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*
Division of Tumor Immunology, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, MA 02115; and
Department of Clinical Immunology and AIDS Research Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan
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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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Crk-associated substrate (Cas)3 lymphocyte-type (Cas-L) (also known as HEF-1) is a 105-kDa cytoplasmic protein that is tyrosine-phosphorylated upon the ligation of ß1 integrin (9, 10). Based on the structural similarity, Cas-L constitutes a Cas family with p130Cas and Efs/Sin (11, 12). Cas-L has 64% of homology to p130Cas and conserved motifs including an N-terminal Src homology (SH)-3 domain, followed by a substrate domain (SD) containing multiple YXXP motifs, which are putative binding sites for SH2-containing proteins, and a C-terminal YDYVHL sequence (9). Our previous study showed that the conserved YDYVHL sequence is the binding site for Src family tyrosine kinases. It has been shown that focal adhesion kinase (FAK) binds to the Cas-L SH3 domain and leads to tyrosine phosphorylation of Cas-L upon the ligation of ß1 integrin, resulting in the association of tyrosine-phosphorylated Cas-L with Crk, Nck, and SHPTP2 (9, 13) through their SH2 domains. These findings support the notion that Cas-L plays an important role as a docking protein in the intracellular signaling pathways through ß1 integrins. Recently, we found that Cas-L is also tyrosine-phosphorylated and forms a complex with Crk and C3G following TCR/CD3 engagement in a FAK-independent manner (14). In T lymphocytes, Cas-L is abundantly expressed, while p130Cas is not detectable (9) and p130Cas is preferentially expressed in adhesive cells. Thus, Cas-L might be a potential downstream molecule in the TCR/CD3-mediated signaling pathways that lead to various T cell immune responses.
In this study, we investigate the involvement of Cas-L in CD3-induced T cell migration on fibronectin (FN) using Boyden chamber assays. The migratory response was induced in the Cas-L-transfected Jurkat cells following the ligation of CD3 and ß1 integrins, whereas parent Jurkat cells that marginally expressed Cas-L did not migrate in the same condition. Furthermore, we note that FN alone provides migratory signals significantly in these Cas-L-transfected cells although the level of migration is lower than that of CD3 plus FN stimulation. The ligation of CD3 and ß1 integrin induces Cas-L tyrosine phosphorylation, which is associated with the migratory behavior. Thus, our findings strongly suggest that Cas-L is a key molecule in T cell migration induced by the ligation of CD3 and ß1 integrin.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human lymphoblastic T cell lines, Jurkat, SUP-T1, and HPB-ALL were obtained from American Type Culture Collection (Manassas, VA) and grown in RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, and gentamicin (50 µg/ml) (complete medium). Human PBMCs were isolated from healthy volunteers by density-gradient centrifugation on Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden). PBMCs were separated into E rosette-positive cells with sheep erythrocytes (15). Contaminating monocytes were depleted by adherence to plastic plates. Further depletion of monocytes was achieved by incubation with 5 mM L-leucine methyl ester hydrochloride. Anti-CD3 (OKT3), anti-CD28 (4B10), anti-CD29 (4B4), anti-CD49d (3G6), and anti-CD49e (2H6) mAbs were described previously (15). Anti-Crk and anti-ZAP70 mAbs were purchased from Transduction Laboratories (Lexington, KY). mAbs against phosphotyrosine (p-Tyr) and c-Myc tag were purchased from Upstate Biotechnology (Lake Placid, NY) and Oncogene Science (Cambridge, MA), respectively. A rabbit polyclonal Ab against Cas-L was described previously (14). Cholera toxin, cytochalasin D, genistein, and wortmannin were obtained from Sigma (St. Louis, MO).
Expression vectors and transfection
cDNAs encoding wild-type Cas-L, the deletion mutant of the SH3
domain (residues 1–61) (Cas-L
SH3) or the SD (residues 63–401)
(Cas-LSD), or the mutant with the substitution of C-terminal YDYVHL
to FDFVHL was described previously (13). The wild-type and
mutant Cas-L cDNAs were c-myc epitope-tagged
(13) and subcloned into the expression vector pBCMG-Hygro
(16). The expression vectors were transfected by
electroporation at 250 V and 960 µF using the Gene Pulser (Bio-Rad,
Hercules, CA). Cells were selected and maintained in the
hygromycin-containing complete medium.
