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Institut National de la Santé et de la Recherche Médicale Unité 503, Centre Européen de Recherche en Virologie et Immunologie, Lyon, France;
Unité Mixte de Recherche 5665, Ecole Normale Supérieure de Lyon, Lyon, France; and
Ludwig Institute for Cancer Research, Melbourne, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cell activation also requires small GTPases from the Rho family proteins that play an important role in the regulation of actin organization and cytoskeletal rearrangements (16, 17). Indeed, morphological changes are crucial all along T cell activation, allowing the cells to migrate through blood vessels, home into lymphoid organs, adhere to target cells, or interact with APCs (18). The actin cytoskeleton also plays a role in T cell activation, permitting the formation of the immune synapse, and the formation of a scaffold for signal transduction molecules (19, 20). GTPases are activated by the recruitment of Tyr-phosphorylated GDP/GTP exchange factors (GEFs)5 to the membrane, allowing their conversion from the inactive GDP-bound to the active GTP-bound state (21). Among GEF, the hemopoietic cell-specific protein Vav is critical for TCR activation (22, 23, 24). It plays a central role in T cell activation-induced actin cytoskeleton rearrangements, leading to a functional interface between APCs and T cells (25). In vitro, Vav catalyzes guanine nucleotide exchange of the Rho family proteins, RhoA (26), Rac (27), and Cdc42 (28). This activity is dependent on its phosphorylation (27, 28), which occurs after TCR stimulation (29), as well as following CD28 costimulation (30, 31). Indeed, CD28 costimulation increases the ZAP-70-mediated Tyr phosphorylation of Vav, and then synergistically induces Rac GDP/GTP exchange that further activates p38 mitogen-activated protein kinase (MAPK) (32).
In this study, we first report that CD46 costimulation leads to a synergistic activation of extracellular signal-regulated kinase (Erk) MAPK, and that indeed the inhibition of this pathway abrogates CD46/CD3 costimulation effect. Furthermore, CD46/CD3 costimulation induces profound changes in cell morphology of human primary T cells, as well as actin relocalization. Importantly, we used fresh primary T cells, and not cell lines, and therefore these results provide new data concerning human T cell biology. We show that CD46 stimulation induces Vav Tyr phosphorylation, and that CD46/CD3 costimulation leads to a synergistic increase of Vav phosphorylation. These results prompted us to investigate whether the GTPases from the Rho family were involved in the signaling cascade induced by CD46/CD3 ligation. We show that Rac is activated upon CD46/CD3 costimulation. These results suggest that the Vav/Rac pathway is a potential candidate for the costimulatory cross-talk between CD46 and TCR. Therefore, CD46 costimulation profoundly affects T cell behavior, and this might be taken in account for its costimulatory effects.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBL were purified from blood of human healthy donors (ETS, Lyon, France) 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 (33).
Antibodies
The mouse mAbs used in this study were 20.6, IgG1 directed against CD46 (7), OKT3, IgG1 directed against CD3 (obtained from the American Type Culture Collection, Manassas, VA), and anti-CD28 Ab (clone CD28.2) that was kindly given by Dr. D. Olive (Marseille, France). Irrelevant IgG1 Abs were purchased at Immunotech, Beckman Coulter (Marseille, France). Affinity-purified rabbit anti-mouse Ig (RaM) were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Anti-Vav Abs, anti-Rac, anti-Erk1/2 MAPK (rabbit Abs), as well as anti-phospho-Erk1/2 were purchased at Upstate Biotechnology (Lake Placid, NY). Rabbit Abs directed to p38 or phospho-p38 were purchased at New England Biolabs (Beverly, MA). Anti-Rho mAb (26C4) was a generous gift from Dr. J. Bertoglio (Paris, France). Anti-Cdc42 mAbs were obtained from Transduction Laboratories (Lexington, KY).
