The widely expressed transmembrane molecule CD46 is the complement regulatory receptor for C3b as well as the receptor for several pathogens. Beside its binding functions, CD46 is also able to transduce signals. We showed that CD46 aggregation on human T cells induces p120CBL and linker for activation of T cells (LAT) phosphorylation. These two proteins are adaptor proteins known to regulate TCR signaling. p120CBL is a complex adaptor protein involved in negatively regulating signaling events, whereas LAT is a transmembrane adaptor protein found in glycolipid-enriched microdomains essential for T cell activation. Therefore, we investigated if a CD46/TCR costimulation would affect T cell activation. Indeed, CD46/CD3 costimulation strongly promotes T cell proliferation. Therefore, we propose that CD46 acts as a potent costimulatory molecule for human T cells.
The transmembrane protein CD46 is a complement regulatory protein ubiquitously expressed that acts as a cofactor for the cleavage of the C3b and C4b complement products by factor I and therefore protects cells from lysis by autologous complement (1). It contains four “short consensus repeats” and a region rich in serine, threonine, and proline (STP region) followed by a transmembrane segment and a short cytoplasmic tail at the C terminus. Two intracellular regions, Cyt1 and Cyt2, are generated by alternative splicing. CD46 is also the receptor for the Edmondston strain of measles virus (MV)3 (2, 3), the human herpes virus 6 (4), the M protein of streptococcus (5), and the pili of Neisseria gonorrhoeae and Neisseria meningitidi (6). Recently, it has emerged that besides its binding functions, CD46 could also transduce signals. First, purified pili trigger a calcium increase in target epithelial cells that is blocked by Abs against CD46 (7). Second, GST fusion proteins, corresponding to the two intracytoplasmic parts of CD46, associate with macrophage kinases, leading to the tyrosine phosphorylation of the CD46 cytoplasmic domains (8). Furthermore, expression of human CD46-Cyt1 in mouse macrophages enhances the production of NO in response to MV infection in presence of IFN-γ (9). Finally, MV-infected monocytes and dendritic cells exhibit a decreased ability to produce IL-12, necessary for activating T cells and NK cells (10, 11). Importantly, the suppression of IL-12 production by monocytes was also observed after C3b stimulation or CD46 cross-linking. All these results argue in favor of active transduction pathways mediated by CD46.
Intracellular signaling involves tyrosine phosphorylation of adaptor proteins. These molecules possess no intrinsic enzymatic function, but mediate protein-protein interactions, and therefore couple biochemical events initiated by cell-surface receptors with more distal signaling pathways. Several newly described adaptor molecules have been shown to be crucial for the regulation of signaling events initiated by lymphocyte Ag receptors (12). Upon activation of the TCR, adaptor proteins such as linker for activation of T cells (LAT) and p120CBL among others play an important role. p120CBL has been identified as a negative regulator of kinases from the ZAP-70/Syk family (13, 14), whereas LAT plays a role in the activation of Ras and phospholipase C γ1 (PLCγ1) and therefore propagates TCR signals toward downstream signaling pathways (15, 16). However, efficient T cell activation also requires costimulatory signals. The CD28/CD80-CD86 receptor/ligand system is one of the dominant costimulatory pathways. CD28-deficient mice require high amounts of Ag and repeated stimulation to initiate T cell responses (17). This costimulatory molecule amplifies the signals transduced upon TCR triggering and therefore allows T cell responses at lower numbers of engaged TCR (18).
In this report, we first analyzed if CD46 aggregation could transduce intracellular signals such as tyrosine phosphorylation. We show that CD46 stimulation leads to p120CBL phosphorylation in human PBL. Second, we show that CD46 stimulation leads to the tyrosine phosphorylation of LAT in human T cells. These results prompted us to analyze the effect of a TCR/CD46 costimulation on T cell proliferation. We observed that CD3/CD46 costimulation induced an hyperproliferation of human T cells. These studies provide the first evidences that CD46 aggregation induces intracellular tyrosine phoshorylation of substrates involved upon TCR signaling and suggest that CD46 is involved in the control of proliferation of human T cells and therefore could act as a costimulatory molecule.
Materials and Methods
PBL were purified from blood of human healthy donors by Ficoll/Hypaque and then Percoll centrifugation. Purified T cells were obtained by immunomagnetic bead depletion of B cells, monocytes, and NK cells as previously described (11).
