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*
Molecular Immunology Unit, Department of Immunology, and
Biology of Cellular Interactions Unit, Institut Pasteur, Paris, France; and
Institut National de la Santé et de la Recherche Médicale, Unit 119, Marseille, France.
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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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B
(10). These data have suggested that CD28 controls a
specific pathway integrating downstream with TCR-generated signals.
However, other observations indicate a modification of this view. Thus,
prolonged Ag presentation can compensate for a deficient T cell
response in CD28-/- mice (11).
These mice show reduced IL-4 production and Th2 polarization (3, 12), which may be explained by a defect in the duration of the
activation signal (13). Moreover, strong stimulatory
conditions through the TCR (e.g., solid-phase bound peptide/MHC
complexes (14)) can induce efficient lymphokine production
and proliferation of primary T cells without CD28 costimulation. These
observations suggest that CD28 provides a quantitative contribution to
increase the intensity and/or the duration of TCR-mediated signals.
More recently, another series of observations indicate that CD28
facilitates early signaling events controlled by the TCR. Indeed, CD28
lowers the threshold of the number of triggered TCRs required for
Ag-induced activation (15) and considerably enhances
tyrosine phosphorylation of TCR-controlled signaling effectors
(16). In addition, CD28 is critical for the formation of
large aggregates of glycolipid-enriched membranes
(GEMs)4
(17), which are required for TCR-mediated activation
(18, 19, 20). Altogether, these observations suggest, on the
one hand, that the TCR is not intrinsically disconnected from some
signaling effectors needed for cytokine gene expression and, on the
other hand, that CD28 may facilitate this task perhaps through an
efficient utilization of effectors shared with the TCR. Indeed, CD28
and the TCR have a number of signaling elements in common (e.g., Src
and Tec family protein tyrosine kinases, PI3K, Vav-1, Cbl, and perhaps
the adapter Grb-2) (21, 22), though some others, like
ZAP-70, LAT, and SLP-76, are activated uniquely through the TCR
(23, 24). One potential candidate contributing to enhance
TCR signaling by CD28 may be Vav-1, which is inducibly phosphorylated
upon CD28 engagement (24, 25). Vav-1 phosphorylation has
been correlated with up-regulation of its exchange activity (through
its Dbl-like, DH domain) for the Rho GTPase family and preferentially
for Rac-1 (26, 27). Rac-1 may provide linkages to multiple
effector pathways, including actin cytoskeletal rearrangements and gene
expression taking place during T cell activation (28, 29).
Indeed, recent studies have revealed that T cells from
Vav-1-/- mice are defective in
activation-dependent actin polymerization, intracellular
Ca2+ rise, and activation of extracellular
signal-regulated kinase (ERK), NF-AT, and NF-B
(30, 31, 32). Consistently, Vav-1 overexpression in Jurkat
cells facilitates activation of NF-AT (33) and NF-B
(34), in combination with SLP-76 (35) and
PKC-
(34), respectively. Moreover, binding of Vav-1 to
SLP-76 has been shown to enhance actin polymerization
(36). We showed previously that the T cell line Jurkat is
a valuable model for investigating the specific role of CD28 in
facilitating TCR triggering under physiological activation conditions
(e.g., staphylococcus enterotoxin E (SEE)-pulsed B7-1-bearing APCs)
(16), and, in the present study, we utilized this system
to ask whether Vav-1 contributes to CD28 signaling. We show that Vav-1
strongly enhanced NF-AT activation when overexpressed in cells
simultaneously stimulated via CD28 and the TCR. Surprisingly, we
observed that CD28 ligation alone in cells overexpressing Vav-1 was
able to drive NF-AT activation, an event normally controlled by the
TCR. This effect required the Src-homology domain 2 (SH2) of Vav-1 and
an increase in tyrosine phosphorylation of SLP-76, suggesting that CD28
acts via Vav-1 to reinforce and integrate signals derived from the TCR.
