IMMUNOBIOLOGY
The Rac activator Tiam1 controls efficient T-cell trafficking and route of
transendothelial migration
Audrey Gérard,1 Rob A. van der Kammen,1 Hans Janssen,2 Saskia I. Ellenbroek,1 and John G. Collard1
Divisions of 1Cell Biology and 2Tumor Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands
Migration toward chemoattractants is a
hallmark of T-cell trafficking and is essential to produce an efficient immune response. Here, we have analyzed the function of the Rac activator Tiam1 in the
control of T-cell trafficking and transendothelial migration. We found that Tiam1 is
required for chemokine- and S1P-induced
Rac activation and subsequent cell migra-
tion. As a result, Tiam1-deficient T cells
show reduced chemotaxis in vitro, and
impaired homing, egress, and contact
hypersensitivity in vivo. Analysis of the
T-cell transendothelial migration cascade
revealed that PKC␨/Tiam1/Rac signaling
is dispensable for T-cell arrest but is
essential for the stabilization of polarization and efficient crawling of T cells on
endothelial cells. T cells that lack Tiam1
predominantly transmigrate through individual endothelial cells (transcellular migration) rather than at endothelial junctions (paracellular migration), suggesting
that T cells are able to change their route
of transendothelial migration according
to their polarization status and crawling
capacity. (Blood. 2009;113:6138-6147)
Introduction
T cells traffic through the body and pass the secondary lymphoid
organs (SLOs), seeking for a cell presenting specific antigens. The
entry of T cells to SLOs (homing) and exit from SLOs (egress) is a
tightly regulated process required for T cells to allow a coordinated
response in time and space.1 The migration of T cells toward
chemoattractants is an essential requirement for their trafficking.
During homing, homeostatic chemokines CCL19, CCL21 (SLC),
CXCL12 (SDF1␣), and CXCL13, which are expressed on high
endothelial venules (HEVs) of peripheral lymph nodes (pLNs), are
essential to attract leukocytes.2 Egress from pLNs is controlled by
the lipid mediator S1P, in part because of its capacity to induce
T-cell migration.3
To home to pLNs and to reach inflammation sites, leukocytes
have to cross a natural barrier corresponding to HEVs or postcapillary venules, which involves a stepwise cascade of events including
rolling, adhesion, and transendothelial migration of leukocytes.4
Additional steps have been recently defined in the transmigration
cascade such as tethering, slow rolling, strengthening of adhesion,
spreading, and crawling (reviewed in Ley et al5). Leukocytes can
use 2 routes to cross the endothelial barrier (ie, the paracellular and
the transcellular route). The most commonly used route is the
paracellular route, when leukocytes transmigrate between endothelial cells at sites of cell junctions.6 During transcellular migration,
leukocytes cross the endothelial barrier by transmigrating directly
through individual endothelial cells. This pathway identified in
various in vitro models7-10 is used by neutrophils and activated
T cells particularly to reach sites of inflammation and to cross the
blood-brain barrier.11
Attractants together with shear flow are essential to trigger
transendothelial migration and cause integrin activation, polarization, directed cell migration, and diapedesis. Small GTPases of the
Rho family, including Cdc42, Rac, and RhoA, regulate and
coordinate the cytoskeleton remodeling required for adhesion,
polarization, and migration of cells.12 Their activation is controlled
by guanine nucleotide exchange factors (GEFs) that promote the
exchange of GDP for GTP and also select upstream and downstream partners.13,14 Rac has been shown to be crucial for transendothelial migration.15 The Rac activator Vav1 is required for T-cell
adhesion and chemotaxis in vitro, but no Vav1-dependent defects in
T-cell trafficking have been observed so far.16,17 Dock2, another
Rac-specific GEF, is crucial for T-cell trafficking,18,19 but the
presence of lymph nodes in Dock2-deficient mice suggests that
other Rac-specific GEFs contribute to T-cell trafficking.