The Abs used in this study were 20.6, IgG1 directed against CD46 (3), and OKT3, IgG1 directed against CD3 (obtained from the American Type Culture Collection, Manassas, VA). Anti-CD28 (CD28.2) was kindly given by Dr. D. Olive (Marseille, France). Irrelevant IgG1 Abs were purchased at Immunotech, Beckman Coulter (Marseille, France). p120CBL (rabbit polyclonal IgG) were purchased at Santa Cruz Biotechnology (Santa Cruz, CA). Anti-LAT Abs were obtained from Upstate Biotechnology (Lake Placid, NY). Affinity-purified rabbit anti-mouse Ig (RαM) were obtained from Jackson ImmunoResearch (West Grove, PA).
Cells were washed twice with RPMI 1640, resuspended in RPMI 1640 at 5 × 106 cells/ml, starved at 37°C for 2 h, and then cooled on ice for 15 min. Cells were then incubated for 15 min on ice with saturating amount of Abs and washed twice with cold RPMI 1640. Cells were then incubated at 37°C with RαM (5 μg/ml) for various periods of time. Finally, cells were lysed in cold lysis buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl, pH 7.6, 5 mM EDTA, 1 mM PMSF, 1 mM iodoacetamide, 5 μM aprotinin, 10 mM NaF, and 1 mM sodium vanadate) for 15 min on ice. After removing insoluble material by centrifugation at 10,000 × g, postnuclear supernatants were either stored at −70°C or immunoprecipitations were directly performed.
Immunoprecipitations and Western blots
Cell lysates were precleared with protein A or G-Sepharose beads (Pharmacia, Piscataway, NJ) and then preincubated with specific Ab for 2 h at 4°C followed by the addition of 50 μl of protein A/G-Sepharose beads for 1 h at 4°C. After 4 washes with lysis buffer, proteins were eluted by boiling with sample buffer (2% SDS, 10% glycerol, 0.1 M Tris, pH 6.8, 0.02% bromophenol blue, with 0.07 M 22-ME), and analyzed by 7.5% SDS-PAGE. Proteins were then transferred to Immobilon-Pô membranes (Millipore, Bedford, MA). Membranes were blocked using 5% BSA for anti-phosphotyrosine (anti-P-Tyr) Abs and with 5% nonfat dried milk for all others Abs in TBS-T (20 mM Tris, pH 7.6, 130 mM NaCl, 0.1% Tween-20) and incubated for 1 h with specific Abs. Immunoreactive bands were visualized by using secondary HRP-conjugated Abs (Promega, Madison, WI) and chemiluminescence (ECL; Amersham, Little Chalfont, U.K.). The membrane was then stripped and reblotted with another Ab. In some experiments, the membrane was directly incubated with another Ab, revealed using secondary alkaline phosphatase-conjugated Abs (Sigma, St. Louis, MO) and chemifluorescence (ECF; Amersham).
A total of 2 × 105 cells were incubated in 96-well plates coated with anti-CD3 (OKT3) (10 μg/ml or as indicated) and/or anti-CD46 (20.6) or anti-CD28 Abs (10 μg/ml), or an irrelevant IgG1, and cultured for various periods of time. Cells were then incubated with 1 μCi [3H]thymidine for 16 h and harvested on 96-filter papers using a Tomtec Instruments cell harvester (Orange, CT). [3H]Thymidine incorporation was measured using a 1450 Betaplate Liquid Scintillation Counter (Wallac, Gaithersburg, MD).
CD46 stimulation leads to intracellular tyrosine phoshorylation
To investigate if CD46 was indeed able to induce intracellular tyrosine phoshorylation, human PBL were stimulated with anti-CD46 mAb, or a control IgG1, for various periods of time and then lysed. Anti-phosphotyrosine (anti-P-Tyr) immunoprecipitations were then performed on cellular lysates and analyzed by Western blot using anti-P-Tyr Abs (Fig. 1). Upon CD46 stimulation, tyros-ine-phosphorylated proteins ranging around 120, 70, 38, and 30 kDa could be immunoprecipitated, as soon as after 3 min of stimulation, whereas such proteins could not be detected in the controls (IgG1-stimulated cells). Therefore, CD46 aggregation on human PBL leads to intracellular tyrosine phosphorylation of several proteins.
CD46 stimulation leads to p120CBL phosphorylation
Among p120 proteins in PBL, p120CBL has been described to be tyrosine phosphorylated under various stimuli such as TCR stimulation (19). Therefore, we investigated if p120CBL was tyrosine phosphorylated under CD46 stimulation. Human PBL were stimulated with anti-CD46 mAb, or a control IgG1, for various periods of time and then lysed. p120CBL was immunoprecipitated and analyzed by anti-P-Tyr immunoblotting. There was a time-dependent increase in the tyrosine phosphorylation of p120CBL (Fig. 2,A, arrow) when PBL were specifically stimulated by 20.6 and not by the IgG1 control Ab. The membranes were then stripped and reprobed with anti-CBL Abs (Fig. 2 B) to confirm that equivalent amounts of p120CBL were loaded in each lane. Therefore, p120CBL phosphorylation was induced by CD46 aggregation in human PBL.