Finally, Vav-1 induced in Jurkat cells substantial alterations in
cortical actin with the formation of lamellipodia and microspikes, a
modification that may also contribute to facilitate TCR and CD28
signaling. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human leukemia Jurkat T cell line (clone J77Cl20) and its derivatives CH7C17 (37), 31.13 (defective for TCR/CD3 surface expression due to the lack of the TCR ß-chain transcript (38)), and ßWT160 (31.13 stably transfected with the TCR ß-chain (38)) were maintained in RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Cergy-Pontoise, France). ßWT160 cultures were additionally supplemented with 2 mg/ml of G418 (Life Technologies). CH7C17 cells reconstituted with CD28 wild-type or a CD28 Del.30 mutant lacking residues 173 to 202 were grown in the same medium supplemented with 4 mg/ml G418, 400 µg/ml hygromycin B (Sigma, St. Louis, MO) and 4 µg/ml puromycin (selective RPMI medium). Dap-3 (Dap) and 5-3.1 L cells expressing HLA-DRB1*0101 (531) (39) were cultured in complete DMEM medium; Dap and 531 cells expressing human B7-1 (Dap-B7 and 531-B7) (39) were cultured in the same medium supplemented with 50 µg/ml hygromycin B. All the cell lines were routinely checked for expression of relevant surface markers by FACS analysis.
Abs and superantigen
The following mouse mAbs were used: anti-phosphotyrosine (4G10) (Upstate Biotechnology, Lake Placid, NY); anti-c-myc (9E10) (Roche Diagnostics, Meylan, France); anti-CD28 (CD28.2) (Coulter Marseille, France); anti-CD3 (OKT3) and anti-HLA-I (W6/32) from America Type Culture Collection (Manassas, VA). Rabbit polyclonal antisera were: anti-VSV-G, previously described (40) and anti SLP-76 (M14), directed against a synthetic peptide (from Neosystem SA, Strasbourg, France) corresponding to the first 12 residues of human SLP-76, generated in our laboratory. Sheep polyclonal anti-SLP76 was from Upstate Biotechnology. Human CTLA4-Ig was kindly provided by Dr. P. S. Linsley (Bristol-Myers Squibb, Seattle, WA). SEE was purchased from Toxin Technology (Sarasota, FL). Cyclosporin A (CsA) was purchased from Novartis (Rueil-Malmaison, France).
Plasmids and constructs
The NF-AT-luciferase (NF-AT-luc) and pSV-ßgal reporter vectors
and pSR
vector containing the VSV-tagged ZAP-70 or ZAP-70KD mutant
were previously described (41). pEF-Bos and pEF-Bos
encoding Flag-tagged SLP-76 (42) were kindly provided by
Dr. D. Cantrell (Imperial Cancer Research Fund, London, U.K.) and
G. A. Koretzky (University of Iowa, Iowa City, IA), respectively.
pEF-Bos expressing N-terminal (33) or C-terminal
(43) myc-tagged Vav-1 were a kind gift of Drs. A. Weiss
(University of California, San Francisco, CA) and M. Deckert (La Jolla
Institute, San Diego, CA), respectively. Vav-1 deletion mutants
SH2/SH3c and SH3c were derived from N-terminal myc-tagged Vav-1
by replacing Trp671 and
Tyr784 codons by a stop codon, respectively,
using Stratagene QuickChange site-directed mutagenesis kit
(Ozyme, Montigny-le-Bretonneux, France) according to the
manufacturer’s instructions. The primers used were:
5'-(p)GTGGGAAGCACAAAGTAGTTTGGCACAGCCAAAG-3' and
5'-(p)CTTTGGCTGTGCCAAACTACTTTGTGCTTCCCAC-3', for SH2/SH3c;
5'-(p)CCTGTCTGTTCATCTCTGATACGCAGGCCCCATGG-3' and
5'-(p)CCATGGGGCCTGCGTATCAGAGATGAACAGACAGG-3', for SH3c. The
entire sequence of the mutants was verified by DNA sequencing.
pHßAPr-1-neo encoding wild type or truncated CD28 (CD28 Del.30) were
described previously (44).
