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The Journal of Immunology, 1999, 163: 5964-5970.
Copyright © 1999 by The American Association of Immunologists

8B4/20, A Private CD43 Epitope on Developing Human Thymocytes, Is Involved in Thymocyte Maturation1

Marina Fabbi2,*, Jens Geginat{dagger}, Micaela Tiso{ddagger}, Dunia Ramarli§, David Parent, Antonio Bargellesi{ddagger} and Eileen Remold-O’Donnell

* Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy; {dagger} FB Biologie Chemie Pharmatie, FU Berlin, Berlin, Germany; {ddagger} Dipartimento Medicinia Sperimentale, Sezione Biochimica, Università di Genova, Genova, Italy; § Azienda Ospedaliera e Università di Verona, Policlinico Borgo Roma, Verona, Italy; and The Center for Blood Research, Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The 8B4/20 Ag is a 120-kDa molecule whose expression on human thymocytes varies according to the differentiation stage: high density on immature CD3-/low thymocytes, reduced density on CD3medium and double-positive thymocytes, and absent on CD3high and single-positive thymocytes and on circulating T cells. In this paper we present immunological and biochemical evidence demonstrating that 8B4/20 Ag is a variant of CD43. We show that 8B4/20-expressing molecules, which are a subset of the CD43 molecules on thymocytes, are heterogeneous in charge, suggesting varying sialylation levels. The 8B4/20 epitope was mapped to the peripherally exposed N-terminal region of CD43, and the 8B4/20 antigenic determinant was characterized by requirement for the sialic acid exocyclic polyhydroxyl side chain, a feature shared with ligands of CD22. Altogether, 8B4/20-CD43 expression pattern and biochemical characteristics suggest its participation in carbohydrate-based interactions in the thymus. We therefore used specific Ab to mimic putative 8B4/20 interactions with natural ligand and examined the effect on isolated thymocytes. Treatment with 8B4/20 had no effect on in vitro apoptosis of isolated thymocytes. In contrast, 8B4/20 ligation enhanced the conversion of isolated thymocytes to differentiated phenotypes. Increased numbers were found in 8B4/20-treated cultures of CD3high and single-positive thymocytes and decreased numbers of CD3-/low and double-positive thymocytes, strongly suggesting that engagement of 8B4/20 delivers a positive signal that favors completion of the thymocyte maturation program. The ability of 8B4/20 mAb to drive thymocyte maturation in vitro suggests that CD43 molecules bearing the 8B4/20 epitope participate in early events of thymic selection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In previous studies of thymic development, we produced a murine mAb 8B4/20 that detects an Ag expressed on human thymocytes and absent on most leukocytes except NK cells and a minor fraction of monocytes (1). The 8B4/20 Ag was characterized as a 120-kDa molecule whose expression level on thymocytes varies inversely with the degree of differentiation of the cell. 8B4/20 is expressed after pro-T cells enter the thymus with the highest levels on early differentiation stages including cells that have not yet acquired TCR (triple-negative thymocytes). Surface density decreases with increasing maturity of the thymocytes, and the Ag is lost before the cells leave the thymus. 8B4/20 Ag is absent on the CD3high thymocytes, which have undergone thymic selection, and is absent on circulating T lymphocytes (1).

In this study, we examined the relationship of 8B4/20 to CD43 (sialophorin/leukosialin). CD43 is also an ~120-kDa Ag on thymocytes (2, 3), but unlike 8B4/20, CD43 is broadly expressed on most leukocytes (4). It is a transmembrane protein with a highly O-glycosylated (~80 O-glycans) (2, 5, 6) and sialylated extracellular region (7, 8, 9), suggesting an unfolded mucin structure as shown by transmission electron microscopy (10). The large expanded structure of CD43 (45 nm in length) (10) together with its high copy number (~150,000 molecules on T lymphoid cells) (5) and the negative charge of multiple sialic acid residues provide a repulsive barrier to the cell surface with potential to interfere with receptor interactions on opposing cells. Indeed, the role of CD43 as an anti-adhesion molecule was demonstrated by transfection and gene-targeting studies, which showed that CD43 prevents cell:cell and cell:matrix interactions involving diverse receptor-ligand pairs (11, 12, 13). CD43 functions, in addition, as a positive adhesion receptor in select systems (including thymocytes) via restricted epitopes (e.g., Ref. 14) (see Discussion). Cross-linking of CD43 with specific mAbs delivers activation signals that vary depending on the cell type including inducing proliferation and activation of T cells (e.g., Refs. 15, 16, 17, 18, 19).

In humans, a single copy gene encodes the single CD43 polypeptide, and all molecular variation results from posttranslational modifications (8, 9). Early studies showed variation of glycosylation resulting in different m.w. isoforms in different cells (3, 6, 20). A well-characterized mAb, T305 (21), reacts with ~135-kDa CD43 on activated T cells and neutrophils and not with ~115-kDa CD43 on resting T lymphocytes (6, 22, 23).

In this study, we present immunological and biochemical evidence that 8B4/20 Ag is a CD43 variant and that 8B4/20-expressing CD43 molecules constitute a subset of the CD43 molecules in thymocytes. We also generate mapping data and chemical characteristics for the epitope recognized by 8B4/20 mAb. In addition, the presence of the 8B4/20 mAb in overnight cultures is shown to enhance the conversion of isolated thymocytes to differentiated phenotypes, suggesting that CD43 molecules expressing the 8B4/20 epitope play a role in thymocyte maturation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells

Human T lymphoblastoid tumor cell lines CEM (24) and MOLT-4 (25) were cultured in RPMI 1640 with 10% heat-inactivated FCS and antibiotics. CEM cells from the American Type Culture Collection (Manassas, VA) were used in Fig. 3Go and Table IGo. CD43-deficient homologous recombinant CEM cells (12) and corresponding wild-type CEM subline A3.01 cells, provided by Dr. B. Ardman (New England Medical Center, Boston, MA), were studied in Figs. 2Go and 4Go. HeLa cells stably transfected with CD43 and CD43-negative wild-type HeLa cells were as described (11).



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  		FIGURE 3. 8B4/20 Ag characterized by enzyme treatments. L10 and 8B4/20 immunoprecipitates from biotin-labeled CEM cells were mock-treated or treated with endoglycosidase-F or sialidase. The precipitates were separated by SDS-PAGE and transferred to nitrocellulose, and biotin-labeled Ag was detected with streptavidin reagent. A positive control for endoglycosidase-F activity was provided by the altered mobility of endogenous IgG or IgM heavy chains (lower panel). Note that the mobility of both L10 and 8B4/20 Ags is unaffected by endoglycosidase-F and substantially decreased by sialidase.

