,
Minoru Fukuda3,
Sumiko Watanabe2 and
Chikao Morimoto
Departments of Clinical Immunology and AIDS Research Center,
1 Hematopoietic Growth Factors, and
2 Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
3 Glycobiology Program, The Burnham Institute, La Jolla Cancer Research Center, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
Correspondence to:
C. Morimoto
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Abstract
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Human CD4 T cells can be divided into two functionally distinct subsets: a CD45RO+ memory subset and a CD45RA+ naive subset. In an attempt to identify novel cell surface molecules on these cells, we have developed a mAb, anti-1D4. The antigen defined by anti-1D4 was preferentially expressed on the memory subset of freshly isolated peripheral CD4 T cells and 1D4+ CD4 T cells functionally corresponded to memory T cells. Retrovirus-mediated expression cloning revealed that the 1D4 antigen is human CD43. Transfection of CHO-leu cells, which stably express human CD43, with core 2 ß-1,6-N-acetylglucosaminyltransferase (C2GnT) conferred expression of the 1D4 antigen and mRNA of C2GnT was detected by RT-PCR only in 1D4+ T cells but not in 1D4 T cells, implying that the 1D4 antigen is composed of core 2-containing O-glycans on CD43. Reactivity with anti-1D4 was completely abolished when cells were treated with neuraminidase, while there remained weak binding of anti-T305, a previously described mAb which also reacts with CD43 modified with core 2-containing O-glycans. Moreover, anti-1D4 markedly reacted with NIH-3T3 cells expressing human CD43 and low levels of endogenous C2GnT, whereas anti-T305 reacted slightly. These results indicate that the 1D4 antigen is distinct from the epitope defined by anti-T305 and anti-1D4 is a more sensitive probe to detect core 2-containing O-glycans than anti-T305. Taken together, our results indicate that core 2-containing O-glycans, whose expression can easily be detected with anti-1D4, are preferentially expressed in the CD45RO+ memory subset of CD4 T cells.
Keywords: CD4, CD43, cell surface molecules, core 2 ß-1,6-N-acetylglucosaminyltransferase, O-glycan, selectin ligand
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Introduction
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The human CD4 T cell population is a heterogeneous collection of lymphocytes having different phenotypic and functional properties. These cells can be divided into functionally distinct and largely reciprocal subsets based on their differential expression of CD45 isoforms (CD45RA and CD45RO) and CD29/VLA ß chain (18). The CD4+CD45RO+CD29high memory (helper inducer) subset responds preferentially to soluble `recall' antigens and provides a marked helper function for B cell IgG production. In contrast, the CD4+CD45RA+ CD29low naive (suppressor inducer) subset responds poorly to recall antigens and lacks the helper function.
CD4+CD45RO+CD29high memory T cells play a key role in host defense as well as triggering and maintaining of inflammation, and these cells preferentially extravasate in sites of chronic inflammation, like in the synovium of rheumatoid arthritis patients (911). Several cell adhesion molecules, including LFA-1, CD2, VLA-4/CD29 and CD26 (1214), are reported to be expressed at increased levels on the memory subset of human CD4 T cells and increased expression of these adhesion molecules may contribute to accumulation of memory T cells in inflamed tissues (9,10,15).
Many cell surface molecules have been defined and characterized by the use of specific mAb. By using this approach, we have identified or characterized several T cell surface molecules, including CD45RA, CD29, CD26, CD27 and CD31, which play important roles in T cell function and activation, and/or can be used as useful cell surface markers to define T cell subpopulations with specific functions (1,2,14,16,17). In the present study, we have shown that a novel restricted CD43 antigen, defined by a mAb, anti-1D4, was preferentially expressed on the memory subset of freshly isolated human peripheral blood CD4 T cells. We have established that the 1D4 antigen is composed of core 2-containing O-glycans on CD43, which have the GlcNAcß1
6 branch attached to the C-6 position of GalNAc and are formed by core 2 ß-1,6-N-acetylglucosaminyltransferase (C2GnT). Moreover, we have shown that the 1D4 antigen is distinct from the epitope defined by anti-T305, a previously described mAb which was developed by immunizing a BALB/c mouse with the cultured human T cell line (18) and reacts with CD43 modified with core 2-containing O-glycans (19,20) and anti-1D4 is a more sensitive probe to detect core 2-containing O-glycans than anti-T305. Our results indicate that core 2-containing O-glycans, which have been proposed to form part of the selectin ligand (2125) and whose expression can easily be detected with anti-1D4, are preferentially expressed in the CD45RO+ memory subset of CD4 T cells.