Cell transfections
Transient transfections were performed by electroporating 107 Jurkat cells in 0.4 ml of RPMI 1640 supplemented with 20% FCS at 260 V, 960 µF, with the indicated amounts of expression vectors together with 10 µg of the NF-AT-luc reporter plasmid and 25 µg of pSV-ßgal plasmid using a Gene Pulser apparatus (Bio-Rad, Ivry sur Seine, France). The total amount of DNA was equalized with empty vector. After 24 h, cells were left unstimulated or stimulated at 37°C for 8 h with confluent 531 or 531-B7 (incubated overnight with or without the indicated amounts of SEE) or with PMA (12.5 ng/ml) and the calcium ionophore A23187 (0.5 µg/ml) (both from Sigma) in flat-bottom 96-well plates. ß-galactosidase and luciferase assays (Promega, Madison, WI) were performed according to the manufacturer’s instructions. Luciferase activity, determined in triplicate samples, was measured using an automated luminometer (Lumat LB9501; EG & G Berthold, Widbad, Germany) and was normalized to the ß-galactosidase values. Results were expressed as the percentage of maximal response (obtained with PMA and A23187) or fold of induction, using as a reference the value of unstimulated cells transfected with empty vector. Stable transfectants expressing CD28 wild type (WT) or CD28 Del.30 were obtained as follows; CH7C17 cells, which do not express CD28, were transfected with 30 µg of pHßAPr-1-neo-CD28WT or pHßAPr-1-neo-CD28 Del.30 as indicated above. After 48 h, cells were placed in 96-well flat-bottom culture plates in selective RPMI medium. Transfectants were analyzed for expression of the T cell markers CD3, CD4, CD28, CD11a/CD18, and CD45 by FACS analysis.
Immunoblot and immunoprecipitation
For determining protein expression, transiently transfected Jurkat cells (5 x 105) were washed in PBS and lysed at 4°C for 30 min in 1% Nonidet P-40 lysis buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM MgCl2, 1 mM EGTA in the presence of inhibitors of proteases and phosphatases: 10 µg/ml leupeptin, 10 mg/ml aprotinin, 1 mM Pefabloc-sc, 50 mM NaF, 10 mM Na4P2O7 and 1 mM NaVO4. Postnuclear lysates were boiled and analyzed on 7% SDS-PAGE. For immunoprecipitation, 1–2 x 107 transfected cells were incubated in suspension at 3:1 Jurkat/531 or 531-B7 ratio at 37°C for 1.5 min, centrifugated, and immediately lysed. Postnuclear lysates were incubated for 2 h with either anti-c-myc (2 µg) or goat anti-SLP-76 (6 µg) preadsorbed to protein A- or protein G-Sepharose, respectively (Pharmacia Biotech, Uppsala, Sweden). Immunoprecipitates were washed twice in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, twice in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and boiled in SDS sample buffer. Immunoblotting and detection of proteins by enhanced chemiluminescence (Amersham Pharmacia Biotech) were performed as described previously (45).