 

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  		Table I. Effect of cell treatments on CD43 epitopes

 


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  		FIGURE 2. 8B4/20 mAb does not bind to CD43-depleted CEM cells. A, The dark flow cytometric profiles show CD43-expressing CEM cells (top panels) and CD43-/- homologous recombinant CEM cells (bottom) stained with L10 (left) or 8B4/20 mAb (right). Isotype control-stained cells are indicated by white profiles. B, CD43-expressing CEM cells and CD43-/- CEM cells lysates were subjected to immunoblotting with L10 (left) and 8B4/20 mAb (right). The 8B4/20 blot was overexposed to show the lack of 120-kDa signal in the CD43-/- CEM cell lysates.

 


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  		FIGURE 4. 8B4/20 mAb precipitates a subset of CD43 molecules of thymocytes. The 8B4/20 and L10 precipitates from thymocyte lysates of three individuals (#1 to #3) were fractionated by isoelectrofocusing and detected by SDS-PAGE and L10 immunoblotting. pH gradient is indicated.

 
Thymocytes, isolated by teasing thymic tissue obtained from children undergoing corrective cardiac surgery, were purified by density centrifugation on Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden).

Antibodies

Murine anti-8B4/20 mAb is an IgM (1). IgG1 isotype anti-CD43 mAbs L10 (26) and T305 (21), provided by Dr. R. I. Fox (Scripps Clinic, La Jolla, CA), react, respectively, with a sialic acid-independent epitope within the distal (N-terminal) region and a sialic acid-dependent epitope proximal to the lipid bilayer (23, 27). Isotype-matched control mAb were from Sigma (St. Louis, MO). Peroxidase-labeled streptavidin and unlabeled, peroxidase-, PE-, and FITC-labeled goat anti-mouse-IgG and anti-mouse IgM Abs were from Southern Biotechnology Associates (Birmingham, AL). PE- and FITC-labeled Abs to CD3, CD4, and CD8 Ags as well as isotype-matched controls were from Becton Dickinson (San Jose, CA).

Immunoblotting

Cell extracts were fractionated by SDS-electrophoresis (28) on 8% polyacrylamide gels under reducing conditions. The separated polypeptides were transferred electrophoretically to nitrocellulose (Hybond-C Extra, Amersham, Little Chalfont, U.K.). Protein Ags were detected by incubating the membrane with mAb (ascites at 1:1000 or purified Ab at 5 µg/ml) in 10 mM Tris-HCl buffer (pH 8.0), 150 mM NaCl, and 0.01% Tween 20 with 2% w/v low fat milk solids), followed by peroxidase-conjugated goat Abs recognizing murine IgG or IgM. When proteins were labeled with biotin (described below), the nitrocellulose membranes were incubated with peroxidase-labeled streptavidin. Peroxidase was revealed by enhanced chemiluminescence (ECL, Amersham) detected by autoradiography (Hyperfilm, Amersham).

Surface labeling and immunoprecipitation

CEM cells (107) in 1 ml of PBS with 1 mM CaCl2, 1 mM MgCl2, and 10 mM glucose were combined as described (29) with sulfosuccinimidyl 6-(biotinamido)hexanoate (Pierce, Rockford, IL) (50 µl of 25 mg/ml in DMSO). After 5 min at ~22°C, an additional 50 µl out, s.c. of biotin solution was added for 10 min. The cells were washed in PBS and lysed with 1% Nonidet P-40 in PBS with 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 1 mM PMSF, and 5 µg/ml leupeptin. The lysates were clarified by centrifugation at 500 x g for 5 min and precleared sequentially with Pansorbin (Calbiochem, La Jolla, CA) and isotype-matched control mAb bound to protein A-Sepharose (Pharmacia Biotech) by means of goat anti-mouse IgG or IgM Abs. The precleared lysates were incubated overnight at 4°C with 8B4/20 or L10 mAb absorbed onto protein A-Sepharose by means of goat anti-mouse IgM or IgG, respectively. Immunoprecipitates were washed with lysis buffer alternating with lysis buffer with 0.5 M NaCl and were extracted for electrophoresis at 100°C for 5 min with SDS- and mercaptoethanol-containing buffer (28).

Enzyme treatments

Crude extracts were incubated for 16 h at 37°C with 1 IU/ml of sialidase (Vibrio cholerae) (Boehringer Mannheim, Indianapolis, IN). CEM cells at 107/ml in Ca2+/Mg2+-free HBSS were incubated with 0.02 IU/ml of sialidase (neuraminidase) (V. cholerae, 20 IU/mg protein) (Calbiochem) for 30 min at ~22°C. MOLT-4 or CEM cells at 1.5 x 107/ml in Ca2+/Mg2+-free HBSS were incubated with 25 µg/ml neutrophil elastase (Elastin Products, Owensville, MO) at 37°C for 10 min. Cell viability was monitored by trypan blue exclusion. Immunoprecipitates from CEM cells were incubated for 16 h at 37°C with 1 IU/ml of sialidase (V. cholerae) (Boehringer Mannheim) in 0.05 M sodium acetate (pH 5.5), 0.15 M NaCl, and 9 mM CaCl2 with 25 mg/ml human serum albumin or with 10 IU/ml of endoglycosidase-F (Calbiochem) in 0.1 M sodium phosphate buffer (pH 6.1), 50 mM EDTA, and 1% Nonidet P-40. For periodate oxidation and borohydride reduction, CEM cells were washed in cold PBS, resuspended at 107/ml in freshly prepared 2 mM sodium periodate in PBS, and incubated on ice in the dark for 15 min. The reaction was stopped by two cycles of dilution and pelleting in cold PBS. The cells were incubated in freshly prepared 20 mM sodium borohydride in PBS for 15 min at ~22°C, washed twice with cold PBS, and prepared for flow cytometry.