Migration assays
Cell migration was assayed using 6.5-mm-diameter Transwell inserts (Costar, Cambridge, MA) with polycarbonate filters (3-µm pore size). The filters were left uncoated or were coated with OKT3 overnight at appropriate concentrations in PBS. After washing, the membranes were coated with human plasma FN (Life Technologies, Gaithersburg, MD) at 5 µg/ml for 2 h at room temperature. Cells were washed and resuspended at 1 x 106 in RPMI 1640 containing 0.6% BSA (BSA medium). For inhibition assays, cells were preincubated for 1 h at 37°C in the presence of mAbs or reagents. The Transwell chambers were inserted into wells filled with 600 µl of the BSA medium, and cells were added to the upper chamber in a final volume of 100 µl. After incubation at 37°C for appropriate duration of time, the filters were removed, fixed in methanol, and stained with May-Giemsa solutions. Cells on the upper side of filters were wiped off. The number of migrated cells on the lower side of the filters (designated as fully migrated cells) was counted microscopically in five high-power fields (HPF) per well at x400 magnification. Each experiment was performed in duplicate wells. The data represent the mean of duplicate wells, and error bars represent the SD.
Adhesion assays
OKT3 mAb (5 µg/ml) and FN (5 µg/ml) were coated on 96-well plates as described for migration assays. The wells were rinsed three times with PBS and blocked for 1 h with 2.5% BSA in PBS at 37°C. Cells were washed, resuspended at 5 x 105 in the BSA medium, and added to each well in a final volume of 100 µl. The plates were centrifuged for 5 min at 500 rpm and incubated for 10 min at 37°C by floatation in a water bath. The plates were washed three times with PBS. The number of adherent cells was quantified by the MTT assay (17). Each data point was calculated from triplicate wells and represents the mean ± SD.
Cell stimulation
Cells were washed three times with RPMI 1640 and then incubated on ice for 15 min with 500 µl of RPMI 1640 containing 10 µg/ml of OKT3 mAb. Following washing with RPMI 1640, cells were incubated with 10 µg/ml of anti-mouse Ig for 2 min at 37°C and subsequently suspended in ice-cold IMDM (Sigma) containing 5 mM EDTA, 10 mM pyrophosphate, 10 mM sodium fluoride, and 0.4 mM sodium vanadate. After centrifugation, cells were lysed in a 1% Nonidet P-40 lysis buffer as described (18). For stimulation with the extracellular matrix, cells were incubated for 1 h on plates coated with poly-L-lysine (Sigma) (5 µg/ml) or FN (5 µg/ml) and then solubilized in the lysis buffer.
Immunoprecipitation and immunoblotting
c-Myc-tagged proteins were immunoprecipitated with 9E10 and protein A-Sepharose (Pharmacia Biotech) as described (9). Immunoprecipitates and lysates were separated by SDS-PAGE and electrophoretically transferred onto the nitrocellulose membranes. Immunoblotting was performed with the indicated primary Abs, a HRP-conjugated anti-mouse or anti-rabbit Ab (Amersham, Arlington Heights, IL), and chemiluminescence reagents (NEN Life Science Products, Boston, MA) as described elsewhere (14). Tyrosine-phosphorylated proteins were detected with 125I-labeled anti-p-Tyr mAb (4G10), followed by autoradiography.
Flow cytometry
Cells were incubated for 30 min at 4°C with saturating concentrations of the indicated mAbs or a control Ab. Subsequently, cells were incubated for 30 min at 4°C with FITC-conjugated anti-mouse Ig, followed by another wash. The cells were suspended in 0.5 ml of PBS containing 1% paraformaldehyde and analyzed on an Epics Elite (Beckman Coulter, Miami, FL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A). It should be noted that
no migration was observed on the filters coated with OKT3 alone in
peripheral T lymphocytes (data not shown). We next analyzed CD3 plus
FN-induced cell migration of various human T lymphoblastic cell lines.
As shown in Fig. 1A, SUP-T1 cells exhibited an increase of
CD3 plus FN-induced cell migration at similar levels to that observed
in peripheral T lymphocytes. In contrast, the migration of Jurkat and
HPB-ALL cells by CD3 stimulation on FN were less than one-tenth in
number compared with peripheral T lymphocytes. Western blotting
analysis confirmed that Jurkat and HPB-ALL cells marginally expressed
Cas-L as described before (9), whereas SUP-T1 cells as
well as peripheral T lymphocytes expressed fair amounts of Cas-L (Fig. 1B). Crk and ZAP70 were expressed at similar levels in all
the cells. These data suggest that Cas-L expression appears to be
correlated with the levels of CD3 plus FN-induced T cell migration.