Immunofluorescence confocal microscopy
Cells transfected with myc-tagged Vav-1 were incubated at 37°C for 5 min then attached to polylysine-coated coverglasses. Cells were then fixed in PBS containing PFA 3.7% and 30 mM sucrose for 10 min at room temperature. After neutralization of aldehydes groups with PBS/NH4Cl 50 mM for 10 min, fixed cells were permeabilized with PBS/saponine (0.05%) for 10 min and incubated with anti-c-myc in PBS/saponine containing BSA 1 mg/ml (permeabilizing buffer) on ice for 30 min. Cells were then washed three times in permeabilizing buffer and incubated with Phalloidin/Texas Red (Molecular Probes, Eugene, OR) and anti-IgG1-FITC on ice for 30 min (Southern Biotechnology Associates, Birmingham, AL). After three washes in permeabilizing buffer and one wash in PBS, coverglasses were mounted on microscope slides in 100 mg/ml Mowiol (Calbiochem, La Jolla, CA), 25% glycerol, 100 mM Tris-HCl, pH 8.5 containing 100 mg/ml Dabco (Sigma). Immunofluorescence analyses were performed with a Zeiss confocal microscope (LSM510) using 63x/1.4 objective lens, at 0.5-µm intervals and sequential acquisitions of FITC and Texas Red emissions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate whether Vav-1 plays a role in CD28-mediated
enhancement of TCR signaling, Jurkat cells
(CD28+) were transiently transfected to
overexpress Vav-1 and subjected to stimulation by SEE-pulsed APCs
bearing or lacking human B7-1. NF-AT-driven gene transcription was
monitored as a read-out of cellular activation
(46). Cells mock transfected with empty vector responded
poorly to SEE presented by APC lacking B7-1 (531) (Fig. 1
A) and, in agreement with
previous results (16), activation was enhanced by 531
cells expressing B7-1 (531-B7). Vav-1 overexpression caused an
augmentation of the response to SEE in the absence of B7-1
costimulation, in keeping with the notion that Vav-1 is a positive
regulator of TCR-directed cellular activation (33).
However, a much greater increase in NF-AT activation (at least 50-fold)
was obtained when cells overexpressing Vav-1 were also subjected to
costimulation by B7-1 (Fig. 1A). This effect was so potent
that 531-B7 cells without SEE sufficed to induce a sizeable increase of
NF-AT activation, which, in part, contributed to the shift in the
dose-response to SEE (Fig. 1A). CD28 ligation was
responsible for it (and, by inference, for the potentiation of the
response to SEE) as CTLA4-Ig, an antagonist of CD28 binding to B7
ligands, completely inhibited NF-AT activation by 531-B7 cells (Fig. 1B). The same results were obtained by using as stimulators
Dap cells expressing B7-1 (Fig. 1B). Anti-CD28.2 mAb caused
a similar NF-AT activation in Jurkat cells overexpressing Vav-1,
whereas a control anti-MHC class I mAb did not (Fig. 1C). CsA completely inhibited CD28/Vav-1 induced NF-AT (Fig. 1D) indicating the implication of the calcineurin pathway.
These data suggested that upon CD28 triggering by B7, Vav-1 was able to
enhance NF-AT activation, an event known to be normally elicited by TCR
rather than CD28 ligation (47). Recent work has shown that
one of the consequences of CD28 triggering is to favor the initiation
and persistence of the most TCR-proximal signaling events (16, 17), thus enhancing TCR-controlled pathways (e.g.,
Ca2+/calcineurin and mitogen-activated protein
kinases) leading to NF-AT activation (16). It was
therefore interesting to consider that Vav-1 may represent a potential
link between CD28 and the TCR signaling pathways and to clarify the
mechanism by which Vav-1, in combination with CD28 engagement, induced
NF-AT activation.
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First, we investigated whether a direct link existed between CD28
signaling capacity and Vav-1 overexpression to induce NF-AT activation.