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Methods
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Antibodies
The anti-1D4 mAb was produced by standard techniques after immunization of a BALB/c J mouse with a phytohemagglutinin-activated human T cell line maintained with IL-2-containing media as previously described (1,2). The isotype of anti-1D4 was determined as IgG2a by a mouse mAb isotyping kit (Amersham, Buckinghamshire, UK). The mAb against CD8 (21Thy2D3) (26), CD14 (322) (27), CD29 (4B4) (2), CD43 with core 2-containing O-glycans (T305) (18) and MHC class II (949) (28) were used as ascites fluid. Purified anti-CD43 (DFT-1), phycoerythrin (PE)-conjugated anti-CD4 (T4), PE-conjugated anti-CD45RA (2H4) and PE-conjugated anti-CD45RO (UCHL-1) were purchased from Coulter Immunology (Hialeah, FL). Control mouse IgG was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).
Preparation of cells
Human peripheral blood mononuclear cells (PBMC) were isolated from healthy donors by Ficoll-Hypaque density-gradient centrifugation (Pharmacia Biotech, Uppsala, Sweden). Unfractionated mononuclear cells were first depleted of monocytes by adherence to plastic plates. T cells (> 90% CD3+) were then purified by E rosetting (1,2). CD4 T cells (>95% CD4+) were obtained from E rosette-positive cells by negative selection with anti-CD8, anti-CD14 and anti-MHC class II using goat anti-mouse IgG-coated immunomagnetic beads (PerSeptive Biosystems, Framingham, MA) (29). B cell and null cell populations were isolated from E rosette-negative cells as previously described (1,2).
Cell lines
T cell lines (Jurkat, H9, Molt4 and PeerIV), Burkitt's lines (Raji and Ramos), an EpsteinBarr virus-transformed B lymphoid cell line (Laz388), a histocytic cell line (U937) and an erythroleukemia cell line (K562) were used in this study. An ecotropic retrovirus packaging cell line BOSC23 (ATCC CRL 11554) (30) was maintained in DMEM containing 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 15 µg/ml hypoxanthine, 2 µg/ml aminopterine, 6 µg/ml thymidine, 250 µg/ml xanthine and 25 µg/ml mycophenolic acid. A murine pre-B cell line Ba/F3 and a murine fibroblast cell line NIH-3T3 were cultured as described (31). The CHO-leu cell line, which stably expresses human CD43, was described previously (32).
Analysis and separation of cells by flow cytometry
Single- or two-color flow cytometric analysis was performed on an Epics XL cell sorter (Coulter Electronics, Hialeah, FL) with System II software as described (29,33). For all samples, lymphocytes were analyzed by selective gating based on the parameters of forward and side scatter. Cell sorting was performed using an Epics Elite cell sorter (Coulter Electronics). In all instances, post-sort viability was >95% by Trypan blue exclusion. Purity of separated T cell subsets was >95%.
Functional assays of T lymphocytes
Proliferative response of separated T lymphocytes to PHA or tetanus toxoid (TT), and helper function of T cells for pokeweed mitogen-induced B cell IgG production were assayed as previously described (1,2).
Immunoprecipitation
H9 cells were surface-labeled with sulfo-NHS-LC-biotin (Pierce, Rockford, IL) and lysed with lysis buffer containing 1% NP-40 (29). Immunoprecipitation was carried out using anti-1D4 and Protein ASepharose beads (Pharmacia Biotech) (29). The immunoprecipitates were separated by SDSPAGE using 6% gel (34) and transferred to PVDF membranes (Millipore, Bedford, MA). After incubation with horseradish peroxidasestreptavidin (Funakoshi, Tokyo, Japan), the electrotransferred membrane was incubated with ECL reagent (Amersham) and then exposed to X-ray films.
Retrovirus-mediated expression cloning
Oligo(dT)- or random-primed cDNA libraries were constructed from the H9 T cell line which constitutively expresses the 1D4 antigen as described (31,35). Recombinant retroviruses were produced and used for infection as previously described (31,35). In brief, BOSC23 cells were transfected with cDNA derived from H9 cells by Lipofectamine reagent (Gibco/BRL). After 2 days, the culture supernatant, containing recombinant retroviruses, was harvested and used for infection of Ba/F3 cells. At 48 h after infection, the cells were subjected to FACS analysis. After three cycles of FACS cell sorting for cells reacting with anti-1D4, 1D4+ cells were cloned. Genomic DNA was isolated from each clone and the integrated cDNA segment was amplified by using primers homologous to retroviral vector pMX (36). The PCR reaction was performed for 38 cycles (30 s at 96°C, 30 s at 58°C, 4 min at 72°C) with the LA PCR kit version 2 (Takara, Kyoto, Japan). The resulting PCR fragments were subcloned into the TA cloning vector pCR2.1 (Invitrogen), and sequenced using the PRISM dye terminator cycle sequencing ready reaction kit (Applied Biosystems, UK) and the ABI 373S sequencing system.
Infection of NIH-3T3 cells with retroviruses was performed as described (31,35).