Flow cytometry

Cells (106) were incubated with mAb (0.5% ascites, 30% culture supernatant) in 200 µl of binding medium (phenol red-free HBSS with 4% FCS) at 4°C for 60 min, washed in cold-binding medium, incubated with 1 µg of FITC-labeled F(ab')2 of goat anti-mouse IgG or IgM in 100 µl of binding medium at 4°C for 30 min, washed in cold PBS, and fixed in 1% formaldehyde in PBS. When a direct staining protocol was used, PE- and FITC-labeled mAb to CD3, CD4, CD8, and isotype-matched controls were diluted in binding medium according to the manufacturer’s instructions and incubated at 4°C for 30 min. Stained cells were washed and fixed and analyzed on a FACScan or FACStar (Becton Dickinson). Nonviable cells were excluded by gating on light scatter profiles. A total of 5000 events were acquired for each sample.

Isoelectrofocusing

Reduced samples were isoelectrofocused as described (30) at 300 V for 17 h and 500 V for 2 h in cylindrical gels containing pH 3.5–10, pH 5–7, pH 6–8, and pH 2.5–4 ampholytes (Pharmacia Biotech) at a ratio of 10:1:1:1.5. The focused samples were separated in a second dimension on 8% polyacrylamide-SDS gels under reducing conditions, and the CD43 region was analyzed by immunoblot.

Treatment of thymocytes and evaluation of apoptosis

Thymocytes (5 x 105/ml) were incubated with 5 µg/ml 8B4/20 mAb, IgM control mAb, or 10 µM dexamethasone (Calbiochem) for 15 h at 37°C in RPMI 1640 with 10% heat-inactivated FCS and antibiotics, after which the cells were harvested, washed in PBS, counted, and processed for surface Ag expression (flow cytometry) and evaluation of apoptosis.

Apoptosis was evaluated by binding of FITC-annexin V (Bender System, Boehringer Ingelheim, Heidelberg, Germany) according to the manufacturer’s instructions and by TUNEL labeling. For the latter (31), thymocytes (107/ml) were incubated in 1% paraformaldehyde in PBS for 15 min at 4°C with gentle shaking, washed twice in PBS, and permeabilized by 70% ethyl alcohol precooled at -20°C. The cells were held at -20°C for 1 day, rehydrated in PBS, and resuspended at 2 x 106 to 50 µl in 45 µl of dUTP-FITC containing reaction buffer and 5 µl of TdT solution (reagents from Boehringer Mannheim). After 1 h at 37°C, the cells were washed three times with PBS and incubated for 30 min at ~22°C in the dark with 0.5 ml of DNA staining solution (5 µg/ml propidium iodide and 100 µg/ml RNase in PBS) and analyzed by a FACStar flow cytometer.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunological relatedness of 8B4/20 Ag and CD43

The putative relationship of 8B4/20 Ag to CD43 was investigated by using L10 mAb, which reacts with various human CD43 isoforms (3, 26). Thymocytes were double stained with 8B4/20 and L10 mAbs and analyzed by flow cytometry. The 8B4/20+ population, although extremely variable in size among individuals (mean 52 ± 20%, range 19–77% of positive cells; n=6), was contained completely within the L10 (CD43) positive population (Fig. 1GoA). Isolated thymocytes were then lysed and immunoprecipitated with 8B4/20 and L10 mAbs and the immunoprecipitates examined by immunoblotting. L10 was found to stain a 120-kDa band in L10 precipitates and 8B4/20 precipitates (Fig. 1GoB, left). The 8B4/20 mAb also stained a 120-kDa thymocyte band in L10 precipitates and in 8B4/20 precipitates (Fig. 1GoB, right). Similar results were obtained when cells of the T lymphoblastoid line CEM were examined (data not shown). The most straightforward explanation for these findings is that 8B4/20 Ag and CD43 are precipitated because they are immunologically related molecules; however, an alternative explanation, namely that 8B4/20 Ag and CD43 are unrelated coprecipitating molecules, could not be excluded.



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  		FIGURE 1. Comparison of 8B4/20 Ag and CD43 by flow cytometry, immunoprecipitation, and immunoblotting. A, Flow cytometric profile of thymocytes double stained with L10 and 8B4/20 mAb. Numbers indicate the percentage of cells in each quadrant. B, Human thymocyte lysates were precipitated with 8B4/20 or L10 mAb, and the precipitates were analyzed by immunoblot. The left four lanes show L10 blots, and the right four lanes show 8B4/20 blots of precipitates of IgG1 isotype control, L10, IgM isotype control, and 8B4/20. Molecular weight marker positions are indicated on the left.

 
Nonhematopoietic cells transfected with CD43 cDNA and lymphoid cells depleted of CD43 by homologous recombination were also used to compare 8B4/20 and CD43. Flow cytometry showed that HeLa cells are negative for L10 and 8B4/20 Ag as expected and that HeLa cells transfected with CD43 were positive for L10 but failed to stain with 8B4/20 mAb (data not shown). Although lack of reactivity of 8B4/20 mAb with transfected HeLa cells could indicate that 8B4/20 Ag is not a CD43 molecule, an alternative explanation is that HeLa cells lack posttranslational enzymes required to generate the 8B4/20 epitope. On examination of CEM cells (CD43+) and CEM cells depleted of CD43, L10 and 8B4/20 mAbs both stained the wild-type cells and both failed to stain the cells from which the CD43 gene had been deleted (Fig. 2Go), consistent with 8B4/20 being a CD43 molecule.

Chemical characteristics of 8B4/20 Ag

CD43, although heavily O-glycosylated, has only one N-linked glycan and, when treated with the N-glycan cleaving enzyme endoglycosidase-F (32), its SDS-PAGE mobility is not altered (33). When immunoprecipitated biotin-labeled 8B4/20 molecules from CEM cells were treated with endoglycosidase-F, SDS-PAGE mobility remained unaltered (Fig. 3Go), suggesting that 8B4/20 is not heavily N-glycosylated, consistent with its being a CD43 molecule.

A more diagnostic biochemical feature of CD43 is the large decrease of SDS-PAGE mobility (increase of apparent m.w.) that results from enzymatic desialylation (26). Whereas mock-treated immunoprecipitated 8B4/20 Ag migrated as expected at 120 kDa, sialidase-treated 8B4/20 immunoprecipitates migrated at an apparent molecular mass of 150 kDa, showing the identical mobility of L10 Ag (Fig. 3Go). The large decrement in its SDS-PAGE mobility following desialylation strongly suggests that 8B4/20 Ag is a CD43-like molecule.

