Evidence for a dual pathway of activation in CD43-stimulated Th2 cells: differential requirement for the Lck tyrosine kinase
Maria J. Fernandez-Cabezudo2,
Camasamudram Vijayasarathy2,
David L. Pflugh3,
Alfred L. M. Bothwell3 and
Basel K. al-Ramadi1
Departments of 1 Medical Microbiology and 2 Biochemistry, Faculty of Medicine and Health Sciences, UAE University, Al Ain, United Arab Emirates
3 Section of Immunobiology, Yale University School of Medicine, New Haven, CT 06520, USA
Correspondence to: B. K. al-Ramadi; E-mail: ramadi.b{at}uaeu.ac.ae
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Abstract
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CD43 is the most abundant cell surface-expressed sialoglycoprotein on T lymphocytes. Despite evidence demonstrating the activation of some signaling components by CD43, the exact function of CD43 in T cell biology remains controversial. In this study, we demonstrate that the sole ligation of CD43 in cloned Th2 cells resulted in cytokine production, cellular proliferation, and upregulation of CD25 and CD69 activation markers. Similarly, cross-linking of CD43 on naive splenic T cells led to a significant proliferative response and an enhancement of the expression of CD25 and CD69 markers. These responses required no additional signals from other T cell molecules, including TCR. In Lck-deficient Th2 cells, however, CD43 ligation led to IL-4 production and an increase in the expression of CD25 and CD69 antigens but, surprisingly, no proliferation. Analysis of signaling pathway components revealed that CD43 associates with the adaptor protein SLP-76 within 30 s of activation. This induces the tyrosine phosphorylation of SLP-76 and promotes the recruitment and phosphorylation of another adaptor, Shc. The formation of this multi-component complex was strictly dependent on Lck. In contrast, comparison of tyrosine phosphorylated proteins in whole extracts of normal and Lck-deficient cells revealed a strikingly similar pattern of phosphorylation involving two major protein bands at 26 and 78 kDa. This suggests that tyrosine kinases other than Lck are activated by CD43 ligation. Taken together, the data support the notion that CD43 ligation may induce a dual pathway leading to the activation of different effector functions in Th2 lymphocytes.
Keywords: costimulatory molecules, protein kinases, T lymphocytes, transcription factors
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Introduction
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CD43 is one of the most abundant, cell surface-expressed, proteins on T lymphocytes with an estimated 11.5 x 105 molecules per cell (1). CD43 is an integral membrane glycoprotein with a cytoplasmic domain of 123 amino acids and a highly glycosylated and sialylated extracellular domain of 239 amino acids. The extracellular domain exhibits a high density of negative charges due to the presence of 8090 sialic acid-bearing O-linked carbohydrate units (2). Post-translational glycosylation results in two major isoforms, 115 kDa isoform expressed on all T lymphocytes and 130 kDa isoform expressed on resting CD8+ as well as activated CD4+ and CD8+ T lymphocytes (3). The high sequence homology of the cytoplasmic domain of CD43 from human, rat and mouse suggested it might have an important role in regulating T cell function. Interestingly, and pointing out the importance of CD43 in lymphocyte function, a defective expression of CD43 by T lymphocytes is reported in the immunologic disorder X-linked WiskottAldrich syndrome, characterized by widespread defects in T lymphocyte function, progressive lymphopenia and thrombocytopenia (4,5).
Over the last 10 years, several reports demonstrated apparently conflicting roles for CD43, including anti-adhesive and pro-adhesive functions (6,7) as well as positive and negative regulation of T cell activation (4,6,810). Ligation of CD43 with mAb induces the generation of second messengers like diacylglycerol and inositol phosphates, calcium mobilization and protein kinase C activation (11). The expression of human CD43 in a murine T-cell hybridoma enhanced the antigen-specific response, an effect that required the cytoplasmic domain of CD43 that becomes phosphorylated upon T cell activation (4,5). Additionally, Pedraza-Alva and co-workers reported that stimulation of human T lymphocytes by anti-CD43 mAb involves the activation of the tyrosine kinase Fyn, and leads to tyrosine phosphorylation of Vav and the activation of MAPK cascade (12,13). Recently, CD43 was shown to be excluded from the immunological synapse during T cell activation, implying that the presence of large molecules such as CD43 is inhibitory to the formation of effective T cellantigen-presenting cell interactions [reviewed in (14)].