CD46 stimulation induces LAT phosphorylation
When anti-P-Tyr immunoprecipitations were performed on CD46-stimulated PBL, a 36- to 38-kDa P-Tyr protein could be oberved (Fig. 1, arrow), whereas such protein could not be detected in the controls (IgG1-stimulated cells). Upon TCR stimulation, a major phosphorylated protein is LAT, previously known as pp36/38. It is a transmembrane adaptor protein that once phosphorylated recruits critical signaling molecules to the membrane. Thus, we investigated if the p36–38 protein observed upon CD46 aggregation could be LAT. Purified human T cells were stimulated with anti-CD46 mAb, or a control IgG1, for various periods of time and lysed. LAT was immunoprecipitated and analyzed by Western blot with anti-P-Tyr Abs (Fig. 3,A). Phosphorylated LAT was detected in specifically CD46-stimulated T cells (arrow). LAT tyrosine phosphorylation was maximal at 3 min and decreased thereafter. The bottom part of the membrane was reblotted with LAT Abs to confirm that equivalent amounts of proteins were loaded in each lane (Fig. 3 B). These results indicated that in human T cells, CD46 stimulation leads to the phosphorylation of LAT adaptor protein.
CD46/CD3 costimulation promotes proliferation of human T cells
CD46 aggregation leads to the tyrosine phoshorylation of two adaptors proteins involved in the regulation of TCR signaling. Therefore, we investigated if CD46 stimulation could modulate T cell activation. Human purified T cells were cultured for 3 or 5 days on 96-wells plates coated with anti-CD46, anti-CD3, both Abs, or CD3 and an irrelevant IgG1 as a control. Proliferation was evaluated by [3H]thymidine incorporation. As shown in Fig. 4,A, CD3 stimulation induced T cell proliferation. The same level of proliferation was observed when the cells were costimulated with anti-CD3 and an irrelevant IgG1. However, CD3/CD46 costimulation induced a 2-fold increased proliferation at day 3 (D3), and this increase was even more drastic at day 5 (D5), reaching a 5-fold increase. Therefore, CD3/CD46 costimulation strongly promotes T cell proliferation, leading to a 5-fold increase of thymidine uptake, as compared with CD3 stimulation alone. We also compared the costimulation effects obtained with CD46 and CD28. Human T cells were cultured for 3 days with various amount of anti-CD3 (ranging from 10 to 1 μg/ml) or/and anti-CD28 or anti-CD46 (10 μg/ml) and proliferation was evaluated (Fig. 4 B). Although for the lower concentrations of anti-CD3 only CD28 has a costimulatory effect, CD46 costimulation was very similar to CD28 for high concentrations of anti-CD3. However, there might be a difference due to different donors because a very similar proliferation was obtained with CD46 and CD28 for one donor even at low doses of anti-CD3 (data not shown).
CD46, first identified as the complement receptor for C3b, was also identified later as the receptor for many pathogenic elements, such as MV, human herpes virus 6, N. gonorrhoeae, N. meningitidi, and the M protein of streptococcus. The hypothesis that CD46 could transduce a signal was suggested when the inhibition of production of IL-12 by MV-infected monocytes could be reproduced by C3b or anti-CD46 Abs (10). Other hints were the induction of a calcium flux after the binding of bacteria pili on CD46 (7), as well as the association of macrophage kinases with GST fusion proteins corresponding to the two intracytoplasmic isoforms (8). However, to our knowledge, no data reported identified substrates phosphorylated upon CD46 triggering. Herein, we show that CD46 ligation in human PBL induces tyrosine phoshorylation of intracellular proteins. We identified one of these proteins as being p120CBL. This protein is the cellular homologue of the transforming protein of the murine Cas NS-1 retrovirus, and is tyrosine phosphorylated in response to a wide variety of cytokines and growth factors and after engagement of Ag receptors on B and T cells (20, 21, 22). Although the exact role of p120CBL in signaling is still unknown, recent data indicate inhibitory effects. Its binds through its N-terminal phosphotyrosine-binding domain to the negative-regulatory P-Tyr323 of Syk and P-Tyr292 of ZAP-70 (23, 24, 25). A role for p120CBL in TCR signaling has been shown with p120CBL-deficient mice. These mice displayed important hemopoietic changes such as modified TCR expression, lymphoid hyperplasia, and hyperphosphorylation of ZAP-70, SLP-76, and LAT, indicative of a role for p120CBL in negatively regulating signaling events that control cell growth (13). Finally, recent studies suggest that p120CBL plays a role in ligand-induced ubiquitination, down-regulation, and/or lysosomal targeting of receptors (epidermal growth factor and platelet-derived growth factor receptors) (26, 27, 28, 29, 30). Therefore, regulation of p120CBL function in vivo may set the threshold for T cell activation by regulating TCR down-regulation. Because CD46 stimulation induced p120CBL phosphorylation, it will be worth studying the ubiquitination and degradation of CD46, since this receptor is down-regulated after MV infection (31). Furthermore, CD46 down-regulation requires a cytoplasmic Tyr-X-X-Leu sequence that resembles known motifs for membrane protein trafficking and receptor signaling (32). One hypothesis might be that p120CBL plays a role in this down-regulation, leading to CD46 ubiquitination and degradation.