Toward this end, a Jurkat clone variant CH7C17 (37) that
had lost expression of CD28 (CD28 Neg) was transfected with wild-type
CD28 (CD28 WT) or a truncated mutant of it (CD28 Del.30) lacking the
sequence coding for the last 30 residues of the intracellular tail of
the protein (44). CD28 Del.30 protein does not promote
Vav-1 tyrosine phosphorylation when expressed in a T cell hybridoma
(25) nor activation of the IL-2 gene when coengaged with
the TCR (44) and similar results were obtained with CH7C17
cells reconstituted with this mutant (our unpublished data). Cell
surface expression of CD28 in CD28 WT and CD28 Del.30 cells was similar
(Fig. 2B, upper
right). Moreover, CD3 (Fig. 2B, lower
right) as well as CD45, CD11a/CD18 (data not shown) were
also expressed at comparable levels in the cell lines utilized. Vav-1
overexpression caused NF-AT activation only in CD28 WT cells in
response to stimulation by 531-B7 cells, whereas no activation occurred
in either CD28 Del.30 cells or CD28 Neg (Fig. 2A,
left). Transfected Vav-1, detected by an anti-myc
epitope Ab was present at comparable levels in each cell line (Fig. 2A, inset). These results were reproduced with
two additional independent transfectants of CH7C17 cells expressing
CD28 wild-type and the Del.30 mutant, respectively (data not shown).
These data are consistent with the idea of a link between CD28
intracellular signaling and Vav-1 to induce activation of NF-AT.
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NF-AT activation is, however, normally undetectable upon ligation
of CD28 alone by B7-bearing APC (Fig. 1
, A and B,
and Ref. 16). Thus, overexpression of Vav-1 might favor
initiation of the TCR-directed signaling cascade by CD28. If this
scenario was correct, then NF-AT activation should be observed only in
the presence of TCR cell surface expression (33) and
should be inhibited by blocking a TCR-proximal signaling event. The
results reported in Fig. 3 show that both
predictions were correct. Indeed, the CD28/Vav-1-induced NF-AT increase
was dramatically reduced in a TCR-negative Jurkat cell (31.13) compared
with the same clone reconstituted for TCR surface expression (WT160)
(Fig. 3A). Furthermore, expression of a kinase-defective
dominant mutant of ZAP-70 (ZAP-70KD) (41) blocked
CD28/Vav-1-induced activation (Fig. 3B). Vav-1
overexpression was similar in all conditions and in all cell lines
tested (Fig. 3, A and B, lower)
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C shows that this
is indeed the case. It is worth noting, however, that the intensity of
SLP-76 phosphorylation is underestimated as only 20–30% of Jur-kat
cells were transfected. Overexpression of Vav-1 allowed a slight basal
increase of SLP-76 phosphorylation (see Fig. 5C).
Importantly, B7-1 stimulation induced a strong increase in the tyrosine
phosphorylation of transfected Vav-1 (Fig. 3C), a
modification thought to correlate with augmented Vav-1 exchange
activity (26, 27). Taken together, these data indicate
that CD28 engagement under conditions of augmented action of Vav-1
(e.g., overexpression) directly potentiates the functioning of
TCR-proximal signaling events.
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Overexpression of Vav-1 could provoke a partial activation level
of TCR-controlled signals (33) and CD28 triggering could
provide an additional input necessary for amplifying NF-AT activation.
If this hypothesis was correct, then overexpression of TCR-controlled
signaling proteins which are placed upstream of, or interact with,
Vav-1 could surrogate overexpression of Vav-1. To this aim, we tested
ZAP-70 and the adapter protein SLP-76, two key elements required for
NF-AT-induced transcriptional activation (48). Similarly
to Vav-1, both proteins are tyrosine phosphorylated following TCR
ligation (49, 50) and when overexpressed in Jurkat cells
they cause an increase of basal (40, 42) and Fig. 1, A and B, and Fig. 4, A and B) as well
as of TCR-driven NF-AT activity (40, 42). Fig. 4, A and B, shows that overexpression of ZAP-70 or
SLP-76 (detected by anti-tag and anti-SLP-76 Abs, respectively, see
small insets in Fig. 4, A and B)
caused a slight NF-AT activation in cells left in medium or in the
presence of 531 cells compared with mock transfected cells. This
increase was of similar magnitude to that measured in Vav-1 transfected
cells. Under these conditions, overexpression of Vav-1, ZAP-70 and
SLP-76 individually potentiated TCR-driven NF-AT activation (data not
shown). However, in contrast to Vav-1, ZAP-70 or SLP-76 were unable to
induce NF-AT activation after CD28 stimulation with 531-B7 cells (Fig. 4, A and B). These results were consistent with
the observation that neither ZAP-70 nor SLP-76 appear to be signaling
proteins dedicated to the CD28 specific pathway (24, 51)
and suggested that CD28 utilizes a unique signaling connection which
together with Vav-1 enhances T cell activation.