Here we have studied the function of Tiam1 in T-cell trafficking
and transendothelial migration. Tiam1 has been identified as a
T-lymphoma invasion and metastasis-inducing gene by retroviral
insertional mutagenesis20 and encodes a GEF specific for Rac.21
Whereas expression of a hyperactive form of Tiam1 induces
invasion of otherwise noninvasive T-lymphoma cells, its actual
function in primary T cells is largely unknown. Here we show that
Tiam1 controls T-cell polarization and is required for chemokineand S1P-induced T-cell migration in vitro and efficient T-cell
homing and egress in vivo. Furthermore, Tiam1-deficient mice are
delayed in contact hypersensitivity, showing the requirement of
Tiam1-dependent polarization and migration for efficient T-cell
response. When crossing the endothelial monolayer, Tiam1deficient T cells switch from paracellular to transcellular migration,
suggesting that T cells can switch their transmigration route
depending on their polarization status.
Submitted July 8, 2008; accepted December 24, 2008. Prepublished online as Blood
First Edition paper, January 12, 2009; DOI 10.1182/blood-2008-07-167668.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2009 by The American Society of Hematology
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Methods
Cell culture and retroviral transduction of T-cell blasts
The murine brain-derived endothelial cell line (bEnd.3; gift from Dr D.
Vestweber, Max-Planck-Institute of Molecular Biomedicine, Münster,
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Germany) was cultured in DMEM supplemented with 10% fetal calf serum
(FCS). T cells were isolated from lymph nodes and spleen of 6- to
12-week-old WT (FVB) and Tiam1⫺/⫺ mice.22 Negative selection was
carried out using a pan T-cell isolation kit (magnetic-activated cell sorting
[MACS]; Miltenyi Biotec, Auburn, CA). T-cell purity was more than 95%
as determined by flow cytometry. T-cell blasts were prepared by culturing
splenocytes derived from WT and Tiam1⫺/⫺ mice with 5 ␮g/mL concanavalin A for 3 days, followed by expansion of nonadherent cells with 25 U/mL
mouse IL-2 (Peprotech, Rocky Hill, NJ). For retroviral transduction of
pIB-GFP-actin (kindly provided by Dr M. F. Krummel, University of
California, San Francisco, CA), virus was prepared using the Phoenix
retrovirus packaging cells as described.23 T-cell blasts (3 ⫻ 106) were
incubated with 1 mL virus-containing supernatant in the presence of
8 ␮g/mL polybrene (Sigma-Aldrich, St Louis, MO) and spin-infected for
2 hours at 800g. A second round of infection was performed after 16 hours
and subsequently cells were allowed to grow for 24 hours. Infection
efficiency was between 30% and 40%.
Antibodies and reagents
The following antibodies were used in immunoblottings and immunofluorescent stainings. Antibodies against PKC␨ (C-20), Tiam1 (C-16), and
PECAM-1 (M-20) were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA). Antibody against Rac1 was from Upstate Technology (Lake
Placid, NY), against phospho-PKC␨/␭ (Thr 410/403) was from Cell
Signaling Technology (Beverly, MA), and against ICAM-1 was from
Chemicon International (Temecula, CA). Antibody against LFA-1 (M17-4)
was provided by E. Roos (The Netherlands Cancer Institute). All the
conjugated secondary antibodies for immunofluorescent staining were
purchased from Invitrogen (Frederick, MD). Recombinant S1P, SLC, and
SDF1␣ were purchased from Peprotech. The PKC␨ pseudosubstrate
inhibitor and Src inhibitor PP2 were purchased from Calbiochem (San
Diego, CA) and Sigma-Aldrich, respectively.
Homing assay
Naive T lymphocytes were fluorescently labeled with 0.5 ␮M green cell
tracker CMFDA or 1.5 ␮M orange cell tracker CMTMR for 2 hours at
37°C. Of each population, 10 ⫻ 106 labeled cells were admixed and
injected intravenously in FVB mice. After 2 hours, recipient mice were
killed, blood was collected, and SLOs were isolated. Percentages of greenand red-labeled adoptively transferred cells were determined by flow
cytometry. The ratio of the input population (input WT cells divided by
input Tiam1⫺/⫺ cells) was used as a correction factor as described.24
A homing ratio was calculated for each organ as followed: (number of
Tiam1⫺/⫺ adoptively transferred cells divided by number WT adoptively
transferred cells) ⫻ (correction factor input). One pLN from each mouse
was snap frozen for immunohistologic analysis.