In addition, no residual biotinylated protein remained at 120 kDa in the sialidase-treated 8B4/20 precipitates (Fig. 3Go), indicating that the CD43-like molecule is the sole surface molecule in 8B4/20 immunoprecipitates. Cumulatively these findings indicate that 8B4/20-reactive molecules are present in L10 precipitates and L10-reactive molecules in 8B4/20 precipitates (Fig. 1GoB) because the molecules are immunologically related. Altogether, the combination of immunological relatedness and shared biochemical features demonstrate that 8B4/20 Ag is a CD43 molecule.

8B4/20 mAb detects a subset of thymocyte CD43 molecules

The broad distribution of CD43 and restricted distribution of 8B4/20 Ag suggest that the latter is an epitope expressed on a subset of CD43 molecules. The extent of sialylation of thymocyte surface molecules increases dramatically during trafficking and maturation in the thymus (34, 35), and this variation is expected to alter charge properties of the molecules. To characterize charge properties, 8B4/20 and L10 precipitates from thymocyte lysates were fractionated by isoelectrofocusing and the Ag detected by immunoblotting with L10. The isoelectrofocusing profiles, shown in Fig. 4Go for thymi from three individuals (#1 to #3), contain multiple CD43 charge variants that may span a wide range of isoelectrofocusing points, consistent with variable sialylation of CD43. The isoelectrofocusing profiles, although reproducible for individual samples (data not shown), vary substantially among individuals (Fig. 4Go). Paired comparisons of 8B4/20 and L10 precipitates indicate that the 8B4/20-expressing thymocyte molecules do not correspond to defined charge species but rather constitute a subset of the differently charged CD43 molecules.

Chemical characterization of the 8B4/20 epitope

To examine whether sialic acid is critical for expression of the epitope, thymocyte lysates were examined after treatment with sialidase. On immunoblotting, L10 stained desialylated CD43, but no bands were detected with 8B4/20 mAb, indicating that the 8B4/20 epitope has a requirement for sialic acid (Fig. 5Go). Likewise, 8B4/20 immunoprecipitates from CEM cells became undetectable by 8B4/20 mAb after treatment with sialidase (data not shown). Sialidase-sensitivity of the 8B4/20 epitope was verified also by flow cytometry, which showed loss of the 8B4/20 epitope in sialidase-treated CEM cells and, as a control, retention of L10 staining (Table IGo).



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  		FIGURE 5. 8B4/20 epitope is sialidase-sensitive. Mock-treated and sialidase-treated thymocyte lysates were subjected to immunoblotting with L10 and 8B4/20 mAbs. Note that L10 stained desialylated CD43, but no bands were detected with 8B4/20 mAb. The 8B4/20 blot was overexposed to show the lack of signal in the sialidase-treated sample.

 
CEM cells were also subjected to mild periodate treatment. Whereas sialidase, which removes entire sialic acid moieties, alters structural and charge properties of molecules, mild periodate treatment converts the nine-carbon sialic acid moiety to a seven-carbon derivative that retains acidic properties (5-acetaido-3,5-dideoxy-L-arabino-2-heptulosonic acid) (36, 37). After periodate treatment, CEM cells bound L10 mAb as anticipated but no longer bound 8B4/20 mAb (Table IGo). This finding indicates that the terminal polyhydroxylated tail of sialic acid participates in the 8B4/20 epitope (see Discussion).

Mapping of the 8B4/20 epitope

To localize the 8B4/20 epitope, we treated 8B4/20-expressing MOLT-4 T lymphoblastoid cells with neutrophil elastase (25 µg/ml) sufficient to cleave and release the N-terminal ~40- to 52-kDa fragment from the linear extracellular region of CD43 (27). The released CD43 fragment contains the L10 epitope, and the residual cell-associated fragment contains the T305 epitope (27). Elastase treatment, which caused loss of >90% of L10-binding sites (Fig. 6Go, left) and did not effect T305 binding (right), led to the loss of 8B4/20-binding sites (90 ± 4% decrease; n = 3) (center), strongly suggesting that the 8B4/20 epitope is located within the N-terminal ~40- to 52-kDa region of CD43.



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  		FIGURE 6. 8B4/20 epitope is released from T-lymphoid cells by neutrophil elastase. Shown are flow cytometric profiles of MOLT-4 cells that were mock-treated (gray profiles) or treated with elastase (white profiles with dark outlines). The cells were stained with L10 (left), 8B4/20 (center), or T305 mAbs (right). Isotype control staining is indicated by dashed profiles. Note that the 8B4/20 epitope, like the L10 epitope and unlike the T305 epitope, is lost from elastase-treated cells. Similar results were obtained for 8B4/20 and L10 staining of elastase-treated CEM cells (data not shown).

 
8B4/20 enhances thymocyte maturation

The expression of 8B4/20-CD43 on early but not late thymocytes, together with previous findings showing that early thymocytes enriched for 8B4/20 expression are more sensitive to both "spontaneous" and induced apoptosis than the 8B4/20- mature ones (38), suggests that these molecules participate in T cell maturational events. To study this putative role, we examined the effects of 8B4/20 ligation on in vitro apoptosis of isolated thymocytes and on the phenotype of the surviving cells. Isolated thymocytes were cultured with 8B4/20 mAb and then examined for apoptosis by TUNEL assay or FITC-annexin V binding. After 15 h with 8B4/20 mAb, the extent of thymocyte apoptosis was not significantly different from the spontaneous apoptosis of thymocytes cultured with control IgM, in contrast to thymocytes cultured with dexamethasone, which had significantly higher numbers of apoptotic cells (Table IIGo). Total surviving cells were also quantified. The fraction of recovered surviving cells varied in the four experiments, ranging from 65% of seeded cells in thymus 3–85% in thymus 1 (mean 78 ± 9%). Within individual experiments, however, the fraction of recovered thymocytes in the 8B4/20 culture differed by <5% from the control IgM culture (data not shown). In two additional experiments, the 8B4/20 mAb was added to the dexamethasone culture, and FITC-annexin V binding was evaluated after 15-h culture. Again, no dramatic effect was induced by the Ab on dexamethasone-induced apoptosis (data not shown). Together, these findings indicate that 8B4/20 mAb does not influence in vitro thymocyte apoptosis.