Cell stimulation
Cells were washed twice with RPMI 1640, resuspended in RPMI 1640 at 5 x 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, washed twice with cold RPMI 1640, and then incubated at 37°C with RaM (5 µg/ml) for various periods of time. Finally, cells were lysed with 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 Na3VO4) for 15 min on ice for Vav immunoprecipitation, or with specific lysis buffer for GTP activity assays (10% glycerol, 50 mM Tris (pH 7.4), 1% Triton X-100, 100 mM NaCl, 2 mM MgCl2, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 mM PMSF). After removing insoluble material by centrifugation at 10,000 x g, supernatants were collected and immunoprecipitations or GTP assays were directly performed.
Immunoprecipitations and Western blots
Cell lysates were precleared with protein A-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-Sepharose beads for 1 h at 4°C. After four 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 2-ME), and analyzed by 8% SDS-PAGE. Proteins were then transferred to Immobilon-P 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 other Abs in TBS-T (20 mM Tris (pH 7.6), 130 mM NaCl, and 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 membranes were then stripped and reblotted with another Ab.
GTPase activity assays by affinity precipitation
The Rac and Rho activity assays were performed as previously described (34, 35). Briefly, to evaluate Rac, Rho, and Cdc42 activity, cell lysates were incubated for 1 h at 4°C with 20 µg of GST-Cdc4/Rac interactive binding domain (CRIB), corresponding to the Rac and Cdc42-binding domain of human PAK1B (kindly obtained from Dr. J. Collard, The Netherlands Cancer Institute, Amsterdam, The Netherlands) (34), GST-RBD (Rho-binding domain of mouse Rhotekin; kindly given by Dr. M. Schwartz, The Scripps Research Institute, La Jolla, CA), and GST-ACK42-Lys34 (minimum Cdc42-binding domain of the ACK1 kinase (36) (kindly provided by Dr. H. Maruta, Ludwig Institute for Cancer Research, Melbourne, Australia), respectively, bound to glutathione-coupled Sepharose beads. After several washes with lysis buffer and elution with sample buffer, bound proteins were then loaded on 8% SDS-PAGE and analyzed by Western blot using anti-Rac, anti-Rho, or anti-Cdc42 Abs.
F-actin localization
Cells (1 x 106) were incubated in 12-well plates with coverslips previously coated with anti-CD3 (10 µg/ml) and/or anti-CD46 or anti-CD28 (10 µg/ml) and cultured for 24 h. Cells were then stained with FITC-phalloidin for 20 min at room temperature and extensively washed with PBS. Coverslips were then mounted with a Prolong antifade kit (Molecular Probes, Eugene, OR). Cells were then analyzed by confocal microscopy (LSM510 microscope; Zeiss, Oberkochen, Germany), using a x63 (numerical aperture 1.4) Zeiss Plan Neo Fluor objective.
Proliferation assay
Cells (2 x105) were incubated in 96-well plates coated with anti-CD3 (OKT3) and/or anti-CD46 or anti-CD28 Abs (10 µg/ml), or an irrelevant IgG1, and cultured for 3 days, in the presence or absence of U0126 (mitogen-activated protein/Erk kinase 1 (MEK1) inhibitor; Promega). Cells were then incubated with 1 µCi of [3H]thymidine for 16 h and harvested on 96-filter papers using a Tomtec Instruments (Orange, CT) cell harvester. [3H]Thymidine incorporation was measured using a 1450 Betaplate liquid scintillation counter (Wallac, Gaithersburg, MD).
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Results |
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We previously reported that CD46/CD3 costimulation strongly
promotes T cell proliferation, as compared with CD3 stimulation alone.
We had also shown that LAT was phosphorylated after CD46 stimulation.
In this study, we further investigated the signaling cascade induced by
CD46 and CD46/CD3 stimulation. We analyzed whether Erk1/2 MAPK could be
activated by CD46 stimulation, since Erk MAPK are involved in the
control of proliferation (37, 38). Human PBL were
stimulated with anti-CD46, anti-CD3, anti-CD3 and
anti-CD46, or anti-CD3 and anti-CD28 as a control
for 1, 10, and 30 min before lysis. An aliquot of each cell
lysate was then analyzed by Western blot using anti-phospho-Erk1/2
Abs (Fig. 1
A), which reflect
Erk1/2 activation. The membranes were then analyzed with
anti-Erk1/2 and anti-actin Abs to estimate the levels of
proteins in each lane (Fig. 1, B and C). A very
faint activation of Erk1/2 could be observed when the cells were
stimulated by anti-CD46 alone or by anti-CD3 stimulation alone.