Transfection of CHO-leu cells with C2GnT cDNA
CHO-leu cells (2x105 cells/well) (32) were cultured in a six-well plate. After 24 h, the cells were transfected with 1 µg/well of pcDNAI-C2GnT (37) and 20 µl/well of Lipofectamine reagent (Gibco/BRL). After incubation for 2 h at 37°C, the medium was replaced with fresh medium. FACS analysis was performed after 2 days from the transfection.
RT-PCR
Total RNA was prepared using TRIzol reagent (Gibco/BRL) and contaminating genomic DNA was digested by incubation with DNase I (Takara). Reverse transcription of 0.35 µg of total RNA was carried out using the RNA PCR kit version 2.1 (Takara) with random hexamers. A 5 µl aliquot of a 1/10 dilution of the resulting cDNA solution was used for amplification by PCR with Takara-Taq (Takara). For detection of human C2GnT mRNA, PCR was performed with: 30 s at 96°C, 30 s at 58°C, 1 min at 72°C, for 45 cycles, using oligonucleotides 5'-ACGTTGCTGCGAAGGAGACT-3' and 5'-TGCCAGAAAAGAGAGGTGTT-3' as the primers. For detection of G3PDH mRNA, PCR was performed with: 30 s at 94°C, 30 s at 60°C, 1 min at 72°C, for 35 cycles, using the human G3PDH primers (Clontech, Palo Alto, CA). PCR products were visualized with 1% agarose gel electrophoresis, followed by staining with ethidium bromide.
Expression of mouse C2GnT was determined semiquantitatively by RT-PCR in general as described above except that the mouse C2GnT primers, corresponding to the human C2GnT primers described above, were used and that PCR was performed for 39 cycles. As an internal control for the RT-PCR analysis, the mouse ß-actin transcripts were amplified from the same cDNA samples as described (38) except that PCR was performed for 24 cycles.
Neuraminidase treatment
Cells were treated with neuraminidase as described by Remold-O'Donnell et al. (39) with some modifications. In brief, H9 cells at 1.5x107 cells/ml in HBSS were incubated for 30 min at room temperature with 2 mM PMSF without or with 0.02 or 0.1 U/ml Vibrio cholerae neuraminidase (Sigma, St Louis, MO). Cells were then washed 3 times and subjected to FACS analysis.
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Results
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Distribution of the 1D4 antigen
In an attempt to identify novel cell surface molecules on human CD4 T cells, we have developed a hybridoma clone, anti-1D4, derived from mouse splenocytes after immunization with a PHA-activated human T cell line maintained with IL-2-containing media. As shown in Fig. 1
(A), the antigen defined by anti-1D4 was present on a subpopulation of human peripheral blood CD4 T cells (39.7 ± 9.5%, n = 8). Therefore, we next examined expression of the 1D4 antigen on CD4+CD45RO+ (CD45RA) memory and CD4+CD45RA+ (CD45RO) naive T cell subsets. Figure 1
(B) shows that the 1D4 antigen was preferentially expressed on the CD45RO+ (CD45RA) memory subset of the freshly isolated human peripheral blood CD4 T cells. More than 50% of CD45RO+ (CD45RA) CD4 T cells expressed the 1D4 antigen, whereas only ~10% of CD45RO (CD45RA+) CD4 T cells expressed this antigen. The 1D4 antigen is also expressed on CD8 T cells, monocytes, null cells, three of four T cell lines, two of three B cell lines and a histocytic cell line U937, but not on peripheral blood B cells (Table 1
). It was also found that stimulation of PBMC with PHA induced expression of the 1D4 antigen and ~90% of lymphocytes expressed the 1D4 antigen after 5 days from a stimulation (data not shown).

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Fig. 1. Preferential expression of the 1D4 antigen on memory CD4 T cells. (A) The antigen defined by anti-1D4 is present on a subpopulation of human peripheral blood CD4 T cells. Two-color analysis of isolated human PBMC was performed using anti-1D4 and anti-CD4. A flow cytometric fluorescence profile stained with control mouse IgG is also indicated. Data are representative of eight separate experiments. (B) The 1D4 antigen is preferentially expressed on the CD45RO+ (CD45RA) memory subset of human CD4 T cells. Two-color analysis of isolated resting CD4 T cells was performed using anti-1D4 and anti-CD45RA or anti-CD45RO. Data are representative of three separate experiments. The actual percentages of cells within each region were as follows: CD45RA+1D4, 51.1%; CD45RA+1D4+, 12.0%; CD45RA1D4, 13.1%; CD45RA1D4+, 23.8%; CD45 RO+1D4, 22.6%; CD45RO+1D4+, 28.1%; CD45RO1D4, 44.2%; CD45RO1D4+, 5.0%.