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To investigate further the mechanism for potentiation of TCR
signaling via CD28 mediated by Vav-1, we examined the effect of
eliminating (or reducing) the interaction with the latter with other
signaling proteins. Previous studies have indicated that the SH2 domain
of Vav-1 is critical for activation of NF-AT (35) and for
the interaction with the adapter protein SLP-76 (23, 35, 52). Fig. 5A shows that
overexpression of a Vav-1 deletion mutant lacking its SH2 and
C-terminal SH3 domain (Vav-1SH2/SH3c) abolished CD28-triggered NF-AT
activity compared with cells transfected with Vav-1 wild-type. In
contrast, a Vav-1 mutant carrying a deletion of the C-terminal SH3 only
(Vav-1SH3c) did not sensibly reduce CD28-mediated NF-AT activity
compared with basal level (531 cells). Control immunoblot shows that
Vav-1 mutants and wild-type proteins were similarly expressed (Fig. 5B). These data indicated that NF-AT activation through CD28
required Vav-1 being engaged with one or more protein partners mainly
through its SH2 domain. Because overexpression of Vav-1 caused an
increase in SLP-76 phosphorylation (Fig. 3C), we asked
whether the SH2 domain of Vav-1 was responsible for it. As shown in
Fig. 5C, basal as well as CD28-induced SLP-76
phosphorylation in cells overexpressing Vav-1SH2/SH3c were lower
than that seen with intact Vav-1 and similar to that of mock
transfected cells. Thus, SLP-76 phosphorylation is an event favored by
the SH2 domain of Vav-1 and is enhanced by CD28 triggering. This slight
phosphorylation of SLP-76 induced by CD28 ligation in mock transfected
cells may be due to endogenous Vav-1 and may reflect a low level of
triggering of the TCR signaling machinery (23).
Vav-1 overexpression induces formation of lamellipodia and microspikes in Jurkat cells
CD28 ligation has been shown to trigger an accumulation of Rho
family GTPases in the T cell contact zone formed with B7-expressing
cells (53). As Vav-1 is a preferential exchange factor for
Rac-1 in vitro (26, 27), it was of interest to examine
whether Vav-1 overexpression could result in Rac-1-induced actin
cytoskeleton changes and obtain additional clues as to the mechanism of
NF-AT activation. Therefore, Jurkat cells were transiently transfected
with Vav-1 and analyzed by confocal microscopy for changes in the
levels of actin polymerization and cell morphology. Fig. 6A shows that cells
overexpressing Vav-1 displayed cellular processes identifiable as
typical lamellipodia and microspikes. These latter structures have been
associated with the activation of the Rho GTPase, Cdc42
(54) and were less frequently observed than lamellipodia
in Vav-1 overexpressing cells. Of note is that no evident morphological
alteration was detected in cells overexpressing SLP-76 (data not
shown). Polymerized actin was strongly increased in these cells
compared with cells negative for Vav-1-myc expression, which remained
relatively round shaped. Lamellipodia were also typically observed in
Jurkat cells transfected with a constitutively active Rac-1V12 mutant
(data not shown). To test whether the loss of NF-AT activation (Fig. 5A) correlated with the absence of actin cytoskeleton
modifications induced by Vav-1, Jurkat cells were transiently
transfected with Vav-1SH2/SH3c (Fig. 6B). However, cells
expressing this mutant showed similar types of morphological
modifications and a similar percentage of modified cells compared with
cells transfected with Vav-1 (see Fig. 6). These results suggest that