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B)
and determined cell migration of those transfectants using the same
assay. Flow cytometry analysis revealed that transfection of Cas-L did
not alter the expression of CD3, CD49d, CD49e, and CD29 (data not
shown). For this purpose, cells were incubated for 2 h on the
filter coated with FN and OKT3. As shown in Fig. 2A, wild-type
Cas-L-transfected Jurkat cells showed a marked increase of CD3 plus
FN-induced cell migration, whereas only slight induction was observed
in the cells transfected with a control vector (Fig. 2A; V1
and V2) or parent Jurkat cells. Similar results were obtained in three
independent clones (Fig. 2A; A1, A11, and A12). It should be
noted that Cas-L transfectants showed a slight increase of migration on
the filters coated with FN alone. The migration of wild-type
Cas-L-transfected Jurkat cells increased in number at higher coating
concentrations of OKT3, whereas vector-transfected cells displayed only
a slight increase of migration even at the concentration of 10 µg/ml
of OKT3 (Fig. 2B), indicating that the Cas-L-mediated cell
migration was induced by OKT3 in a concentration-dependent manner. In
time course experiments using the filters coated with OKT3 and FN, the
migration of wild-type Cas-L-transfected Jurkat cells were observed in
2 h after incubation and was further increased in number after 4-h
incubation (Fig. 2C). In contrast, the kinetics of migration
was slow in vector-transfected cells, showing a 6-fold decrease in
number compared with wild-type Cas-L-transfectants in 4 h after
incubation (Fig. 2C). As previously mentioned, no migration
was observed on the filters coated with OKT3 alone in the transfectants
even at 4 h. Furthermore, neither OKT3 plus type I collagen nor
OKT3 plus BSA could induce Cas-L-mediated cell migration (data not
shown). These results indicate that the expression of Cas-L plays an
important role in T cell migration following the ligation of CD3 and
that, in addition to the stimulation of CD3, ligation of
ß1 integrin is required for this migration. The
ligation of CD28 can provide signals into the parent Jurkat cells as
well as Cas-L transfectants in TCR/CD3-mediated IL-2 production
(19). We next determined whether CD28 stimulation may
substitute for CD3 stimulation in Cas-L-mediated migration on FN. As
shown in Fig. 2D, the wild-type Cas-L transfectants failed
to migrate on filters coated with FN and anti-CD28 mAb, indicating
that the signal from CD28 alone is insufficient for T cell migration
on FN.
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,
anti-CD49e mAb, as well as anti-CD29 mAb, strongly blocked cell
migration (
70% inhibition as compared with a control mAb). In
contrast, anti-CD49d mAb showed 30% inhibition in cell
migration. These data indicate that VLA-5, with a partial contribution
by VLA-4, is mainly involved in CD3 plus FN-induced T cell
migration.
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, the ligation of CD3
induced cell adhesion to FN in vector control and wild-type
Cas-L-transfected cells. However, there was no significant difference
in their binding ability to FN with or without CD3 stimulation. Similar
results were obtained in the adhesion assays at the time period of 20
and 60 min (data not shown). These findings indicate that
Cas-L-mediated cell migration on FN induced by the ligation of CD3 is
not due to the change of the integrin avidity to FN.
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SH3), the SD-deleted
mutants (Cas-LSD), and the mutants with the substitution of
C-terminal YDYVHL to FDFVHL (Cas-LF) were created and stably expressed
in Jurkat cells. Comparable amounts of CD3, CD49d, CD49e, and CD29 were
expressed on all the transfectants (data not shown). As shown in Fig. 5A, Cas-LSD failed to
promote the CD3 plus FN-induced cell migration, whereas Cas-LSH3 and
Cas-LF induced significant increases in cell migration at similar
levels to that was observed in wild-type Cas-L. Western blotting
analysis with an mAb to p-Tyr revealed that Cas-LSD was
significantly diminished in tyrosine phosphorylation following the
ligation of CD3, although the transfectants expressed comparable
amounts of proteins (Fig. 5B). In contrast, Cas-LSH3 and
Cas-LF, as well as wild-type Cas-L, were tyrosine-phosphorylated after
CD3 cross-linking. These results indicate that the Cas-L SD, but not
the C-terminal YDYVHL sequence or SH3 domain, plays an important role
in CD3 plus FN-induced cell migration and that the tyrosine
phosphorylation of the SD is also critical for this migration.