Egress assay
Assessment of egress from pLNs was performed essentially as described.25
Naive T lymphocytes were fluorescently labeled using 0.5 ␮M CMFDA or
1.5 ␮M CMTMR for 2 hours at 37°C. Of each population, 25 ⫻ 106 cells
were admixed in 200 ␮L PBS and injected intravenously in FVB mice.
Twenty-four hours after lymphocyte transfer, L-selectin blocking Ab
(Mel-14 [Biolegend, San Diego, CA], 100 ␮g/mouse) was injected intravenously to prevent further homing. Mice were killed 0, 12, and 24 hours after
mAb injection and pLNs were isolated. The relative numbers of adoptively
transferred T cells were determined using flow cytometry. For each organ,
absolute numbers were also deduced. A pLN retention ratio was determined
for each time point as the ratio of (number of recovered Tiam1⫺/⫺
T lymphocytes divided by number of recovered WT lymphocytes) ⫻ correction
factor for the input population ratio. Twenty-four hours after adoptive
transfer, some of the mice were killed and pLNs were snap frozen for
immunohistologic analysis.
Contact hypersensitivity
WT and Tiam1⫺/⫺ mice were immunized by applying 0.5% FITC solubilized in 1:1 acetone/dibutyl pthalate (400 ␮L) on their shaved abdomens.
Tiam1 REGULATES T-CELL TRAFFICKING
6139
After 6 days, baseline ear thickness was measured and 10 ␮L 0.5% FITC
solution was applied to both left ear surfaces. Acetone/dibutyl pthalate
solution was applied on the right ear as negative control. On days 1 and 4
after challenge, the thickness of each ear was measured. The data are
expressed as changes in ear thickness (ear thickness 24 hours after
elicitation minus baseline ear thickness). From each mouse, one of the ears
was snap frozen for immunohistologic analysis. From each experiment,
2 mice were killed 3 days after exposure of the abdomen to hapten, and
draining LNs were isolated. Single-cell suspensions were counted, stained
with anti-CD11c conjugated with APC (BD Biosciences, San Diego, CA),
and analyzed by flow cytometry. Animal experiments were performed
strictly according to the laws of the Netherlands and were approved by the
Animal Commission of The Netherlands Cancer Institute.
Histology
For homing and egress experiments, 10-␮m sections of frozen pLNs were
fixed for 10 minutes in 2% paraformaldehyde at room temperature (RT),
washed in PBS, blocked with FCS, and stained with anti-PNAd Ab
(MECA-79 antibody; BD Biosciences). As a secondary Ab, biotinylated
anti–rat Ig was used, followed by Cy5-conjugated streptavidin. For contact
hypersensitivity experiments, 5-␮m sections of frozen ears were acetonefixed for 10 minutes, washed in PBS, blocked with FCS, and stained with
biotinylated anti-CD3 (145-2C11 antibody; BD Biosciences), followed by
Cy5-conjugated streptavidin. The number of T cells infiltrating the inflamed
ears was quantified as the number of CD3⫹ cells present per field using a
10⫻/0.4 NA phase-contrast objective. All sections were analyzed using a
Leica confocal microscope (Heidelberg, Germany).
Transmigration under shear flow
Experiments were performed essentially as described.26 Briefly, bEnd.3
cells were plated at confluence on 30-mm coverslips coated with fibronectin
(50 ␮g/mL in PBS) and stimulated for 16 hours with TNF␣ (500 U/mL).
The bEnd.3 cell monolayers were subsequently overlaid with SDF1␣
(2 ␮g/mL) for 5 minutes. After extensive washing, coverslips were
mounted on a laminar flow chamber (C&L Instruments, Hershey, PA).