the absence of NF-AT activation with the Vav-1 SH2 deletion mutant
(Fig. 5A) was not due to lack of Rac-1 activation. Rather,
the interaction of Vav-1 with other proteins, likely to include SLP-76,
is a prerequisite for exerting its function upon CD28 triggering.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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D) or by SEE/MHC and B7 stimulation (unpublished data) were
entirely CsA-sensitive. These data suggest that Vav-1 contributes to
calcineurin-dependent signals from CD28 and are consistent with the
critical role of Vav-1 in Ca2+ response upon T
cell activation (31, 32). It has been reported that CD28
does not contribute significantly to the control of NF-AT in Jurkat
cells activated by superantigen and B7-expressing APC
(59). This contrasts with our observation that the
addition of CTLA4-Ig or the absence of B7 on the APC abolished NF-AT
activation in cells overexpressing or not Vav-1 (16) and
Fig. 1B). The apparent discrepancy between these results may
be due to the dose and/or potency of enterotoxins (SED vs SEE) used as
well as the dependency of the Jurkat clones utilized on CD28
costimulation. Our data agree with the observation that CTLA4-Ig
inhibited completely NF-AT induced transcription in Ag and
B7-costimulated primary T cells (60).
A surprising finding of our investigation was that upon overexpression
of Vav-1, CD28 ligation alone was sufficient to activate pathways
controlled by the TCR (e.g., Ca2+/calcineurin and
MAP-kinases/AP-1 needed for NF-AT activation). These particular
stimulatory conditions allowed activation of NF-AT by borrowing the
TCR’s most proximal signaling components (e.g., ITAMs/ZAP-70/SLP-76).
These findings indicate the existence of an early connection between
the TCR and CD28 pathways for signal amplification, as previously
suggested (16), and that Vav-1 plays a key role in it. The
precise mechanism that forced activation of the TCR signaling pathway
in the absence of a direct TCR engagement is unclear at present. One
potential limitation of our findings is that Jurkat cells have a high
background of tyrosine phosphorylation compared with normal T cells.
This might result in the lowering of the activation threshold and thus,
exacerbation of the observed phenomena. However, our data offer a few
explanations that can fit with recent advances in the molecular
understanding of T cell activation. Two important modifications were
detected as a consequence of Vav-1 overexpression: a strong increase in
actin polymerization associated with the appearance of lamellipodia and
microspikes (a likely consequence of increased activated Rac-1 and,
perhaps, Cdc42, respectively) and a basal increase in SLP-76
phosphorylation which was further potentiated when CD28 was ligated by
B7. Recent data have shown that, upon engagement, CD28 associates with
specialized membrane microdomains, called GEMs, and that it can
contribute, together with the TCR, to form large patches of GEMs
(17, 61). GEMs appear to be essential for
concentration/assembly of TCR signaling components including Vav-1 and
SLP-76 (18, 19, 20) and the formation of large
TCR/CD28-induced GEMs is likely to be important for signal
stabilization (17). Vav-1 may modify, through cortical
actin changes, the spatial segregation of TCR signaling components
(e.g., Lck/ITAMs and, possibly, protein tyrosine phosphatases
(PTPases)) to the point of facilitating triggering when it is recruited
via CD28. Indeed, Vav-1 overexpression causes a low NF-AT activation
without TCR engagement (Ref. 33 , and the present work).