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  		Table II. 8B4/20 mAb does not induce apoptosis in isolated human thymocytes

 
When examined for surface marker expression, viable thymocytes harvested after 15 h with 8B4/20 mAb included a substantially smaller number of cells that were CD3- (Fig. 7GoA, M1 region) and a greater number of cells that were CD3high (region M4) compared with cultures with control IgM. An increase was noted also in the fraction of CD3medium cells (region M3) in the 8B4/20-treated cultures. This pattern of change was observed in thymi from six of six individuals; the mean 8B4/20-induced decrease of CD3- cells was -11 ± 6% (range 4–19%), and the mean increase of the CD3high population was +10 ± 3% (range 3–14%).



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  		FIGURE 7. 8B4/20 mAb triggers maturation of human thymocytes in vitro. Flow cytometric profiles of thymocytes after 15-h culture with control IgM (left panels) or 8B4/20 mAb (right panels). A, Harvested thymocytes were stained with CD3-FITC; the table indicates the percentage of cells in each marker region. B, Harvested thymocytes were double stained with CD4-FITC and CD8-PE; the table indicates the percentage of cells in each quadrant. One thymus representative of six is shown. C, Harvested thymocytes were double stained with CD3-FITC and 8B4/20 mAb, followed by PE-labeled goat anti IgM serum. The table indicates the percentage of cells in each quadrant. One thymus representative of three and different from the one in A and B is shown.

 
Double staining showed that thymocytes cultured with 8B4/20 mAb significantly decreased the number of CD4+CD8+ double-positive cells and increased the number of CD4 and CD8 single-positive cells relative to cultures with control IgM (Fig. 7GoB). The 8B4/20-induced increase was greater for CD4+ cells than CD8+. This pattern of change was observed in six of six thymi; the mean decrease of double-positive cells was -9 ± 4%, and the mean increase of single-positive cells was +11 ± 4% (+8 ± 4% for CD4+ and +3 ± 1% for CD8+). 8B4/20 expression relatively to CD3 after culture with the Ab was evaluated by further addition of 8B4/20 mAb followed by PE-goat anti-IgM and CD3-FITC. As shown in Fig. 7GoC, the surface density of 8B4/20 Ag decreased over culture (-45 ± 6% 8B4/20 positive cells, n = 5), and the resulting phenotype was consistent with progression of maturation because mature CD3high thymocytes are 8B4/20-. These findings strongly suggest that 8B4/20-CD43 molecules function in thymocyte maturation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Immunological and biochemical findings identified 8B4/20 as an epitope on human thymic CD43 molecules. The immunological comparisons were made with L10, the mAb used in the original definition of CD43 (39). L10 was found to react with molecules precipitated by 8B4/20, and 8B4/20 reacts with L10-precipitated molecules (Fig. 1GoB). L10 and 8B4/20 mAbs both stained wild-type CD43+ CEM cells, and both failed to stain CEM cells depleted of CD43 by homologous recombination (Fig. 2Go). Biochemical characteristics shared by 8B4/20 Ag and L10 Ag (CD43) include an atypical large decrease of SDS-PAGE mobility (increase of apparent m.w.) on removal of sialic acid (Fig. 3Go). 8B4/20 Ag, like CD43, is sensitive to low levels of neutrophil elastase (Fig. 6Go) and insensitive to endoglycosidase-F (Fig. 3Go). On the other hand, 8B4/20-expressing molecules and L10-expressing molecules are not identical because the former show a differentiation-dependent, primarily thymic distribution, and the latter are broadly expressed on leukocytes.

Isoelectrofocusing showed that 8B4/20 molecules constitute a subset of the thymocyte molecules reactive with L10, and also that 8B4/20-CD43 molecules are heterogeneous in charge, suggesting variable content of sialic acid (Fig. 4Go). Previous studies showed that developmentally regulated CD43 epitopes result from posttranslational events primarily glycosylation (40). For thymocyte CD43, two enzymatic activities are known to be important: 1) core 2 ß 1–6-N-acetylglucosaminyl transferase (C2GnT), expressed in early but not late thymocytes (14), which generates branched O-glycans (6), and 2) sialyltransferase, which determines the extent of sialylation (3, 41).

C2GnT is required to generate the T305 epitope on thymic CD43 (42). Because T305 and 8B4/20 are similarly distributed (on immature thymocytes and not on CD3high cells) (14, 21), we mapped 8B4/20 with respect to T305. The mapping data localized 8B4/20 to the N-terminal region of CD43, thereby distinguishing this epitope from T305, which is located proximal to the phospholipid bilayer (Fig. 6Go). It is not known whether 8B4/20 requires core 2 glycosylation; however, its expression on strains of CEM cells expressing primarily low m.w. CD43 and negative for T305 together with its absence on neutrophils, which are strongly positive for T305 (23), suggest that 8B4/20 does not require core 2 glycosylation. Thus, 8B4/20 and T305 represent two distinct glycosylation-dependent CD43 structures expressed in a developmentally regulated fashion on thymocytes.

The other enzymatic activity known to be relevant for CD43 epitopes is sialyltransferase. Several sialyltransferases are expressed in human thymus, some of which show differentiation-dependent expression (43, 44). In particular, increased expression of Gal-ß-1,3GalNAc {alpha}2,3-sialyltransferase in mature thymocytes correlates with increased sialylation of O-glycans (43). Sialylation was found to be essential to the 8B4/20 epitope, as recognition by its mAb was abrogated by sialidase treatment (Fig. 5Go and Table IGo) and also by mild oxidation conditions that specifically truncate the exocyclic polyhydroxyl side chain of sialic acid without destroying the acidic moiety (36, 45) (Table IGo). Despite the strict epitope requirement for a sialic acid moiety, the level of 8B4/20 expression is high on immature thymocytes, which are minimally sialylated, and low on mature thymocytes, which are heavily sialylated. The decrease of 8B4/20 expression as thymocytes mature could reflect decrease of an enzyme required for synthesis of the epitope. Alternatively, decrease of 8B4/20 could result from epitope "masking" due to extensive sialylation. The latter possibility is suggested by analogy with peanut agglutinin (PNA) binding epitopes, which also decrease as thymocytes mature (34, 35). Binding sites for PNA, consisting of Gal-GalNac epitopes (46), are masked to varying extent on CD43 molecules and can be exposed by enzymatic desialylation (26, 41).