Previously, our laboratory described the derivation of Lck-deficient transfectants of a non-transformed, cloned, Th2 cell line by using Lck-specific antisense RNA (15). Using these transfectants, we demonstrated the central role played by the T cell-specific protein tyrosine kinase, Lck, in Th2 cell activation, co-stimulation and growth (1517). In the present report, we utilized a mAb (designated R2/60) known to react with both isoforms of CD43 (18) to study CD43 signaling and function in normal as well as Lck-deficient cells. Our findings demonstrate that the sole triggering of CD43 by R2/60 leads to the activation of Th2 cells as assessed by several functional and biochemical events. Evidence is presented that signaling via CD43 proceeds through a dual pathway involving Lck-dependent as well as Lck-independent components, leading to the activation of different effector functions in Th2 cells.
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Methods
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Cell lines and immunologic reagents
The parental Th2 clone D10.G4.1 (hereafter referred to as D10) expresses an
ß TCR which specifically recognizes a peptide derived from conalbumin in the context of the murine I-Ak MHC class II molecule (19). Lck-deficient transfectants of D10 were derived using a vector containing a 400 bp-fragment of murine Lck cloned in the antisense orientation, as previously detailed (15). One of the transfectant clones, which has been extensively characterized (1517), was used in the present study. Lck-deficient cells were maintained by biweekly stimulation with cognate antigen plus syngeneic feeder cells in the presence of IL-1, IL-2 and IL-4 lymphokines. For all experiments, T cells were used in the resting phase 1014 days after the last round of activation. Splenic T cells from C57BL/6 mice were isolated by negative selection using biotinylated mAbs to CD19, CD11b and CD86 (all from PharMingen, San Diego, CA) and streptavidin-conjugated magnetic beads (New England BioLabs). Flow cytometric analysis confirmed that the purified T cells were >92% Thy-1+. Human rIL-1
was from Genzyme (Cambridge, MA) and mouse rIL-2 and rIL-4 from BioSource International (Camarillo, CA). The following mAbs were used: R2/60, anti-CD43 (8); YCD3-1, anti-CD3
(20); H57-597, anti-TCR Cß (21); 11B11, anti-IL-4 (22). All mAbs were affinity purified from hybridoma culture supernatants on Protein GSepharose (Pharmacia Biotech, Piscataway, NJ) or used as supernatants. For biochemical analysis, antibodies specific to Lck, SLP-76 or Shc were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), anti-CD43 mAb (S7 mAb; rat IgG2a) from PharMingen and anti-phosphotyrosine 4G10 mAb from Upstate Biotechnology (Lake Placid, NY).
Flow cytometry
Expression of CD43 on parental and Lck-deficient D10 cells was determined by staining viable T cells with R2/60 followed by FITC-conjugated goat anti-rat IgM secondary antibody (Southern Biotechnology Associates, Birmingham, AL). As a negative control, a rat IgM mAb specific to TCR Vß14 (clone 14.2) was used (note that D10 cells are Vß8.2+). To study the expression of T cell activation markers, cloned T cells were pre-incubated with R2/60 or control mAb (at 10 µg/ml) for 15 min at room temperature followed by the addition of goat anti-rat IgM (at 10 µg/ml). After a 16 h incubation at 37°C, cells were stained with biotin-conjugated mAbs specific to CD69 or CD25, or CD4 (PharMingen) followed by phycoerythrin-conjugated Streptavidin (Southern Biotechnology Associates), following established procedures (15). Upregulation of CD69 and CD25 was also assessed using purified splenic T cells activated for 16 h with cross-linked R2/60 mAb or control rat IgM (clone R4-22, PharMingen). Cells were analyzed by two-color staining with mAbs specific to CD4 and CD69 or CD25. Cells were analyzed on a FACScan (Becton Dickinson, Mountain View, CA). Data collected from 15 000 cells were analyzed using CELLQUEST software.
T cell proliferation assay
Antibody-coated plates were prepared by coating the wells of flat-bottom microtiter plates with 50 µl of R2/60 or H57-597 mAb diluted in PBS to the indicated concentration. After incubation for 2 h at 22°C, the plates were blocked for 30 min with 5% FCSPBS and washed before addition of T cells (104 cells/well) and culture in DMEM supplemented with 5% FCS, gentamycin, glutamine, non-essential amino acids, essential amino acids, sodium pyruvate, HEPES and 2-ME (GibcoBRL, Paisley, UK). Purified splenic T cells were plated at 5 x 104 cells/well in the presence of R2/60 or control rat IgM (clone R4-22, PharMingen). As a control, anti-CD3 (145-2C11) was added at 1 µg/ml. Cross-linking was achieved by the addition of F(ab')2 goat anti-rat IgM (Jackson ImmunoResearch) at 5 µg/ml. In some assays, anti-IL-4 neutralizing antibody was also added at a final concentration of 20 µg/ml. Cell proliferation was assessed after a 72 h incubation following a pulse with 1 µCi of [3H]thymidine for the last 18 h of culture.