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, cell migration was blocked by
wortmannin, inhibitors of phosphatidylinositol 3-kinase, in a
dose-dependent manner. Cholera toxin, an ADP-ribosylating agent of the
Gs protein, was previously reported to inhibit T cell migration toward
FN (21). Cholera toxin also inhibited Cas-L-mediated cell
migration induced by CD3 plus FN stimulation. These data suggest that
the phosphatidylinositol 3-kinase and the Gs proteins are involved in
this cell migration. Cytochalasin D, an inhibitor of actin
polymerization, and genistein, an inhibitor of tyrosine
phosphorylation, also showed significant inhibitory effects on cell
migration, suggesting that actin polymerization and tyrosine
phosphorylation are important for Cas-L-mediated cell migration induced
by the ligation of CD3 and ß1 integrin.
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A; 31.8 ± 3.3 vs
11.4 ± 3.0/HPF at 4 h, p < 0.01),
suggesting that ligation of ß1 integrin alone
could provide migratory signals into cells through Cas-L. To further
define the mechanism of FN induced cell migration,
ß1 integrin-mediated cell migration by FN was
determined in Jurkat cells expressing Cas-L mutants. In the case of
transfectants of Cas-LSH3 and Cas-LSD mutants, only slight
migration comparable to vector control cells was observed (Fig. 7B). Notably, both of the mutants failed to be
tyrosine-phosphorylated by FN stimulation (Fig. 7C). In
contrast, Cas-LF as well as wild-type Cas-L could promote FN-dependent
cell migration, and these proteins were tyrosine-phosphorylated by FN
stimulation. These results indicate that, like the Cas-L SD, the Cas-L
SH3 domain is necessary for FN-dependent T cell migration, and that
Cas-L tyrosine phosphorylation is a critical event for cell migration.
Taken together, these findings suggest that FN not only provides
adhesion sites for T cells but also induces intracellular signals to
Cas-L through ß1 integrin, leading to T cell
migration.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies using T lymphocytes from TCR transgenic mice that can be tracked in vivo following specific Ag stimulation have shown that Ag-specific T lymphocytes change in their localization through the body in response to foreign Ags (2). Kedl and Mescher (5) demonstrated that CD8+ T cells recognize initial Ags in lymphoid organs such as draining LNs and spleen, resulting in the access of the sensitized cells to Ag deposition sites in the periphery. Moreover, CD4+ T cells have been shown to move from paracortical T zones to B cell follicles following Ag stimulation (7). These observations indicate that Ag stimulation through TCR/CD3 provides migratory signals for T cells. It has been well documented that the Ag-induced locomotion of lymphocytes can be regulated by changes of adhesion molecules in the expression levels and the affinity with the ligand (1, 2, 8, 23). Our studies demonstrated that, following CD3 cross-linking, Cas-L transfectants, but not vector controls, showed significant increases in cell migration through ß1 integrins, although both cells were increased in their binding activity to FN. Moreover, PMA enhanced cell adhesion to FN, but did not enhance migration through FN even at higher concentrations in Cas-L transfectants (data not shown). Therefore, it is concluded that interactions between ß1 integrins and FN are critical for CD3-induced T cell migration, but that the change of the integrin avidity is not sufficient for the cell migration.
FAK is a 125-kDa cytoplasmic protein that plays an important role in
ß1 integrin-mediated signaling pathways
(24, 25). FAK is localized to focal adhesions in adherent
cells and exhibits tyrosine kinase activity (26, 27). Upon
the ligation of ß1 integrin, FAK is activated
and tyrosine-phosphorylated (28). Recently FAK has been
reported to promote integrin-mediated cell migration in adherent cells
(29), indicating that FAK also appears to be involved in T
cell migration. However, our results showed that the Cas-LSH3
mutant, which fails to bind FAK, could promote T cell migration induced
by the ligation of CD3 and ß1 integrin. Our
previous study has shown that the Cas-LSH3 is
tyrosine-phosphorylated after the ligation of CD3 (14),
suggesting that Cas-L is tyrosine-phosphorylated after the ligation of
CD3 in a FAK-independent manner. Because FAK is not tyrosine
phosphorylated after the ligation of CD3 (14), our results
strongly suggest that FAK may not be involved in signal transduction
pathways that induce T cell migration following the ligation of
CD3.