A situation possibly related to 8B4/20 expression exists in germinal center B cell interactions in which developmentally regulated expression of the sialic acid-requiring epitope CDw75 on surface molecules such as IgM regulates binding of the cells to the germinal center specific lectin CD22 (45, 47, 48). Interestingly, both the CDw75 and 8B4/20 epitopes require the exocyclic polyhydroxyl side chain of sialic acid. In the B cell situation, interactions of the CDw75 ectodomain, which provide the cell with information on its microenvironmental context, are physiologically relevant to the regulation of other signaling pairs, fine-tuning the strength and quality of the Ag receptor signal (reviewed in Ref. 49). The developmentally regulated sialylation-dependent 8B4/20 epitope might similarly provide developing T cells with the surface structure required to regulate intrathymic localization and/or the delivery of intracellular signals via binding to an unidentified counterreceptor.

Altogether, its expression pattern and biochemical characteristics suggest the participation of 8B4/20-CD43 in carbohydrate based interactions of developing thymocytes. Discrete stages of thymic development are distinguishable by sequential phenotypic changes: from the immature triple-negative (CD4-CD8-CD3/TCR-) to double-positive (CD4+CD8+CD3/TCR-/low/medium) to the immunocompetent mature single-positive (CD4+CD8-CD3/TCRhigh or CD4-CD8+CD3/TCRhigh) thymocytes. T cell repertoire selection occurs primarily at the double-positive stage and is driven by both positive and negative selection signals (50, 51).

Carbohydrate-mediated interactions have a major role, at least for negative (apoptotic) selection mechanisms. Galectin-1, a lectin product of thymic epithelium, interacts with galactose-bearing receptors on immature thymocytes. Interaction with galectin-1 induces apoptosis of immature thymocytes in vitro and is thought to deliver a second apoptotic signal to developing thymocytes, which synergizes with CD3-TCR engagement (52). Prominent among the thymocyte surface molecules that bind galectin-1 is the T305 variant of CD43 (14).

To characterize the function of the 8B4/20 variant of CD43, we assumed that its interaction with natural ligand could be mimicked by specific Ab, and we therefore investigated whether mAb addition to cultures of isolated thymocytes would alter the rate of in vitro apoptosis or the phenotype of the surviving cells. The presence of the 8B4/20 mAb in overnight cultures did not induce apoptosis of isolated thymocytes above the spontaneous levels occurring in control cultures (Table IIGo).

In contrast, 8B4/20 mAb increased the conversion of thymocytes to differentiated phenotypes. The fraction of CD3- cells was decreased in 8B4/20 cultures, the fraction with high CD3 expression increased substantially and the CD3med fraction increased slightly (Fig. 7Go). These changes in CD3 expression in 8B4/20-treated thymocytes were accompanied by a decrease of double-positive cells and an increase of single-positive cells. In the absence of increased cell death, the finding of higher numbers of CD3high cells and single-positive cells strongly suggests that 8B4/20 ligation delivers a positive signal that favors completion of the thymocyte maturation program. These findings together with the higher expression of 8B4/20 on CD3-/low cells suggest that 8B4/20-CD43 participates in the early events of thymic selection, where it might deliver information on the microenvironmental context permitting CD3/TCR up-regulation. Assuming these effects as physiological, then a dual role for CD43 in T cells emerges: as carrier of epitopes interacting with ligand(s) relevant for maturation signals in thymocytes and as regulator of cell surface interactions during activation in circulating T cells (53). A role for 8B4/20-CD43 surface molecules in influencing the balance between negative and positive selection is consistent with the current model of intrathymic development, in which a series of overlapping signals provided by growth factors, cytokines, and adhesion molecules integrate the effects of T cell Ag receptors engagement, guiding thymocyte survival, expansion, and differentiation.

Taken together, these findings demonstrate that select CD43 thymocyte molecules carry a differentiation epitope, 8B4/20, that is structurally and functionally distinct from the previously described T305 epitope. Biochemical characterization indicates that 8B4/20-CD43 could serve as ligand for carbohydrate-based interactions to deliver cues to the developing thymocyte on its microenvironmental context. The ability of 8B4/20 mAb to drive thymocyte maturation in vitro strongly suggests that 8B4/20-CD43 molecules participate in early events of thymic selection, conducive to thymocyte differentiation.


   Acknowledgments
 
We thank B. Ardman (Department of Hematology-Oncology, New England Medical Center, Boston MA) for performing immunofluorescence on CD43 stably transfected HeLa cells and for providing CD43-depleted CEM cell lines; R. Fox for providing T305 Ab; M. E. Cosulich for helpful discussion; and Istituto "G. Gaslini" Cardiac Surgery, for providing thymus specimens.


   Footnotes
 
1 This work was supported by grants from Consiglio Nazionale delle Ricerche (strategic project: Cell Cycle and Apoptosis), and Ministero dell’Universitá e della Ricerca Scientifica e Tecnologica (project: Regulatory Signals in Lymphocyte Life and Death) (to A.B.), and National Institutes of Health Grants AI29880 and AI39574 (to E.R.-O.). J.G. was recipient of a Deutscher Akademischer Austauschdienst fellowship. Back

2 Address correspondence and reprint requests to Dr. Marina Fabbi, Centro Biotecnologie Avanzate, Largo R. Benzi 10, 16132 Genova, Italy. E-mail address: Back