Production and measurement of IL-4
T cells were cultured at 105 cells/well in 24-well plates, stimulated with 10 µg/ml of R2/60, YCD3-1, or as a control, 14.2 mAb, and cross-linked with 10 µg/ml goat anti-rat Igs (IgM/IgG/IgA) secondary antibody (Southern Biotechnology Associates). After an incubation of 48 h at 37°C, cell-free supernatants were collected and analyzed for IL-4 content by a specific ELISA (Endogen, Cambridge, MA).
Activation of T cells, immunoprecipitation and immunoblotting
T cells (36 x 106 per group) were incubated in 0.2 ml of ice-cold DMEM for 15 min at 4°C with R2/60 or control 14.2 mAb at 10 µg/ml. Additional cross-linking was achieved by adding goat anti-rat IgM (10 µg/ml) after which the cells were incubated at 37°C for the indicated times. Cells were then washed twice in ice-cold PBS and whole cell extracts were prepared in 1% (v/v) Triton X-100 lysis buffer, as previously described (15,17). For immunoprecipitations, clarified cell lysates (prepared from 68 x 106 cells per group) were incubated with the indicated antibody overnight at 4°C. Immune complexes were harvested with protein ASepharose after a 4 h or overnight incubation at 4°C. After extensive washing, the beads were resuspended in reducing sample buffer and boiled for 5 min. The immunoprecipitates were resolved on 10% SDSPAGE, transferred to PVDF membranes, blocked with 5% nonfat milk in TBS-T (50 mM TBS, pH 7.5, 0.05% Tween 20) and immunoblotted with the indicated antibody in TBS-T. After three washings in TBS-T, membranes were incubated with appropriate HRP-conjugated secondary antibody and developed using SuperSignal substrate (Pierce, Rockford, IL). For direct western blotting, equal amounts of whole cell extracts were resolved on 10% SDSPAGE, immunoblotted with 4G10 mAb, and developed as above.
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Results
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Immobilized anti-CD43 mAb induces an IL-4-dependent proliferative response in Th2 cells
Stimulation of T cells by immobilized or cross-linked TCR or CD3-specific mAb is a useful means of studying T cell activation without the need for accessory cells. To study the effect of CD43 ligation on Th2 cells, D10 cells were activated in microtiter wells coated with R2/60 (anti-CD43) or H57-597 (anti-TCR Cß) mAb. As shown in Fig. 1(A), stimulation with immobilized R2/60 resulted in a dose-dependent proliferative response by D10 cells. The fold-increase in the proliferative response was 36x and 107x in the presence of 2.5 and 25 µg/ml immobilized R2/60 mAb, respectively. The R2/60-driven response was consistently weaker than that observed in anti-TCR-stimulated cells. In contrast, while the addition of soluble anti-TCR mAb had consistently little effect on D10 cell proliferation, soluble anti-CD43 mAb (used at relatively high concentrations e.g. 25 µg/ml) resulted in a low, but significant, level of cellular proliferation, typically representing 2030% of the level observed with immobilized/crosslinked R2/60 mAb (data not shown). D10 cells stimulated with an isotype-matched control mAb (clone 14.2, rat IgM) failed to exhibit any proliferation (data not shown). The anti-CD43-induced proliferation of D10 cells was reduced by 210-fold in the presence of a neutralizing mAb to IL-4 (clone 11B11), suggesting that the response was dependent on endogenously produced IL-4 (Fig. 1B). The effect of CD43 ligation in naive, splenic T lymphocytes was next examined. The data (Fig. 1C) demonstrate that the sole ligation of CD43 on naive T cells also led to a significant level of proliferation. The degree of proliferation was dependent on the dose of R2/60 mAb and, similar to what was observed with D10 T cells, was lower than that induced by anti-CD3 stimulation (Fig. 1C).