Previous studies have shown that the ligation of
ß1 integrin triggers T cell motility (21, 22). We showed that interactions between
ß1 integrins and FN trigger migratory signals,
which are mediated by Cas-L. The immobilized CS1 domain, the ligand for
VLA-4, also triggered Cas-L-mediated T cell migration, which was
inhibited by an mAb against VLA-4 (data not shown). These data indicate
that migratory signals can be induced by ß1
integrin through Cas-L. In contrast to the results of CD3 plus
FN-induced migration, the Cas-LSH3 failed to promote T cell
migration and was not tyrosine-phosphorylated upon the ligation of
ß1 integrin. We previously reported that Cas-L
binds to the FAK C terminus through the Cas-L SH3 domain and is
tyrosine-phosphorylated by FAK upon the ligation of
ß1 integrin (9, 13). These
findings indicate that the FAK-Cas-L interaction is important for the
signaling pathway in T cell migration triggered by the ligation of
ß1 integrin alone. Taken together, Cas-L may
receive signals through both TCR and ß1
integrins in FAK-independent and -dependent manners, respectively,
which results in tyrosine phosphorylation of Cas-L to transfer
migratory signals to downstream signaling molecules.
It has been reported that ß1 integrins provide costimulatory signals to CD3-dependent T cell proliferation and IL-2 production (30, 31, 32, 33, 34). We reasoned that the ligation of ß1 integrin by FN would mediate costimulation to activate cells, resulting in an increase of T cell migration induced by CD3 stimulation. However, we observed no increase of Cas-L-mediated T cell migration by stimulation with anti-CD3 plus anti-CD28 mAbs (data not shown), which also can induce IL-2 production in the same cells (19). The results indicate that the costimulatory signal that can activate T cells is insufficient to T cell migration, suggesting that ß1 integrins may provide specific signals for T cell migration in addition to costimulatory signals in T cells. Because Cas-L was significantly tyrosine-phosphorylated upon the ligation of either CD3 or ß1 integrin, the two receptors can independently provide sufficient migratory signals through Cas-L. The CD3 plus FN-induced T cell migration occurred within 2 h, while ß1 integrin-induced migration was observed 4 h after incubation. Cas-L is rapidly tyrosine-phosphorylated and dephosphorylated quickly after the ligation of CD3. In contrast, tyrosine phosphorylation of Cas-L after the ligation of ß1 integrin occurs slowly and continues stably (14, 35). These findings suggest that TCR and ß1 integrins may induce cell migration in different phases; TCR plus ß1 integrin signals are mainly for an early phase, and ß1 integrin signal alone for a late phase in the T cell migratory process. These differences in the time courses, of which mechanism is still unclear, made it difficult to evaluate tyrosine phosphorylation or cell migration at the same time point. It should be noted that there remains a possibility that difference in the time frame of examination might have influenced our experimental results.
Our study showed that the Cas-LSD mutant failed to promote cell
migration. It is conceivable that the SD appears to provide binding
sites for downstream molecules leading to migratory signal
transduction. Crk, an adopter protein that consists of SH2 and SH3
domains, is one of the possible binding molecules to the SD of Cas-L
(36). v-Crk was originally reported as the
oncogene that can transform mammalian cells such as fibroblasts
(36). Crk has been reported to bind to several signaling
molecules including C3G, Dock180, and c-abl through the Crk SH3 domain
(37, 38, 39). Crk may regulate cytoskeletal changes through
these molecules. Alternatively, overexpression of Crk with Cas-L
results in tyrosine phosphorylation of Cas-L without any stimulation
(9), suggesting that Crk may play a role in the regulation
of Cas-L phosphorylation. We reported that Nck also binds to
tyrosine-phosphorylated Cas-L (9). Nck is an adapter
protein consisting of one SH2 domain followed by three SH3 domains
(40). This molecule has been reported to bind the
p21-activated kinase that regulates actin reorganization through the
second Nck SH3 domain (41, 42) and to the Wiskott-Aldrich
syndrome protein (WASP) encoded by the gene affected in patients with
Wiskott-Aldrich syndrome (WAS) (43). Actin polymerization
following CD3 cross-linking is impaired in leukocytes from the WAS
patients (44), indicating that WASP appears to be involved
in reorganization of actin cytoskeleton. Therefore, Cas-L may be
involved in cytoskeletal changes of migrating cells through the
Nck-WASP or Nck-PAK1 interaction. It should be noted that the deletion
in Cas-LSD is rather huge (residues 63–401) compared with other
mutants. Therefore, the possibility should be taken into account that
conformational change due to this huge deletion might affect the signal
transducing ability of Cas-LSD as well as that abolishment of
tyrosine phosphorylation in the SD itself might shut off the binding of
putative downstream signal-transducing molecules such as Crk and Nck
through their SH2 domains.