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Proliferative response and helper function for B cell IgG production of 1D4+ and 1D4 populations of CD4 T cells
Human CD4+CD45RO+ memory (helper inducer) T cells are known to respond preferentially to recall antigens such as TT (2,6). Since our results indicated that the 1D4 antigen is preferentially expressed on the CD45RO+ memory subset of the CD4+ T cells, we utilized anti-1D4 to subdivide CD4+ T cells into 1D4+ and 1D4 populations, and examined their proliferative response to TT. As shown in Fig. 2
(A), CD4+1D4+ cells strongly responded to TT, while CD4+1D4 cells poorly responded to TT. In contrast, a proliferative response to a mitogen (PHA), which was examined as a control, was somewhat lower in the CD4+1D4+ cell population, as compared to the CD4+1D4 cell population.

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Fig. 2. CD4+1D4+ T cells functionally correspond to memory T cells. (A) Proliferative responses of unfractionated CD4+, CD4+1D4+ and CD4+1D4 subpopulations to PHA and TT. Results are representative of five experiments performed. Data are expressed as the mean ± SEM of triplicate samples. Media alone were <1000 c.p.m. Treatment of CD4 T cells with anti-1D4 and goat anti-mouse Ig had no effect on these proliferative responses (not shown). (B) Comparison of helper function provided by CD4+1D4+ and CD4+1D4 cells for pokeweed mitogen-induced B cell IgG production. Results are representative of three experiments performed. Data represent the mean of triplicate samples. SEM was <15%. Treatment of CD4 T cells with anti-1D4 and goat anti-mouse Ig had only a marginal effect on the helper function (not shown).
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Next, we examined the helper function of the 1D4+ and 1D4 populations for pokeweed mitogen-induced B cell IgG production. As shown in Fig. 2
(B), CD4+1D4+ cells provided strong helper function for B cell IgG production, whereas CD4+1D4 cells provided poor helper function. These results demonstrate that CD4+1D4+ cells functionally correspond to the CD45RO+ memory (helper inducer) subset of CD4 T cells.
Biochemical characterization of the 1D4 antigen
In order to examine the nature of the 1D4 antigen, the antigen was immunoprecipitated with the anti-1D4 mAb from cell lysates of H9 cells whose cell surface molecules were labeled with biotin. Separation of the labeled immunoprecipitates by SDSPAGE, followed by blotting with horseradish peroxidasestreptavidin revealed a single band with a mol. wt of 135 kDa under reducing conditions (Fig. 3A
). SDSPAGE under non-reducing conditions gave a single band with a similar electrophoretic mobility (Fig. 3D
). Immunoprecipitation with an anti-CD29 mAb (anti-4B4) yielded a molecule which showed a slightly different electrophoretic mobility as compared to that of the 1D4 antigen (Fig. 3B
). These results indicate that the mol. wt of the 1D4 antigen is different from those of other cell surface molecules such as CD45RO, LFA-1, CD2, LFA-3, CD26 and CD29, which have been reported to be expressed at increased levels or preferentially on the CD45RO+ memory subset of CD4 T cells (2,6,12,14).

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Fig. 3. Immunoprecipitation of the 1D4 antigen. Biotin-labeled H9 cells were lysed in lysis buffer and immunoprecipitation was carried out with anti-1D4 (A and D), anti-CD29 (anti-4B4; B) or control mouse IgG (C). Immunoprecipitates were analyzed on 6% SDSPAGE under reducing (AC) or non-reducing (D) conditions, followed by blotting with horseradish peroxidasestreptavidin. The positions of mol. wt markers are indicated on the left.
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Expression cloning of the 1D4 antigen
Since the 1D4 antigen appeared to be different from the previously established molecules which are expressed preferentially or at increased levels on memory CD4 T cells, we next focused on isolating cDNA encoding this molecule using a retrovirus-mediated expression cloning system (31,35). Murine Ba/F3 cells were infected with recombinant retrovirus stock derived from H9 cell cDNA libraries and several 1D4+ cell clones were isolated after three cycles of FACS cell sorting with the anti-1D4 mAb. Genomic DNAs were prepared from two independent 1D4+ cell clones, which were derived from either an oligo(dT)- or random-primed cDNA library. The integrated cDNA segments were amplified by PCR using the retrovirus vector primers, TA cloned and sequenced. Surprisingly, it was found that the nucleotide sequences of both cDNAs encoding the 1D4 antigen were completely identical to that of the cDNA encoding human CD43, as the result of homology search against DDBJ/EMBL/GenBank database (data not shown). Therefore, a 1.4 kbp PCR fragment, which was obtained from one of the two independent 1D4+ cell clones, was further subcloned into the pMX vector, yielding a plasmid pMX-CD43-ra4. BOSC23 cells were transfected with the plasmid pMX-CD43-ra4 and the resulting recombinant retrovirus containing CD43-ra4 was used for infection of Ba/F3 cells. The infected cells were confirmed to express the 1D4 antigen by FACS analysis. As shown in Fig. 4
, Ba/F3 cells infected with the pMX-CD43-ra4 retrovirus showed specific reactivity with both DFT-1 (a commercially available anti-CD43 mAb, Fig. 4e
) (40) and anti-1D4 (Fig. 4f
), whereas these mAb did not react with control Ba/F3 cells (Fig. 4b and c
respectively). Several irrelevant mAb, including anti-CD29, did not react with Ba/F3 cells infected with the pMX-CD43-ra4 retrovirus (data not shown). Furthermore, immunoblot analysis using whole cell lysate from H9 cells showed that DFT-1 detected a band with the same electrophoretic mobility as that detected by anti-1D4 (data not shown). Form these results, we concluded that the 1D4 antigen is human CD43.