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Fig. 1. CD43 ligation induces cell proliferation in an IL-4-dependent manner. (A) D10 cells were stimulated with immobilized anti-CD43 or anti-TCR Cß mAb at the indicated concentrations. Cell proliferation was assessed after a 72 h incubation. (B) Parallel sets of culture were set up with D10 cells stimulated with immobilized anti-CD43 mAb in the presence or absence of 11B11, a neutralizing anti-IL-4 mAb. Each data point represents the mean ± SEM of triplicate cultures. Asterisks denote statistically significant differences between (A) anti-TCR and anti-CD43 stimulated cultures or (B) anti-CD43 stimulated cultures in the presence or absence of 11B11 mAb (**P < 0.01; ***P < 0.001). The data are representative of six independent experiments. (C) Purified splenic T cells were stimulated in triplicate with cross-linked anti-CD3 (1 µg/ml) or anti-CD43 mAb at the indicated final concentrations. The Control group represents the level of proliferation observed in cultures of T cells stimulated with cross-linked, control rat IgM antibody (at a final concentration of 10 µg/ml). Cells were incubated for a total of 72 h and pulsed with tritiated thymidine for the final 18 h. Each data point represents the mean ± SEM of triplicate cultures. The data are representative of two independent experiments.
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Anti-CD43 stimulation delivers an independent activating signal in Th2 cells
Previous reports suggested that triggering through CD43 provides a costimulatory signal to T cells (8,18). We therefore tested whether R2/60 mAb could provide a costimulatory signal to D10 T cells stimulated with a submitogenic dose of anti-TCR Cß mAb. The results show that stimulation of D10 cells with co-immobilized anti-TCR (at 2 µg/ml) and anti-CD43 (at 25 µg/ml) induced a level of proliferation that was indistinguishable from that observed with cells treated with anti-CD43 mAb alone (Fig. 2). Thus, CD43 cross-linking can deliver an independent proliferative signal to Th2 cells.

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Fig. 2. CD43 ligation does not costimulate anti-TCR-induced proliferation of D10 cells. Anti-TCR Cß mAb was immobilized alone or in conjunction with anti-CD43 mAb at the indicated concentrations. D10 cells were plated at 1 x 104 cells per well, incubated for 72 h, and pulsed with [3H]thymidine for the final 18 h of culture. Each data point represents the mean ± SEM of triplicate cultures. The data are representative of three independent experiments.
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CD43-induced proliferation is dependent on functional Lck kinase
The ability of R2/60 mAb to stimulate the proliferation of Lck-deficient D10 cells was next studied. As shown in Fig. 3(A), R2/60 failed to induce any response in Lck-deficient cells. Both parental and Lck-deficient D10 cells express high levels of CD43 (Fig. 3B), ruling out a decrease in CD43 expression as the cause of the non-responsiveness of Lck-deficient cells. To examine the relationship between CD43 and Lck, the state of Lck tyrosine phosphorylation, a correlate of its kinase activity (23), was studied by western blotting. Figure 3(C) shows the level of tyrosine-phosphorylated Lck before and after CD43 ligation. There was a basal level of Lck phosphorylation in non-stimulated D10 cells, which is in accordance with previous data (15), but the level increased within 30 s of CD43 ligation and remained elevated for up to 10 min. As expected, there was no phosphorylated Lck detected in Lck-deficient cells. These data suggest that CD43-induced proliferation requires a functional Lck, and that CD43 ligation results in tyrosine phosphorylation of Lck.

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Fig. 3. CD43-induced proliferation is dependent on functional Lck kinase. (A) Parental and Lck-deficient D10 cells (1 x 104 cells/well) were stimulated with immobilized R2/60 and proliferation was assessed 72 h later. Each data point represents the mean ± SEM of triplicate cultures. Asterisks denote statistically significant differences between the responses of parental and Lck-deficient D10 cells (***P < 0.001). The data are representative of four independent experiments. (B) Flow cytometric analysis of CD43 expression on parental and Lck-deficient D10 cells. Cells were stained with R2/60 or isotype-matched control mAb followed by FITC-conjugated goat anti-rat IgM. The data are representative of six independent experiments. (C) Immunoblot of tyrosine phosphorylated Lck in parental or Lck-deficient D10 cells following CD43 ligation. Cells were incubated with R2/60 and cross-linked with goat anti-rat IgM for the indicated times. Equal amounts of proteins (50 µg/lane) were run on 10% SDSPAGE and immunoblotted with 4G10 antibody. The data are representative of three independent experiments.