In addition, recent studies demonstrated that p130Cas, a homologue of Cas-L, is involved in ß1 integrin-mediated cell migration of adherent cells (45). p130Cas-knockout mice showed severely impaired actin stress fiber formation in their fibroblasts. These findings indicate that p130Cas is involved in actin-based cell motility of adherent cells such as fibroblasts (46). Cas-L, being abundantly expressed in T cell, seems to be involved in T cell migration by similar mechanisms to p130Cas in adherent cells, although further studies are required to elucidate the migratory signaling pathways through Cas-L or p130Cas.
In conclusion, our results showed that Cas-L can regulate T cell migration in response to signals from TCR/CD3 and/or ß1 integrin. Furthermore, the present study strongly suggests that Cas-L plays a critical role as a docking protein regulating the association of signaling molecules to promote T cell migration.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Satoshi Iwata, Division of Tumor Immunology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115. E-mail address:
3 Abbreviations used in this paper: Cas, Crk associated substrate; Cas-L, Cas lymphocyte-type; SH, Src homology; FN, fibronectin; FAK, focal adhesion kinase; p-Tyr, phosphotyrosine; SD, substrate domain; Cas-LSH3, SH3 domain-deleted mutant of Cas-L; Cas-LSD, SD-deleted mutant of Cas-L; Cas-LF, Cas-L mutant with the substitution of C-terminal YDYVHL to FDFVHL; HPF, high-power fields; WASP, Wiskott-Aldrich syndrome protein; VLA, very late Ag; .
Received for publication March 29, 1999. Accepted for publication July 22, 1999.
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4-integrins in lymphocyte homing to mucosal tissues in vivo. J. Immunol. 152:3282.[Abstract]
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) or FN (5
µg/ml) plus OKT3 (5 µg/ml) (
). The results are expressed as the
number of fully migrated cells on the lower side of the filters. The
numbers of cells were counted in five microscope fields. Bars represent
the mean ± SD of duplicate samples. Data are representative of
two independent experiments. B, Expression of Cas-L in T
cells and transfectants. Whole-cell lysates (50 µg/lane) were
subjected to Western blotting with indicated Abs.
). The results are expressed as the number of fully migrated cells
on the lower side of the filters. The numbers of cells were counted in
five microscope fields. Bars represent the mean ± SD of duplicate
samples. Data are representative of at least two independent
experiments.
), or BSA plus OKT3 (5 µg/ml) (
). The adherent
cells were quantified by the MTT assay. Each data point was calculated
from triplicate wells and represents the mean ± SD. Data are
representative of three independent experiments.
) and vector-transfected (
)
Jurkat cells were incubated on porous filters coated with FN (5
µg/ml). The results are expressed as the number of fully migrated
cells on the lower side of the filters. The numbers of cells were
counted in five microscope fields. Each data point represents the
mean ± SD of duplicate samples in two independent clones. Data
are representative of two independent experiments. B, Cells were incubated for 4 h on
porous filters coated with FN (5 µg/ml). The results are expressed as
the relative number of fully migrated cells to the number observed in
vector-transfected cells. The numbers of cells were counted in five
microscope fields. Bars represent the mean ± SD of duplicate
samples in two independent clones. Data are representative of three
independent experiments. C, Cells stimulated with FN (5
µg/ml) or poly-L-lysine (PLL, 5 µg/ml) coated on plates
for 1 h were lysed and then immunoprecipitated with 9E10. The
immunoprecipitates were analyzed by Western blotting with
anti-Cas-L Ab, followed by anti-pTyr. Data are representative
of three independent experiments.