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Fig. 4. Expression of CD43 and the 1D4 antigen on Ba/F3 cells infected with the retrovirus containing the isolated cDNA. Ba/F3 cells were infected with the pMX-derived retrovirus as a negative control (ac) or with the pMX-CD43-ra4-derived retrovirus (df) and then stained with mouse IgG (a and d), DFT-1 (b and e) or anti-1D4 (c and f). Data are representative of two separate experiments.
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CD43 (leukosialin, sialophorin) is a major cell surface sialoglycoprotein present on all human leukocytes except resting B cells and is heavily glycosylated by ~80 highly sialylated mucin-type O-linked oligosaccharide chains (39,4144). Although CD43 is expressed on almost all peripheral blood T lymphocytes, the structure of O-linked oligosaccharides attached to CD43 is characteristic for each cell lineage and for different stages of differentiation or activation (45,46). Therefore, the 1D4 antigen appeared to be a unique carbohydrate antigen on human CD43.
The 1D4 antigen is composed of core 2-containing O-glycans
Previously, Fox et al. have reported a mAb, anti-T305, which was developed by immunizing a BALB/c mouse with the cultured human T cell line and reacts with a small population of human peripheral blood T cells (18). This mAb recognizes CD43 modified with GlcNAcß1
6-branched, core 2-containing O-glycans (19,20), whose expression is substantially increased when T cells are activated (46) and is dependent on the presence of C2GnT. Since the 1D4 antigen appeared to be a unique carbohydrate antigen on CD43, which is expressed on a subpopulation of human peripheral blood T cells, we investigated whether expression of the 1D4 antigen is dependent on C2GnT. Figure 5
shows that, neither anti-1D4 (Fig. 5c
) nor anti-T305 (Fig. 5b
) reacted with CHO-leu cells (32), which stably express human CD43 but no detectable amounts of C2GnT, when they were transfected with a control plasmid (pcDNA1.1/Amp). However, transfection of CHO-leu cells with pcDNAI-C2GnT conferred reactivity with both anti-1D4 (Fig. 5g
) and anti-T305 (Fig. 5f
). DFT-1 reacted with both control CHO-leu cells (Fig. 5d
) and CHO-leu cells transfected with pcDNAI-C2GnT (Fig. 5h
) in a similar fashion. These results indicate that expression of the 1D4 antigen is dependent on C2GnT and that the 1D4 antigen is composed of GlcNAcß1
6-branched, core 2-containing O-glycans attached to human CD43 backbone.

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Fig. 5. Transfection of CHO-leu cells with C2GnT confers expression of the 1D4 antigen. CHO-leu cells, which stably express human CD43 but no detectable level of C2GnT, were transfected with a control plasmid (pcDNA1.1/Amp) (ad) or pcDNAI-C2GnT (eh). After 2 days, cells were stained with mouse IgG (a and e), anti-T305 (b and f), anti-1D4 (c and g) or DFT-1 (d and h). Data are representative of two separate experiments.
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In order to confirm that expression of the 1D4 antigen is dependent on C2GnT, we separated human peripheral blood T lymphocytes into 1D4+ and 1D4 populations, and examined the expression of C2GnT mRNA by RT-PCR in each population. Figure 6
shows that the C2GnT mRNA was detected only in the 1D4+ population but not in the 1D4 population, while mRNA of G3PDH, a house-keeping gene used as a control, was detected in both 1D4+ and 1D4 populations. Taken together, these results clearly indicate that the 1D4 antigen is composed of GlcNAcß1
6-branched, core 2-containing O-glycans attached to human CD43. Therefore our results show that core 2-containing O-glycans are preferentially expressed on the CD45RO+ memory subset of human resting CD4 T cells.

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Fig. 6. Expression of C2GnT in human 1D4+ T cells. Human peripheral blood T lymphocytes were separated into 1D4+ and 1D4 populations with a flow cytometer, and the total RNAs were isolated. Expression of C2GnT or G3PDH was detected by RT-PCR as described in Methods. RT, reverse transcriptase; the `+' and `' indicate whether RT was added or not to the RT reaction. The separated subpopulations are indicated at the top.