However, CD46/CD3 costimulation induced a more dramatic activation of
Erk1/2 (1 and 10 min, arrow). As a control, we also show that CD3/CD28
costimulation induced a strong Erk activation that was more important
than the one induced by CD3/CD46 costimulation. Therefore, CD46
costimulation induced a synergistic activation of Erk1/2 MAPK.
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, similarly to CD28, addition of U0126
inhibits CD46 costimulation in a dose-response manner, indicating that
stimulation of Erk1/2 is indeed required for CD46 costimulation.
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Furthermore, besides promoting proliferation, CD46/CD3
costimulation also induced morphological changes of human T cells, as
depicted on Fig. 3, in which pictures
have been taken from representative cell cultures. Indeed, whereas
CD3-stimulated or CD28/CD3-costimulated cells grew in clonal suspension
with apparent homotypic aggregates, CD46/CD3-costimulated cells were
not aggregated and adhered to the substratum. In presence of
anti-CD46 alone, the cells were similar to the unstimulated cells
(data not shown). Therefore, CD46 costimulation induces signals that
modulate adhesion of human T cells. Furthermore, when observed at high
magnification (Fig. 4A),
CD46/CD3-costimulated cells appeared spread on the bottom of the well
and exhibited membrane protusions such as lamellipodia and filipodia
(arrows). This effect was not observed when the cells were costimulated
by CD28/CD3, although they were clearly stimulated, as observed by the
enlargement of the cells compared with CD3 stimulation alone (Fig. 4A). These cells remained mainly spherical, although few
cells presented a flatter aspect than CD3-stimulated cells, and some of
them were attached to the substratum. To better characterize the
morphological changes induced after CD46/CD3 costimulation, we analyzed
the actin organization after FITC-phalloidin staining. Cells cultured
with either anti-CD3, anti-CD3 and anti-CD46, or
anti-CD3 and anti-CD28 for 24 h were analyzed by
FITC-phalloidin staining and confocal microscopy. Whereas actin was
detected at the periphery of CD3-stimulated cells, it was found mostly
polarized at one edge in CD28/CD3-stimulated cells. In
CD46/CD3-costimulated cells, actin was found in lamellipodia-like
structures, as well as in thin protusions resembling long
retraction fibers (arrow) usually seen at the rear edge of highly
motile cells. Furthermore, CD46/CD3-costimulated T cells were spread on
the substratum and presented a completely different morphology than
CD3- or CD28/CD3-costimulated T cells that remained mostly spherical
(Fig. 4B).
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CD46 stimulation leads to Vav phosphorylation
Vav has been shown to play a crucial role in T cell activation, as
well as being a GEF for the small GTPases of the Rho family that are
also involved in cytoskeletal rearrangements. As we have previously
shown that CD46 plays an important role in T cell activation, and that
morphological changes occur after costimulation, we investigated
whether Vav was phosphorylated either after CD46 stimulation alone or
after CD46/CD3 costimulation, and after CD28/CD3 costimulation as a
control. Human PBL were stimulated with anti-CD46 mAb,
anti-CD3, both Abs, as well as with anti-CD28 and anti-CD3
for 3 min at 37°C, and then lysed. Vav was immunoprecipitated and
analyzed by anti-P-Tyr immunoblotting. Although CD46 as well as CD3
stimulation induced Vav tyrosine phosphorylation (Fig. 5A, arrow), CD46/CD3
costimulation led to a more dramatic increase of Vav phosphorylation.
The same result was obtained after CD28/CD3 costimulation, as already
described (32). The membrane was then stripped and
reprobed with anti-Vav Abs (Fig. 5A) to confirm that
equivalent amounts of Vav protein had been loaded in each lane.