Received for publication March 22, 1999. Accepted for publication September 21, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Fabbi, M., M. Tiso, R. M. R. Gangemi, A. Favre, P. Demartini, A. Bargellesi-Severi. 1994. A novel 120-kDa antigen shared by immature thymocytes and long-term-activated T cells. Eur. J. Immunol. 24:1.[Medline]
  2. Brown, W. R. A., A. N. Barclay, C. A. Sunderland, A. F. Williams. 1981. Identification of a glycophorin-like molecule at the cell surface of rat thymocytes. Nature 289:456.[Medline]
  3. Remold-O’Donnell, E., C. Zimmerman, D. Kenney, F. S. Rosen. 1987. Expression on blood cells of sialophorin, the surface glycoprotein that is defective in Wiskott-Aldrich syndrome. Blood 70:104.[Abstract/Free Full Text]
  4. Horejsi, V., H. Stockinger. 1997. CD43 workshop panel report. T. Kishimoto, and H. Kikutani, and A. E. G. Kr. von dem Borne, and S. M. Goyert, and D. Y. Mason, and M. Miyasaka, and L. Moretta, and K. Okumura, and S. Shaw, and T. Springer, et al eds. Leukocyte Typing VI: White Cells Differentiation Antigens 494. Garland, New York.
  5. Remold-O’Donnell, E., III A. E. Davis, D. Kenney, K. Ramakrishnan-Bhaskar, F. S. Rosen. 1986. Purification and chemical composition of gpL115, the human lymphocyte surface sialoglycoprotein that is defective in Wiskott-Aldrich syndrome. J. Biol. Chem. 261:7526.[Abstract/Free Full Text]
  6. Carlsson, S. R., M. Fukuda. 1986. Isolation and characterization of leukosialin, a major sialoglycoprotein on human leukocytes. J. Biol. Chem. 261:12779.[Abstract/Free Full Text]
  7. Killeen, N., A. N. Barclay, A. C. Willis, A. F. Williams. 1987. The sequence of rat leukosialin (W3/13 antigen) reveals a molecule with O-linked glycosylation of one third of its extracellular amino acids. EMBO J. 6:4029.[Medline]
  8. Shelley, C. S., E. Remold-O’Donnell, III A. E. Davis, G. A. P. Bruns, F. S. Rosen, M. C. Carroll, A. S. Whitehead. 1989. Molecular characterization of sialophorin (CD43), the lymphocyte surface sialoglycoprotein defective in Wiskott-Aldrich syndrome. Proc. Natl. Acad. Sci. USA 86:2819.[Abstract/Free Full Text]
  9. Pallant, A., A. Eskenazi, M.-G. Mattei, R. E. K. Fournier, S. R. Carlsson, M. Fukuda, J. G. Frelinger. 1989. Characterization of cDNAs encoding human leukosialin and localization of the leukosialin gene to chromosome 16. Proc. Natl. Acad. Sci. USA 86:1328.[Abstract/Free Full Text]
  10. Cyster, J. G., D. M. Shotton, A. F. Williams. 1991. The dimensions of the T lymphocyte glycoprotein leukosialin and identification of linear protein epitopes that can be modified by glycosylation. EMBO J. 190:893.
  11. Ardman, B., M. A. Sikorski, D. E. Staunton. 1992. CD43 interferes with T-lymphocyte adhesion. Proc. Natl. Acad. Sci. USA 89:5001.[Abstract/Free Full Text]
  12. Manjunath, N., R. S. Johnson, D. E. Staunton, R. Pasqualini, B. Ardman. 1993. Targeted disruption of CD43 gene enhances T lymphocyte adhesion. J. Immunol. 151:1528.[Abstract]
  13. Manjunath, N., M. Correa, M. Ardman, B. Ardman. 1995. Negative regulation of T-cell adhesion and activation by CD43. Nature 377:535.[Medline]
  14. Baum, L. G., M. Pang, N. L. Perillo, T. Wu, A. Delegeane, C. H. Uittenbogaart, M. Fukuda, J. J. Seilhamer. 1995. Human thymic epithelial cells, express an endogenous lectin, galectin-1, which binds to core 2 O-glycans on thymocytes and T lymphoblastoid cells. J. Exp. Med. 181:877.[Abstract/Free Full Text]
  15. Mentzer, S. J., E. Remold-O’Donnell, M. A. V. Crimmins, B. E. Bierer, F. S. Rosen, S. J. Burakoff. 1987. Sialophorin, a surface sialoglycoprotein defective in the Wiskott-Aldrich syndrome, is involved in human T lymphocyte proliferation. J. Exp. Med. 165:1383.[Abstract/Free Full Text]
  16. Axelsson, B., Y. Youseffi-Etemad, S. Hammarstrom, P. Perlmann. 1988. Induction of aggregation and enhancement of proliferation and IL-2 secretion in human T cells by antibodies to CD43. J. Immunol. 141:2912.[Abstract]
  17. Alvarado, M., C. Klassen, J. Cerny, V. Horejsi, R. E. Schmidt. 1995. MEM-59 monoclonal antibody detects a CD43 epitope involved in lymphocyte activation. Eur. J. Immunol. 25:1051.[Medline]
  18. Sperling, A. I., J. M. Green, R. L. Mosley, P. L. Smith, R. J. DiPaola, J. R. Klein, J. A. Bluestone, C. B. Thompson. 1995. CD43 is a murine T cell co-stimulatory receptor that functions independently of CD28. J. Exp. Med. 182:139.[Abstract/Free Full Text]
  19. Fanales-Belasio, E., G. Zambruno, A. Cavani, G. Girolomoni. 1997. Antibodies against sialophorin (CD43) enhance the capacity of dendritic cells to cluster and activate T lymphocytes. J. Immunol. 159:2203.[Abstract/Free Full Text]
  20. Carlsson, S. R., H. Sasaki, M. Fukuda. 1986. Structural variations of O-linked oligosaccharides present in leukosialin isolated from erythroid, myeloid, and T-lymphoid cell lines. J. Biol. Chem. 261:12787.[Abstract/Free Full Text]
  21. Fox, R. I., M. Hueniken, S. Fong, S. Behar, I. Royston, S. K. Singhal, L. Thompson. 1983. A novel cell surface antigen (T305) found in increased frequency on acute leukemia cells and in autoimmune disease states. J. Immunol. 131:762.[Abstract]
  22. Piller, F., F. LeDeist, K. I. Weinberg, R. Parkman, M. Fukuda. 1991. Altered O-glycan synthesis in lymphocytes from patients with Wiskott-Aldrich syndrome. J. Exp. Med. 173:1501.[Abstract/Free Full Text]
  23. Remold-O’Donnell, E., D. Parent. 1994. Two proteolytic pathways for down-regulation of the barrier molecule CD43 of human neutrophils. J. Immunol. 152:3595.[Abstract]
  24. Foley, G. E., H. Lazarus, S. Farber, B. G. Uzman, B. A. Boone, R. E. McCarthy. 1965. Continuous culture of human lymphoblasts from peripheral blood of a child with acute leukemia. Cancer 18:522.[Medline]