We also show that CD46 aggregation induces LAT phosphorylation. This transmembrane adapter protein is expressed in T cells, NK cells, mast cells, and platelets (15). It is phosphorylated by ZAP-70/Syk family kinases (15, 33) and then recruits an array of critical signaling molecules to the membrane (PLCγ1, Grb2, Grap, p85, phosphatidylinositol 3-kinase, SOS, and SLP-76). Mutation of the Grb2 Src homology 2 binding sites leads to an inhibition of the TCR-mediated NF-AT/AP-1 activation (15). Furthermore, studies using the LAT-deficient J.CAM2 Jurkat T cell line have shown that LAT is essential in coupling the TCR to PLCγ1-Ca2+ and Ras signaling pathways (16). Interestingly, LAT is targeted to glycolipid-enriched microdomains (GEMs, also called rafts), and this is required for an efficient LAT phosphorylation and recruitment of critical signaling molecules (34, 35). TCR stimulation results in the enrichment in GEMs of critical signaling molecules, including p23 and P-Tyr forms of ZAP-70 and PLCγ1, suggesting an important role for membrane compartmentation in T cell activation (36). Because CD46 aggregation leads to LAT phosphorylation, it would be interesting to analyze the localization of CD46 and its potential targeting to the GEMs, as well as the recruitment of other signaling proteins. Indeed, the costimulatory effects of CD28 on T cell activation are due to a redistribution of GEMs at the site of TCR engagements, resulting in an increased and more stable tyrosine phosphorylation of the substrates, including LAT (37). One might hypothesize that CD46 plays a similar role. In CD46-stimulated human T cells, tyrosine-phosphorylated proteins ranging around 120, 70, 38, and 30 kDa could be immunoprecipitated (Fig. 1). The 120-kDa protein corresponds to p120CBL, whereas the 38-kDa protein is LAT. The other proteins haven’t been identified yet.
Therefore, CD46 aggregation induces the tyrosine phoshorylation of two adaptor proteins, p120CBL and LAT, respectively involved in the negative regulation or positive amplification of TCR signaling. These results prompted us to examine the role of CD46 on T cell activation. Proliferation assays showed that CD3/CD46 costimulation promotes T cell proliferation as compared with the proliferation induced by TCR triggering alone. This result suggests that the LAT cascade is more likely activated and leads to a further activation of the cells. Therefore, we propose that CD46 acts as a costimulatory molecule for T cell activation. It has not been clearly defined if CD46 could recognize C3b bound to pathogens. However, if CD46 only recognizes unbound C3b, deposited at the cell membrane, a T cell will be specifically stimulated through its TCR and through CD46 by the excess of C3b. Therefore, CD46 triggering will induce a massive proliferation of specific T cells. This would result in an expansion of specific T cells, allowing a rapid elimination of the pathogen. Thus, one can imagine that CD46 aggregation in human T cells leads to a positive signaling pathway through the phosphorylation of LAT. This signal would then be turned off by p120CBL phosphorylation that will lead to ubiquitination and degradation of the receptor.
Our result demonstrate that CD46 stimulation induces tyrosine phosphorylation of crucial substrates in T cells and that, more importantly, it could act as a potent costimulatory molecule for human T cells.
We are grateful to Dr. N. Bonnefoy, Professor J. P. Revillard, and Dr. V. Lotteau for helpful discussions and thank J. Marie, P. O. Vidalain, and S. Desforges for their help.
This work was supported by Institut National de la Santé et de la Recherche Médicale, Association pour la Recherche sur le Cancer, Ligue Nationale contre le Cancer, Programme de Recherche Fondamentale en Microbiologie et Maladies Infectiouses et Parasitaires, and Région Rhone-Alpes.
Abbreviations used in this paper: MV, measles virus; LAT, linker for activation of T cells; RαM, rabbit anti-mouse Ig; P-Tyr, phosphotyrosine; PLCγ1, phospholipase Cγ1; GEM, glycolipid-enriched microdomain.