Therefore, Vav phosphorylation is induced by CD46 and CD3 aggregation
in human PBL, but CD46/CD3 costimulation has a synergistic effect on
Vav phosphorylation.
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B).
Whereas the same level of phosphorylation could be observed after 3 min
of stimulation under both stimuli, CD28/CD3 costimulation induced a
stronger effect on Vav phosphorylation at 10 min. CD46 costimulation induces Rac activation
Once Tyr-phosphorylated, Vav is a GEF for the Rho family proteins
Rac, Rho, and Cdc42 that are involved in TCR activation. We
therefore investigated whether Rac, Rho, and Cdc42 could also be
stimulated after CD46/CD3 costimulation. To analyze Rac, Rho, and Cdc42
activities, cells were stimulated for 3 min with CD3 and/or CD46 or
CD28/CD3 as a control, and their cell lysates were then pulled down
with glutathione-Sepharose beads coupled with GST-fusion
proteins containing the binding domain of each GTPase: GST-CRIB,
GST-RBD, and GST-ACK42 fusion proteins, to precipitate the respective
GTP-bound GTPases. Proteins were then eluted and analyzed by Western
blot using anti-Rac (Fig. 6A), anti-Rho A (Fig. 6B), or anti-Cdc42 Abs (Fig. 6C). As a
loading control, the membranes were stripped and reblotted with
anti-GST Abs, as indicated. Furthermore, an aliquot of each cell
lysate was also loaded on the gel to quantify the amount of each
GTPase. The ratio of GTP/GDP-bound proteins was then determined after
densitometry analysis. Although a basal level of Rac.GTP could be
detected, a 2.5-fold increase was observed when cells were costimulated
by CD46/CD3 or after CD28/CD3 costimulation (densitometric values,
representative of three independent experiments). However, no
activation of Rho or Cdc42 was detected after CD46/CD3 costimulation,
whereas both GTPases were activated after CD28/CD3 costimulation, as
already described (32, 39). CD46 stimulation alone had a
very mild effect on Rac activation compared with CD46/CD3 costimulation
(Fig. 6A, right panel). Therefore, CD46
costimulation differs from CD28 costimulation that induces activation
of each GTPase tested, whereas only Rac is activated by CD46
costimulation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(PKC) that has been recently shown to play an important role in the
CD28/CD3-induced stimulation of MAPK (44). After CD28/CD3
stimulation, Vav promotes PKC translocation from the cytosol to the
membrane and induces its activation in a pathway that is dependent on
Rac and on actin cytoskeleton reorganization. The authors described too
very recently that translocation of PKC in the rafts was also
required for T cell activation (45). Since we report that
Vav and Rac are activated and that actin reorganization is observed
after CD46/CD3 costimulation, one can imagine that a similar
recruitment of PKC occurs after CD46 engagement. Finally, it has
been recently reported that TCR and Rac activation also led to protein
kinase B/AKT activation (46). The authors report
that Rac is implicated in this pathway, connecting the TCR with the
lipid kinase phosphatidylinositol 3-kinase. Further investigations will
tell whether PKC and AKT are also activated under CD46
costimulation. Activation of T cells requires actin cytoskeleton for changing their cellular shape and migration that will allow the formation of the immune synapse (for review, see Refs. 20 and 47). In this study, we show that CD46 stimulation induces Vav phosphorylation, and Vav is implicated in the cytoskeletal reorganization mediated by the TCR (48, 49). Vav might possibly be activated by LAT since it has been reported that phosphorylated LAT plays a role in the recruitment of Vav to the membrane of T cells, enabling Vav to activate Rac (50). Indeed, we show that Rac, a member of the Rho family, is activated by CD46/CD3 costimulation. More importantly, CD46/CD3 costimulation has a synergistic effect on Vav phosphorylation, suggesting a synergistic effect on its activation (27, 28). CD46 activation might act like CD28 by increasing the ZAP-70-mediated Tyr phosphorylation of Vav and then synergistically induces Rac GDP/GTP exchange (32). Although an activation of p38 by CD28/CD3 costimulation has been reported (32), we could not observe any increase of p38 activity after CD46/CD3 ligation compared with CD3 stimulation alone (data not shown).