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CD43 ligation induces tyrosine phosphorylation of CD43 and association with SLP-76
To study the possible interaction between CD43 and intracellular signaling components, CD43 was immunoprecipitated from R2/60-stimulated cells, run on 10% SDSPAGE and blotted with anti-phosphotyrosine mAb (Fig. 4, top panel). Cross-linking with R2/60 induced tyrosine phosphorylation of CD43 within 30 s, with phosphorylation level peaking at 5 min (lanes 58). Stimulation of D10 cells with control mAb gave no detectable response (data not shown). Phosphorylation of CD43 was clearly dependent on Lck kinase as it was not observed in Lck-deficient cells (lanes 14). Two other proteins of 76 and 88 kDa were co-precipitated with CD43. Since it was previously reported that SLP-76 is associated with CD43 signaling (13), the blot was re-probed with antisera specific to SLP-76. The results (Fig. 4, lower panel) revealed a low level of constitutive association between CD43 and SLP-76 in non-stimulated D10 cells (lower panel, lane 5). However, after CD43 ligation, the level of CD43-associated SLP-76 increased by 6-fold (lower panel, lanes 68) and that, in turn, led to its tyrosine phosphorylation, reaching a maximal level by 5 min (top panel, lanes 68). In Lck-deficient cells, there was also a low level of SLP-76 associated with CD43 constitutively (lower panel, lane 1) and this increased by
3-fold after R2/60 cross-linking (lanes 24). However, there was no phosphorylation of SLP-76 observed in these cells at any time. It is intriguing to note that there appears to be an ordered sequence of tyrosine phosphorylation of the three proteins in CD43 precipitates, with CD43 being phosphorylated first, followed by SLP-76 and finally the 88 kDa protein (top panel). The identity of the 88 kDa protein is unknown. These results suggest that CD43 ligation induces the phosphorylation of CD43 and promotes CD43-SLP-76 association leading to tyrosine phosphorylation of SLP-76 in an Lck-dependent manner.

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Fig. 4. Ligation of CD43 induces tyrosine phosphorylation of CD43 and increased recruitment and phosphorylation of SLP-76 adaptor. Parental and Lck-deficient D10 cells were stimulated with cross-linked R2/60 for the indicated times. Cell lysates were immunoprecipitated with anti-CD43 (S7 mAb), resolved on SDSPAGE, and blotted with 4G10 mAb (top panel). The membrane was stripped and re-probed with anti-SLP-76 antibody (bottom panel). Protein molecular weight markers (in kDa) are indicated. Data are representative of three independent experiments.
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CD43 ligation induces Shc phosphorylation in an Lck-dependent manner
To further investigate the components of CD43 signaling pathway, SLP-76 was immunoprecipitated, resolved on SDSPAGE and probed with anti-phosphotyrosine mAb. As can be seen, SLP-76 was tyrosine phosphorylated after cross-linking with R2/60 (Fig. 5, top panel), but not with the control 14.2 mAb (data not shown). Moreover, a second protein of 52 kDa co-precipitated with SLP-76 and was also tyrosine phosphorylated. The phosporylation of the 52 kDa protein was first observed at 5 min but reached highest level at 10 min after CD43 ligation (Fig. 5, top panel, lanes 68). Based on published reports, we reasoned that the 52 kDa protein might correspond to the adaptor protein Shc (13,24). Re-probing the blot with anti-Shc antibody confirmed the identity of the 52 kDa protein as Shc (Fig. 5, lower panel). Shc was associated with SLP-76 constitutively in both parental and Lck-deficient D10 cells (lower panel, lanes 18). CD43 cross-linking did not increase SLP-76-Shc association, but rather it induced the tyrosine phosphorylation of Shc in a time-dependent manner. Importantly, Shc phosphorylation was not observed in Lck-deficient cells, suggesting that it requires the prior phosphorylation of SLP-76 protein (top panel, lanes 14). Taken together, our data suggest that CD43 ligation induces the formation of a complex consisting of CD43, SLP-76 and Shc proteins, which are tyrosine phosphorylated in a time-dependent manner by the action of the Lck kinase.

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Fig. 5. Lck-dependent tyrosine phosphorylation of Shc following CD43 ligation. Parental and Lck-deficient D10 cells were stimulated with cross-linked R2/60 for the indicated times. Cell lysates were immunoprecipitated with anti-SLP-76 antibody, resolved on SDSPAGE, and blotted with 4G10 (top panel). The membrane was stripped and re-probed with anti-Shc antibody (bottom panel). Protein molecular weight markers (in kDa) are indicated. Data are representative of three independent experiments.