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The 1D4 antigen is distinct from the epitope defined by anti-T305
Since it was suggested that the 1D4 antigen is composed of core 2-containing O-glycans on CD43, we examined further the relationship between the 1D4 antigen and the structure recognized by anti-T305. We first examined the effect of removal of cell surface sialic acids by neuraminidase treatment on the reactivity with anti-1D4 and that with anti-T305. As shown in Fig. 7
, binding of anti-1D4 to H9 cells was reduced as the concentration of neuraminidase increased and was completely abolished at 0.1 U/ml (Fig. 7d,
i and n). Binding of DFT-1, whose epitope was reported to be neuraminidase sensitive (47), was also abolished after neuraminidase treatment (Fig. 7b,
g and l). In contrast, there remained weak but significant binding of anti-T305 even when cells were treated with 0.1 U/ml of neuraminidase (Fig. 7o
), which was comparable to that seen at a neuraminidase concentration of 0.02 U/ml (Fig. 7j
), although binding was reduced as compared to the control (Fig. 7e
). The reactivity with anti-4B4 (anti-CD29), used as a control, was unaffected by these neuraminidase treatments (Fig. 7c,
h and m). Similar results were obtained when human PBMC were used instead of H9 cells (data not shown). These results suggest that the structures recognized by anti-1D4 and by anti-T305 are different, although both contain sialic acids (see Discussion).

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Fig. 7. Effect of neuraminidase treatment on the reactivity with anti-1D4 and anti-T305. H9 cells were treated without (ae) or with 0.02 (fj) or 0.1 (ko) U/ml neuraminidase as descried in Methods. After extensive washing, the cells were stained with mouse IgG (a, f and k), DFT-1 (b, g and l), anti-4B4 (c, h and m), anti-1D4 (d, i and n) or anti-T305 (e, j and o).
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We next compared the reactivity of anti-1D4 and anti-T305 using murine NIH-3T3 cells, in which infection efficiency of pMX vectors has been reported to be nearly 100% (35). As shown in Fig. 8
(A), NIH-3T3 cells infected with pMX-CD43-ra4 retrovirus alone (NIH-3T3-CD43 cells) showed marked reactivity with anti-1D4 (Fig. 8A,
c), whereas these cells showed only low reactivity with anti-T305 (Fig. 8A,
d). Infection efficiency of NIH-3T3 cells with pMX-CD43-ra4 retrovirus was ~95%, as judged by the reactivity with DFT-1 (Fig. 8A,
b). These three mAb did not react with mock-infected NIH-3T3 cells (data not shown). When NIH-3T3 cells were co-infected with pMX-CD43-ra4 and pMX-C2GnT retroviruses, remarkable reactivity with anti-T305 was observed (Fig. 8A,
h). Co-infection of NIH-3T3 cells with pMX-CD43-ra4 and pMX-C2GnT retroviruses further increased reactivity with anti-1D4 (Fig. 8A,
g), as compared to the NIH-3T3-CD43 cells (Fig. 8A,
c). NIH-3T3 cells infected with pMX-C2GnT retrovirus alone were not reactive with either anti-1D4, anti-T305 or DFT-1 (data not shown). When the expression of endogenous mouse C2GnT was examined by semiquantitative RT-PCR, NIH-3T3-CD43 cells expressed relatively low levels of endogenous mouse C2GnT, as compared to Ba/F3 cells (Fig. 8B
), which showed marked reactivity with both anti-1D4 and anti-T305 when they were infected with pMX-CD43-ra4 retrovirus (data not shown). In contrast, expression of ß-actin, a house-keeping gene used as a control, was relatively comparable. These results further suggest that the 1D4 antigen and the T305 antigen are composed of distinct O-linked carbohydrate chains on CD43 although both contain core 2 structures.

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Fig. 8. Reactivity of anti-1D4 and anti-T305 with NIH-3T3 cells expressing human CD43 and low levels of endogenous C2GnT. (A) Reactivity of anti-1D4 and anti-T305 with NIH-3T3 cells infected with the pMX-CD43-ra4 retrovirus alone (NIH-3T3-CD43 cells) or with the pMX-CD43-ra4 and the pMX-C2GnT retroviruses. Murine NIH-3T3 cells were infected with the pMX-CD43-ra4 retrovirus alone (ad) or with the pMX-CD43-ra4 and the pMX-C2GnT retroviruses (eh), and were stained with mouse IgG (a and e), DFT-1 (b and f), anti-1D4 (c and g) or anti-T305 (d and h). These antibodies did not react with mock-infected NIH-3T3 cells and cells infected with pMX-C2GnT virus alone (not shown). (B) Expression of C2GnT in NIH-3T3-CD43 cells and Ba/F3 cells. Expression of endogenous mouse C2GnT or mouse ß-actin was detected by semiquantitative RT-PCR as described in Methods. RT, reverse transcriptase; the `+' and `' indicate whether RT was added or not to the RT reaction. The cells used are indicated at the top.