  25. Minowada, J., T. Onuma, G. E. Moore. 1972. Rosette-forming human lymphoid cell lines. I. Establishment and evidence for origin of thymus-derived lymphocytes. J. Natl. Cancer Inst. 49:891.
  26. Remold-O’Donnell, E., D. Kenney, R. Parkman, L. Cairns, B. Savage, F. S. Rosen. 1984. Characterization of a human lymphocyte surface sialoglycoprotein that is defective in Wiskott-Aldrich syndrome. J. Exp. Med. 159:1705.[Abstract/Free Full Text]
  27. Remold-O’Donnell, E., D. Parent. 1995. Specific sensitivity of CD43 to neutrophil elastase. Blood 86:2395.[Abstract/Free Full Text]
  28. Laemmli, U. K.. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680.[Medline]
  29. De Rossi, G., D. Zarcone, F. Mauro, G. Cerruti, C. Tenca, A. Puccetti, F. Mandelli, C. E. Grossi. 1993. Adhesion molecule expression on B-cell chronic lymphocytic leukemia cells: malignant cell phenotypes define distinct disease subsets. Blood 81:2679.[Abstract/Free Full Text]
  30. O’Farrell, P. H.. 1975. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250:4007.[Abstract/Free Full Text]
  31. Gorczyca, W., J. Gong, B. Ardelt, F. Traganos, Z. Darzynkiewicz. 1993. The cell cycle related differences in susceptibility of HL60 cells to apoptosis induced by various antitumor agents. Cancer Res. 53:3186.[Abstract/Free Full Text]
  32. Tarentino, A. L., C. M. Gomez, Jr T. H. Plummer. 1985. Deglycosylation of asparagin-linked glycans by peptide:N-glycosidase F. Biochemistry 24:4665.[Medline]
  33. Wilson, A. P., C. C. Rider. 1992. Evidence that leukosialin, CD43, is intensely sulfated in the murine T lymphoma line RDM-4. J. Immunol. 148:1777.[Abstract]
  34. Reisner, Y., M. Linker-Israeli, N. Sharon. 1976. Separation of mouse thymocytes into two subpopulations by the use of peanut agglutinin. Cell. Immunol. 25:129.[Medline]
  35. Sharon, N.. 1983. Lectin receptors as lymphocyte surface markers. Adv. Immunol. 34:213.[Medline]
  36. Van Lenten, L., G. Ashwell. 1971. Studies on the chemical and enzymatic modification of glycoproteins: a general method for the tritiation of sialic acid-containing glycoproteins. J. Biol. Chem. 246:1889.[Abstract/Free Full Text]
  37. Schauer, R.. 1982. Chemistry, metabolism and biological functions of sialic acids. Adv. Carbohydr. Chem. Biochem. 40:131.[Medline]
  38. Tiso, M., R. Gangemi, A. Bargellesi Severi, S. Pizzolitto, M. Fabbi, A. Risso. 1995. Spontaneous apoptosis in human thymocytes. Am. J. Pathol. 147:434.[Abstract]
  39. Borche, L., F. Lozano, R. Vilella, J. Vives. 1987. CD43 monoclonal antibodies recognize the large sialoglycoprotein of human leukocytes. Eur. J. Immunol. 17:1523.[Medline]
  40. Fukuda, M.. 1991. Leukosialin, a major O-glycan-containing sialoglycoprotein defining leukocyte differentiation and malignancy. Glycobiology 1:347.[Abstract/Free Full Text]
  41. Brown, W. R. A., A. F. Williams. 1982. Lymphocyte cell surface glycoproteins which bind to soybean and peanut lectins. Immunology 46:713.[Medline]
  42. Bierhuizen, M. F. A., K. Maemura, M. Fukuda. 1994. Expression of a differentiation antigen and poly-N-acetyllactosaminyl O-glycans directed by a cloned core 2–1,6-N-acetylglucosaminyltransferase. J. Biol. Chem. 269:4473.[Abstract/Free Full Text]
  43. Gillespie, W. G., J. C. Paulson, S. Kelm, M. Pang, L. G. Baum. 1993. Regulation of {alpha}2,3-sialyltransferase expression correlates with conversion of peanut agglutinin (PNA)+ to PNA- phenotype in developing thymocytes. J. Biol. Chem. 268:3801.[Abstract/Free Full Text]
  44. Baum, L. G., K. Derbin, N. L. Perillo, T. Wu, M. Pang, C. H. Uittenbogaart. 1996. Characterization of terminal sialic acid linkages on human thymocytes: correlation between lectin-binding phenotype and sialyltransferase expression. J. Biol. Chem. 271:10793.[Abstract/Free Full Text]
  45. Sgroi, D., A. Varki, S. Braesch-Andersen, I. Stamenkovic. 1993. CD22, a B cell-specific immunoglobulin superfamily member, is a sialic acid-binding lectin. J. Biol. Chem. 268:7011.[Abstract/Free Full Text]
  46. Swamy, M. J., D. Gupta, S. K. Mahanta, A. Surolia. 1991. Further characterization of the saccharide specificity of peanut (Arachis hypogea) agglutinin. Carbohydr. Res. 213:59.[Medline]
  47. Stamenkovic, I., D. Sgroi, A. Aruffo, M. S. Sy, T. Anderson. 1991. The B lymphocyte adhesion molecule CD22 interacts with leukocyte common antigen CD45RO on T cells and {alpha}2,6 sialyltransferase, CD75, on B cells. Cell 66:1133.[Medline]
  48. Leprince, C., K. E. Draves, R. L. Geahlen, J. A. Ledbetter, E. A. Clark. 1993. CD22 associates with the human surface IgM-B cell antigen receptor complex. Proc. Natl. Acad. Sci. USA 90:3236.[Abstract/Free Full Text]
  49. Cyster, J. C., C. C. Goodnow. 1997. Tuning antigen receptor signaling by CD22: integrating cues from antigens and the microenvironment. Immunity 6:509.[Medline]
  50. von Boehmer, H.. 1994. Positive selection of lymphocytes. Cell 76:219.[Medline]
  51. Page, D. M., L. P. Kane, T. M. Onami, S. M. Hedrick. 1996. Cellular and biochemical requirements for thymocytes negative selection. Semin. Immunol. 8:69.[Medline]
  52. Perillo, N. L., C. H. Uittenbogaart, J. T. Nguyen, L. G. Baum. 1997. Galectin-1, an endogenous lectin produced by thymic epithelial cells, induces apoptosis of human thymocytes. J. Exp. Med. 185:1851.[Abstract/Free Full Text]
  53. Sperling, A. I., J. R. Sedy, N. Manjunath, A. Kupfer, B. Ardman, J. K. Burkhardt. 1998. TCR signaling induces selective exclusion of CD43 from the T cell-antigen-presenting cell contact site. J. Immunol. 161:6459.[Abstract/Free Full Text]



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