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Lck-independent activation of Th2 cells
It has been reported that CD43 signaling in human T cells is dependent on Fyn kinase (12). Our data so far pointed to Lck as the principal kinase involved in CD43-induced activation in murine Th2 cells. In order to better understand the reason(s) behind this apparent discrepancy, we analyzed the potential role of CD43 in inducing other Th2 effector functions, namely the production of IL-4 and upregulation of activation markers. T cells were stimulated with cross-linked anti-CD43, anti-CD3, or control IgM mAb, and culture supernatants were collected at 48 h and analyzed for IL-4 content. As shown in Fig. 6, ligation of CD43 or CD3 resulted in the production of large amounts of IL-4 (>1.0 ng/ml per 105 cells) in D10 cells. Surprisingly, similar treatment of Lck-deficient cells also led to the secretion of IL-4 in quantities that were indistinguishable from those observed in D10 cells. Thus, CD43-dependent stimulation of IL-4 secretion in Th2 cells appears to be independent of Lck. Furthermore, CD3-triggered IL-4 secretion by Th2 cells is also independent of Lck, as was previously reported (15).

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Fig. 6. CD43 Ligation induces IL-4 production in parental and Lck-deficient D10 cells. Cells were stimulated for 48 h with R2/60 (anti-CD43), YCD3-1 (anti-CD3 ), or as a control, 14.2 (anti-Vß14) mAb, and cross-linked with goat anti-rat Igs secondary. Cell-free supernatants were analyzed for IL-4 content by a specific ELISA. The data represent the mean values ± SEM of two independent experiments.
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Upon activation through the TCR/CD3 complex, T cells rapidly upregulate the surface expression of a variety of proteins, including CD25, CD69 and CD154. We therefore assessed the potential regulation of CD25 and CD69 expression by CD43. Cells were stimulated for 1620 h with cross-linked R2/60, following which cells were stained with mAbs to CD69 and CD25 antigens, and as a control, CD4 protein. The data indicated that CD43 ligation led to the upregulation of CD69 and CD25 proteins on both parental and Lck-deficient D10 cells (Fig. 7A). The expression of CD69 was upregulated by 2-fold and 2.4-fold in D10 and Lck-deficient cells, respectively (Fig. 7A, middle panels). Similarly, the expression of CD25 was increased by 1.7-fold and 3.6-fold after CD43 ligation (Fig. 7A, bottom panels). In contrast, CD4 expression in non-stimulated and CD43-stimulated cells was virtually unchanged (Fig. 7A, top panels). Upregulation of CD25 and CD69 was also studied using purified splenic T cells (Fig. 7B). Incubation with anti-CD43, but not control, mAb resulted in marked increases (3.9-fold and 7.1-fold, respectively) in the expression of CD69 and CD25 antigens on 9096% of normal splenic T cells. These findings suggest that CD43 triggering leads to T cell activation, as assessed by increased expression of CD69 and CD25 antigens, and that this response occurs independently of Lck kinase.

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Fig. 7. CD43 ligation upregulates the expression of CD25 and CD69 antigens by a Lck-independent pathway. (A) D10 cells and their Lck-deficient derivatives were stimulated with cross-linked R2/60 or isotype-matched control mAb for 16 h, washed, and stained with specific mAbs to CD25, CD69 or CD4. Dashed lines indicate background staining with secondary antibody alone, solid lines staining of control-stimulated cells, and filled histograms staining of CD43-stimulated cells. The data are representative of four independent experiments. (B) Purified splenic T cells were stimulated as described above and double-stained with mAbs specific to CD4 and CD25 or CD4 and CD69. The percentage of cells within each quadrant is shown in the dot plots.
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The fact that we observed CD43-induced activation events in Lck-deficient Th2 cells suggests either these events occur independent of tyrosine phosphorylation or, more likely, that they utilize a second pathway involving other tyrosine kinases, such as Fyn (12). To address this issue, we analyzed the total tyrosine phosphorylation pattern in parental and Lck-deficient D10 cells following CD43 triggering. Figure 8 shows an anti-phosphotyrosine blot of the total extracts. Three prominent, tyrosine phosphorylated proteins, p26, p56 and p78, were observed in D10 cells following CD43 ligation. Of these proteins, two (p26 and p78) were also phosphorylated, in a CD43-dependent manner, in Lck-deficient cells. The degree of phosphorylation of p26 and p78 proteins was 2- to 5-fold lower in Lck deficient cells compared to D10 cells. These results suggest that CD43 ligation induces tyrosine phosphorylation of some signaling components through a second pathway that does not involve Lck. Whether this second pathway is responsible for the CD43-mediated effector functions observed in Lck-deficient cells remains unknown at present.