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In support of this, anti-T305, even above the saturation concentration, did not compete with anti-1D4 for binding to peripheral blood lymphocytes (PBL) (data not shown). Furthermore, the percentages of 1D4+ cells in PBL, peripheral blood T cells and CD4 T cells were constantly higher than those of T305+ cells, and only 16% of CD4+CD45RO+ T cells were reactive with anti-T305 under the conditions where ~55% of CD4+CD45RO+ T cells were reactive with anti-1D4. Taken together, these results indicate that the 1D4 antigen is distinct from the epitope defined by anti-T305, and anti-1D4 may be used as a novel and sensitive probe to detect core 2-containing O-glycans in CD43+ cells, which enabled us to elucidate the preferential expression of core 2-containing O-glycans on the CD45RO+ memory subset of human CD4 T cells.
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Discussion
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In the present study, we have shown that a novel restricted CD43 antigen, defined by a mAb, anti-1D4, was preferentially expressed on the memory subset of freshly isolated resting human peripheral blood CD4 T cells. We have established that the 1D4 antigen is composed of core 2-containing O-glycans on CD43, which have the GlcNAcß1
6 branch attached to the C-6 position of GalNAc and are formed by C2GnT. The results indicate that core 2-containing O-glycans are preferentially expressed on the CD45RO+ memory CD4 T cell subset.
Human CD4+CD45RO+ memory T cells have been reported to preferentially extravasate in sites of chronic inflammation, like in the synovium of rheumatoid arthritis patients, and to be involved in triggering and maintaining of inflammation (911). Several cell adhesion molecules, including LFA-1, CD2, VLA-4/CD29 and CD26, have been reported to be expressed at increased levels on the memory subset of human CD4 T cells (1214). However, little is known about selectin ligands, which are involved in extravasation of memory T cells in inflammatory sites, except cutaneous lymphocyte-associated antigen (CLA) on skin-homing memory T cells, which has been shown to be an inducible carbohydrate modification of P-selectin glycoprotein ligand (PSGL)-1 mediated by fucosyltransferase VII (4851). Several reports have indicated that core 2-containing O-glycans form part of the selectin ligand (2125). Therefore, the preferential expression of core 2-containing O-glycans on memory CD4 T cells, which was shown in this study, may explain, at least in part, the mechanism of preferential recruitment of these cells to inflammatory sites via selectin-mediated adhesion pathways. In support of this, we have observed that the percentage of 1D4+ cells in synovial fluid T lymphocytes from rheumatoid arthritis patients was significantly higher, as compared to that in peripheral blood T lymphocytes from healthy donors (Mukasa et al., in preparation).
Previous studies have indicated that activated (e.g. with OKT3 and IL-2 for 96 h) human T lymphocytes express increased levels of C2GnT activity and the resulting core 2-containing O-glycans, whereas isolated resting peripheral blood T cells express negligible levels (46). Our study, presented here, expanded the understanding of expression of core 2-containing O-glycans on a larger subset of T cells, specifically the memory CD4 T cells, which constitute ~50% of the resting human peripheral blood CD4+ T lymphocytes (18). In this study, we immediately used freshly isolated human peripheral blood T lymphocytes for experiments without any activation and we used anti-MHC class II mAb, which can eliminate activated T lymphocytes, when we isolated CD4 T cells by negative selection. Moreover, we observed only negligible levels of CD25 expression on isolated PBL, when >50% of the cells expressed the 1D4 antigen (data not shown). Therefore, it is unlikely that the 1D4+ T cells were `activated' T cells. To our knowledge, this is the first report which describes the difference in carbohydrate structures on CD43 between naive and memory subsets of human resting CD4 T cells.
Although the 1D4 antigen is preferentially expressed on the CD45RO+ (CD45RA) subset, we also observed some CD45RAhigh(CD45RO)1D4high cells (Fig. 1B
). These cells represented only 23% of the cells in the gate of lymphocytes. It was conceivable that the CD45RAhigh1D4high subpopulation might belong to NK cells because this subpopulation did not produce IL-2 (<0.6 ng/ml) after stimulation with phorbol myristate acetate and ionomycin, while CD45RAhigh1D4, CD45RA1D4 and CD45RA1D4+ cells produced fair amounts of IL-2 (10.517.5 ng/ml) (Mukasa et al., unpublished). We have also observed that a significant population of the CD45RO+ memory CD4 T cells actually express little or no 1D4 antigen (Fig. 1B
). Although functional differences between 1D4+CD45RO+ cells and 1D4CD45RO+ cells have not been demonstrated, it is conceivable that 1D4+CD45RO+ cells have enhanced memory function as compared to 1D4CD45RO+ cells. Consistent with this hypothesis, we have observed that proliferative response to a memory antigen (TT) and helper function for B cell IgG production of the 1D4 CD4 T cells, which should contain 1D4CD45RO+ CD4 T cells in addition to most CD45RA+ naive CD4 T cells, were far weaker, as compared to the 1D4+ CD4 T cells, which should be mainly composed of 1D4+CD45RO+ CD4 T cells (Fig. 2
).