CD46/CD3 costimulation induced drastic morphological changes of human T cells. Costimulated cells do not grow in homotypic aggregates, but stick to the bottom of the wells. This effect appears to be specific to CD46 since CD28/CD3-costimulated T cells also mainly grow within aggregates, although some cells attend to stick after longer time of culture (data not shown). Regulation of adhesion of human T cells is essential for the immune response. Indeed, circulating lymphocytes have to adhere to components of the extracellular matrix at sites of inflammation and in lymphoid tissues. This requires a regulation of the integrins’ affinity for their ligands, resulting in an alteration of cell spreading. It has been shown that spreading of T cells is specifically dependent on Rac (51). The authors showed that expression of an activated mutant of Rac triggered a dramatic spreading of Jurkat T cells. The Rac-induced spreading is accompanied by cytoskeletal rearrangements, and no effect was observed with Rho- or Cdc42-activated mutants. Similarly, we found that CD46 did not induce any activation of these two last GTPases, only Rac is activated, and we indeed observed a spreading of the cells. Actin localization in purified human T cells after CD46/CD3 costimulation also showed a reorganization of actin within the cells. Importantly, our results were obtained with primary T cells and not Jurkat cells, and therefore importantly reinforce a role for T cell spreading in the immune system. Moreover, it has been reported that in Jurkat T cells, Vav participates in the regulation of cytoskeletal organization, and that its phosphorylation is strongly dependent on adhesion to fibronectin (52). Furthermore, Vav overexpression enhances the formation of lamellipodia and increases the cellular growth rate in an adhesion-dependent manner. When both the TCR and CD46 are engaged, a transduction pathway leads to Vav and Rac activation. One can imagine that CD46 costimulation modulates T cell spreading at sites of inflammation and lymphoid tissues. Indeed, integrin ligation induces the activation of the Erk1/2 MAPK in different cells (53, 54, 55) as well as in T cells (56, 57). Furthermore, recent studies report a role of Erk activation in the modulation of affinity of integrins (58, 59). It will be interesting to analyze now the effects of CD46 on the avidity and affinity of different integrins, as well as the potential migration of T cells costimulated by CD46 and CD3.
Our results demonstrate that CD46 costimulation induces a drastic Vav Tyr phosphorylation and activates the small GTPase Rac. Furthermore, we show drastic morphological changes and actin reorganization in human primary T cells following costimulation. Therefore, CD46/CD3 costimulation profoundly affects T cell behavior. These biochemical events suggest that the Vav/Rac pathway is involved in the cross-talk between TCR and CD46, leading to cytoskeletal and morphological changes, and might help to better understand the way CD46/CD3 costimulation occurs in T cells. Finally, these data could help to define more precisely T cell adhesion function.
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Acknowledgments |
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Footnotes |
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2 Y.Z. and O.D. contributed equally to this work.
3 Current address: Department of Adult Oncology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, MA 02115. E-mail address: Anne_Astier{at}dfci.harvard.edu
4 Address correspondence and reprint requests to Dr. Anne Astier, Institut de la Santé et de la Recherche Médicale Unité 503, Immunobiologie Fondamentale et Clinique, Centre Européen de Recherche en Virologie et Immunologie, 21 avenue Tony Garnier, 69365 Lyon Cédex 07, France. E-mail address: astier{at}cervi-lyon.inserm.fr
5 Abbreviations used in this paper: GEF, GDP/GTP exchange factor; anti-P-Tyr, anti-phosphotyrosine; CRIB, Cdc42/Rac interactive binding domain; Erk, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein/Erk kinase; PKC, protein kinase C; RaM, rabbit anti-mouse Ig; RBD, Rho-binding domain.
Received for publication December 14, 2000. Accepted for publication October 3, 2001.
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