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Fig. 8. Total tyrosine phosphorylation pattern of parental and Lck-deficient D10 cells following CD43 ligation. Cells were incubated with R2/60 and cross-linked with goat anti-rat IgM for the indicated times. Equal amounts of proteins (50 µg/lane) were run on 10% SDSPAGE and immunoblotted with 4G10 antibody. Protein molecular weight markers (in kDa) are indicated. The data are representative of three independent experiments.
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Discussion
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CD43 has been described as a coreceptor molecule involved in different and often contradictory functions, such as enhancement/inhibition of T cell activation (4,6,810) and positive/negative regulation of T cell homing and adhesion (7,25). The inhibitory functions ascribed to CD43 have in general been attributed to the repulsive nature of its extracellular domain arising from the negatively charged sugar groups (6,26). The negative influence of CD43 on T cell activation was shown by the demonstration that T cells of CD43-deficient mice were hyper-responsive to antigenic stimulation (6). However, this view has been challenged by a recent report showing that T cells of CD43 null mice, backcrossed for eight generations onto the C57BL/6 background, behave normally (27). Another recent report demonstrated that failure to displace CD43 from the immunological synapse had no inhibitory effect on T cell activation (28). Moreover, available evidence indicates that the function of CD43 in the different cell types is dependent on its cytoplasmic domain (4,29). Uncovering the molecular interactions involved in CD43 signaling pathway is key to understanding the mechanisms that underlie its many functions.
In this report, we provide evidence that CD43 may induce effector functions via dual signaling pathways. A robust proliferative response was observed following CD43 ligation in parental, but not Lck-deficient, D10 cells. Similarly, CD43 cross-linking induced the tyrosine phosphorylation of CD43, SLP-76 and Shc proteins in D10 cells, but not in Lck-deficient cells. On the other hand, CD43 ligation induced the secretion of IL-4, upregulation of activation markers, and tyrosine phosphorylation of at least two prominent proteins in both cell lines, suggesting that alternative tyrosine kinases may be activated in response to CD43 triggering. Taken together, these data suggest that cell activation through the CD43 molecule may well utilize more than one signaling pathway.
Our present findings suggest that Lck plays a crucial role in CD43 signaling pathway. Tyrosine phosphorylation of CD43 cytoplasmic domain, presumably one of the earliest steps in the pathway, as well as the phosphorylation of adaptor proteins SLP-76 and Shc, required a functional Lck enzyme. It is proposed that Lck may well be the earliest kinase to be activated following CD43 cross-linking. This is akin to the role of Lck in TCR/CD3 signaling pathway. We and others have demonstrated that Lck-deficient T cells have a block in TCR signaling pathway as evidenced by the lack of phosphorylated CD3
chain and ZAP-70 kinase and the absence of calcium flux (15,30). Recent evidence suggests that Lck kinase acts upstream of Fyn and that the activity of the latter kinase is regulated by the former (31). Moreover, using high-speed time-lapse microscopy to visualize green fluorescent protein (GFP)-tagged lck, Ehrlich and co-workers recently demonstrated that, in addition to being associated with immunological synapses, Lck also exists in intracellular pools ready to be translocated to mature synapses as required, thereby insuring the continuous propagation of the signal transduction pathway (32). The available evidence, therefore, supports the notion that Lck plays a predominant role in signaling through both the TCR/CD3 complex and CD43 molecule.