Anti-T305 (18), which also recognizes human CD43 modified with core 2-containing O-glycans (19,20), has been known for years. However, the preferential expression of core 2-containing O-glycans on the memory subset of human T lymphocytes has not been elucidated. This may be partly because this mAb reacts with an even smaller population of human peripheral blood T lymphocytes and is a less sensitive probe to detect core 2-containing O-glycans, as compared to anti-1D4 (Fig. 8
). We have actually observed that anti-T305 also preferentially reacted with the memory subset of human CD4 T cells, although the percentage of T305+ cells was much lower than that of 1D4+ cells. Thus, ~17% of CD45RO+ (CD45RA) memory CD4 T cells expressed the T305 antigen, whereas only ~5% of CD45RO (CD45RA+) naive CD4 T cells expressed this antigen. Our results (Figs 7 and 8
) indicate that the 1D4 antigen and the T305 antigen are on distinct portions of the CD43 molecule. It is possible that the region on CD43 which is recognized by anti-1D4 may be more accessible to C2GnT in the Golgi complex and readily modified with the GlcNAcß1
6 branch even when the C2GnT activity is low, as compared to that recognized by anti-T305. We have also shown that neuraminidase (0.1 U/ml) treatment of H9 cells completely abolished the reactivity with anti-1D4, while there remained weak but significant binding of anti-T305 under the same conditions (Fig. 7
). These results indicate that anti-1D4 may recognize the disialosyl form of core 2-containing O-linked oligosaccharide chain, NeuNAc
2
3Galß1
3(NeuNAc
2
3Galß1
4GlcNAcß1
6)GalNAc, whereas anti-T305 may recognize a monosialosyl form and also have some affinity for the asialo form (20,45). Data about the reactivity of anti-T305 shown by Piller et al. are consistent with this hypothesis (20). In any case, since C2GnT is generally difficult to detect at the protein level (52), anti-1D4 may be used as a novel and sensitive probe to detect core 2-containing O-glycans in CD43+ cells, which enabled us to elucidate the preferential expression of core 2-containing O-glycans on the CD45RO+ memory subset of human CD4 T cells.
Although the anti-1D4 mAb does not inhibit binding of H9 cells to tumor necrosis factor-
-activated human umbilical vein endothelial cells (Mukasa et al., unpublished), there is still a possibility that core 2-containing O-glycans on CD43 are involved in binding to selectins because the core 2-containing O-glycan(s) which is not recognized by anti-1D4 may be involved. Sawada et al. have previously reported that soluble CD43 that contained sialyl Lex termini could inhibit E-selectin-mediated binding of high metastatic colonic carcinoma cells to mouse endothelial cells (23). Alternatively, core 2-containing O-glycans on other mucin-type glycoproteins may contribute to binding to selectins. It has been reported that PSGL-1, which is expressed on all human T cells (53), is involved in binding of human T cells to P-selectin (54,55). Since C2GnT enzyme activity has been shown to be critical for PSGL-1 binding to P- (and E-) selectin (24,25), it is likely that PSGL-1 is properly modified by C2GnT on only memory T cells and mediate binding of memory T cells to P- (and E-) selectin. It will be interesting to determine if these core 2-containing O-glycans and/or C2GnT enzyme can be novel therapeutic targets for human inflammatory diseases such as rheumatoid arthritis.
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Acknowledgments
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We thank Dr Hiroshi Kobayashi, Dr Hitomi Taguchi and Kio Nakamaru for flow cytometric analysis. We also thank Dr Tohru Takashi, Yasuo Kita, Atsushi Sato and Tomoe Higashida (Daiichi Pharmaceutical Co.) for helpful discussion and technical assistance. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and NIH grants CA33000, AR33713 and AI29530.
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Abbreviations
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C2GnT | core 2 ß-1,6-N-acetylglucosaminyltransferase |
PBL | peripheral blood lymphocytes |
PBMC | peripheral blood mononuclear cells |
PE | phycoerythrin |
PHA | phytohemagglutinin |
PSGL | P-selectin glycoprotein ligand |
TT | tetanus toxoid |
 |
Notes
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Transmitting editor: T. Saito
Received 14 August 1998,
accepted 26 October 1998.
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