Under physiological conditions of CD28 ligation, Vav-1 membrane
recruitment/phosphorylation, an event shown to be sustained and
effective (24, 25), is likely to contribute to cortical
actin rearrangements through a high local enrichment of activated
Rac-1. Indeed, recent observations have indicated that Vav-1 as well as
Rho family proteins are concentrated beneath the T cell area contacting
the APC
(53)5,
where intense changes in membrane morphology and dynamics take place
even before intracellular Ca2+ increase
(62, 63, 64). It has been reported that SLP-76 overexpression
in Jurkat cells induces an increase in polymerized actin
(36). Interestingly, overexpression of SLP-76 did not lead
to CD28-induced NF-AT activation (Fig. 5B), nor to the
formation of morphological changes like Vav-1 did (data not shown),
suggesting that major modifications of cortical actin were necessary to
connect CD28 to the TCR signaling components. However, actin
rearrangement per se was insufficient as activated Rac-1 (Rac-1V12)
transfected in Jurkat cells induced formation of lamellipodia similar
to Vav-1 (data not shown), but not CD28 (or TCR)-mediated enhancement
of NF-AT activation. Rather, our data indicate that an intact
intracellular tail of CD28 and the presence of the SH2 domain of Vav-1
were required for NF-AT activation. The lack of CD28 intracellular tail
resulted in no tyrosine phosphorylation of overexpressed Vav-1 (data
not shown) and it was likely to affect activation of Src and
Tec protein tyrosine kinases from CD28 (65, 66, 67, 68)
contributing overall to a defect in NF-AT activation. Moreover, a key
step in the signaling leading to NF-AT activation appears to be the
functional cooperation of Vav-1 with SLP-76 (35), an event
which is likely to be mediated by the SH2 domain of Vav-1 (23, 35). The defect in augmentation of tyrosine phosphorylation of
SLP-76 and NF-AT activation in the absence of Vav-1 SH2 domain (Fig. 5)
further suggests a correlation between these two events. Recent data by
Arudchandran et al. (69) indicate that Vav-1 membrane
recruitment and SLP-76 phosphorylation show some dependency on the SH2
domain of the former. Although membrane binding and Rho GTPases
activation should have been achieved by the Vav-1SH2/SH3c mutant
(possibly through the PH domain (70)) as indicated by the
cortical actin modifications (Fig. 6B), perhaps SLP-76
recruitment was missing or strongly altered with consequences for both
Ca2+ increase and MAP kinase activation
(71). The possibility remains that the SH2 domain of Vav-1
is necessary for a polarized cortical actin change beneath the engaged
receptors and that this may be also critical for NF-AT activation. In
light of our data, we would like to propose that engagement of CD28 may
provide a critical input of activated Vav-1, which, via Rac-1 (and
perhaps Cdc42), will initiate modifications of cortical actin necessary
for facilitating TCR signaling. Vav-1 will contribute to integrate
signals for nuclear activation when it interacts with SLP-76, which
under physiological activation conditions, is controlled through the
TCR (24, 72).
Our immunofluorescence microscopy analysis indicates for the first time that Vav-1 is able to generate in T cells morphological alterations reminiscent of the activation of Rac-1 and Cdc42 proteins. These modifications are likely to be polarized by CD28 at the contact area between the T cell and the APC and may contribute to the intense membrane dynamics and formation of the supramolecular activation clusters (62, 64, 73) and ultimately, to prolonged signaling. In conclusion, our data provide first evidence for an early mechanistic link between CD28 and the TCR for signal amplification mediated by Vav-1.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Frédérique Michel, Molecular Immunology Unit, Department of Immunology, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France.
3 Current address: Università "La Sapienza," Dipartimento di Biologia Cellulare e dello Sviluppo, Via degli Apuli 1, 00185 Rome, Italy.
4 Abbreviations used in this paper: GEM, glycolipid-enriched membrane; ERK, extracellular signal-regulated kinase; SEE, Staphylococcus enterotoxin E; CsA, cyclosporin A; SH2, Src homology domain 2; WT, wild type.
5 A. Roumier, F. Niedergang, A. Dautry-Varsat, and A. Alcover. Relocalization of ezrin and Vav to the site of contact of T cells with APCs upon T cell activation: evidence for the interaction between these two proteins. Submitted for publication.
Received for publication May 1, 2000. Accepted for publication July 17, 2000.
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References |
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50–75% of Vav-1-expressing
cells.