Tyrosine phosphorylation promotes the formation of docking sites for SH2-containing adaptors, such as SLP-76, Shc and Grb-2, thereby linking upstream receptor activation to downstream enzymatic effectors, such as the guanine exchange factor Vav, PLC-
and MAPKs. Moreover, the recruitment of signaling components to the activation site helps in the amplification and diversification of the signal. In this report, we demonstrate that CD43 is tyrosine phosphorylated within 30 s of antibody cross-linking in an Lck-dependent manner. The recruitment and tyrosine phosphorlayion of the SLP-76 adaptor protein was also observed within 30 s of activation. Although we detected a low level of constitutive association between CD43 and SLP-76 in control cells, this association was increased following CD43 ligation, reaching a maximum at 5 min of activation. A similar increase in the degree of CD43-SLP-76 association was also observed in Lck-deficient cells, suggesting that tyrosine phosphorylation of CD43 per se is not required for this association. Similarly, a constitutive association between SLP-76 and Shc was observed in control mAb-treated parental and Lck-deficient D10 cells. Following CD43 ligation, phosphorylation of SLP-76 and Shc occurred only in the presence of Lck, reaching a maximum at 5 and 10 min of activation, respectively. Phosphorylation of SLP-76 occurs in T and B lymphocytes following activation through TCR and BCR, respectively, as well as in myeloid cells stimulated via Fc
RI (3335). The kinase responsible for SLP-76 phosphorylation is thought to be ZAP-70, a Syk family tyrosine kinase (36). Whether ZAP-70 is activated by CD43 cross-linking was not addressed in this study. However, we have previously demonstrated that TCR-mediated tyrosine phosphorylation of ZAP-70 is deficient in our Lck-deficient cell line (15). Our data are consistent with published reports indicating that CD43 ligation on T cells induces tyrosine phosphorylation of SLP-76 and Shc proteins (13).
Previous reports have highlighted the function of CD43 as a co-stimulatory molecule, generally enhancing the proliferation of T cells when added with sub-optimal concentrations of anti-CD3 mAb (8,9). However, under the conditions used in this study, there was no evidence of a synergistic effect when cells were activated with anti-TCR and anti-CD43 mAbs. This suggests that in murine Th2 cells, activation through CD43 proceeds independently of the TCR/CD3 complex and is in line with a previous report using primary human T cells (11). Nevertheless, our data cannot completely rule out the possibility that CD43 may function as a costimulatory molecule under other combinations of stimulatory conditions or antibody concentrations. For example, in this study, we have used anti-TCR Cß mAb in the costimulation assays while in the study by Sperling and colleagues (8) an anti-CD3 mAb was used. The lack of apparent costimulatory activity may simply reflect differences in the affinity of these two mAbs. Importantly, anti-CD43 stimulation of purified splenic T cells also resulted in a dramatic upregulation of CD25 and CD69 antigens, as well as a significant proliferative response, underscoring the physiological relevance of the findings in normal T cell biology. Despite the fact that the overall level of CD43-induced stimulation was not high, we believe it is very significant considering it is observed in the absence of any costimulatory stimulus. The fact that CD43 ligation on Th2 cells induces IL-4 production independent of a TCR signal is intriguing. A specific receptor for CD43 is yet to be identified. Recently, T cells expressing invariant TCR were shown to secrete IL-4 independent of TCR engagement (37). This type of activation might be helpful in innate immunity. However, the importance of CD43-driven IL-4 production in Th2 cells remains unclear. We are currently examining the generalizability of this phenomenon in different T lymphocyte populations.
The CD43-mediated activation of IL-4 secretion, but not proliferation, in Lck-deficient T cells is similar to their previously characterized response to TCR-induced stimulation (15,17). The exact reason(s) for this differential response is not fully understood, but one contributing factor may be related to the increased susceptibility of Lck-deficient cells to apoptosis in the absence of exogenous growth factors (16). Other factors, such as the utilization of different pathways and the requirement for different signaling thresholds for activation may also play a role. It is known for example that unlike the production of IL-2, secretion of IL-4 does not require any costimulatory signals (38). Furthermore, stimulation of Th2 cells by altered peptide ligands, a weak form of TCR agonists, results in IL-4 secretion in the absence of proliferation (39).
In conclusion, we propose that CD43 ligation on Th2 cells leads to the induction of a dual signaling pathway, distinguished by their differential dependence on the expression of functional Lck enzyme. These dual signaling pathways may be responsible for the activation of different effector functions in Th2 lymphocytes.
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Acknowledgements
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We are grateful to C. A. Janeway (Section of Immunobiology, Yale University School of Medicine) for providing serologic reagents and G. Bashir and A. Ullah for excellent technical assistance. This work was funded by a grant from the Terry Fox Fund for Cancer Research (#98/03) to B. K. al-R. Additional support was provided by grant NP/03/10 from the Research Grants Committee of the Faculty of Medicine and Health Sciences, United Arab Emirates University to M. J. F.-C.
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Notes
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Transmitting editor: C. Terhorst
Received 10 April 2004,
accepted 11 June 2004.
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References
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