T-cell blasts or naive T cells were untreated or treated with 2 ␮M PP2
and/or 2 ␮M PKC␨ inhibitor for 1 hour. After washing, T cells were
resuspended at 0.5 ⫻ 106/mL in flow buffer (20 mM Hepes, 132 mM NaCl,
6 mM KCl, 1 mM MgSO4, 1.2 mM KH2PO4, 5 mM glucose, 1 mM CaCl2,
0.5% BSA). Perfusion of T cells into the flow chambers was performed at
0.25 dyne/cm2 for 2 minutes over the monolayer to allow accumulation of
leukocytes. The flow rate was then increased to 2 dyne/cm2 (physiologic
shear flow) for a period of 15 minutes. Images were recorded at 1 frame per
10 seconds using a 20⫻/0.4 NA phase-contrast objective with a microscopelinked CCD camera (DFC 350 FX; Leica). Motion analysis was performed
manually. Lymphocytes arrested during the accumulation phase and
resisting detachment from the endothelial surface were subdivided into
3 categories: (1) Lymphocytes that remained stationary throughout the
assay were considered stationary. (2) Lymphocytes that crawled at least
2 cell diameters without detaching or transmigrating were considered
crawling. (3) Lymphocytes that underwent stepwise darkening in phase
contrast were considered transmigrating cells (diapedesis). Finally, T cells
that showed a leading and trailing edge after 15-minute tracking were
considered polarized, independently of their migration status.
Arrest, locomotion, and polarization under shear flow
ICAM-1 proteins (5 ␮g/mL) were coated on coverslips OVN at 4°C. T-cell
blasts were resuspended at 0.5 ⫻ 106/mL in PBS⫹/⫹ (PBS containing
1 mM CaCl2 and 1 mM MgCl2), and perfused through the flow chamber at
2 dyne/cm2. All flow experiments were conducted at 37°C and were
recorded at 1 frame per 5 to 10 seconds with a 10⫻/0.4 NA phase-contrast
objective and a microscope-linked CCD camera (DFC 350 FX; Leica).
Adherent cells were defined as cells remaining attached to the substrate for
the complete flow period. The number of adherent cells per field was
quantified over time. Migration analysis on purified ligands was conducted
during the same time. Cells that showed after 10 minutes a leading and
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GÉRARD et al
Figure 1. Tiam1 is required for chemokine- and S1P-induced Rac
activation and chemotaxis. (A) Lymphocytes from WT and Tiam1⫺/⫺
mice were either nonstimulated or stimulated with 250 ng/mL SDF1␣ for
the indicated time period. Subsequently Rac activity was determined. (Top
panel) Tiam1. (Bottom panels) GTP-bound Rac and total Rac. Sizes are
indicated in kilodaltons. Representative results of 3 independent experiments are shown. (B) Chemotaxis of WT and Tiam1⫺/⫺ T lymphocytes
toward SDF1␣ (100 ng/mL) was measured in transwells. After 1 hour, cells
present in the lower chamber were counted. Results are derived from
3 independent experiments and presented as the percentage of the input
cells. Error bars indicate SD; p indicates P value. (C) Lymphocytes from
WT and Tiam1⫺/⫺ mice were either nonstimulated or stimulated with
250 ng/mL SLC for the indicated time period. Subsequently Rac activity
was determined. (Top panel) Tiam1. (Bottom panels) GTP-bound Rac and
total Rac. Sizes are indicated in kilodaltons. Representative results of
2 independent experiments are shown. (D) Chemotaxis of WT and
Tiam1⫺/⫺ T lymphocytes toward SLC (100 ng/mL) was measured in
transwells. After 1 hour, cells present in the lower chamber were counted.
Results are derived from 2 independent experiments and presented as the
percentage of the input cells. Error bars indicate SD; p indicates P value.
(E) Lymphocytes from WT and Tiam1⫺/⫺ mice were either nonstimulated
or stimulated with 150 nM S1P for the indicated time period. Subsequently
Rac activity was determined. (Top panel) Tiam1. (Bottom panels) GTPbound Rac and total Rac. Sizes are indicated in kilodaltons. Representative results of 3 independent experiments are shown. (F) Chemotaxis of
WT and Tiam1⫺/⫺ T lymphocytes toward S1P (15 nM) was measured in
transwells. After 1 hour, cells present in the lower chamber were counted.
Results are derived from 3 independent experiments and presented as the
percentage of the input cells. Error bars indicate SD; p, P value.
trailing edge and GFP-actin accumulation at the leading edge were
considered polarized, independently of their migration status.
1% osmiumtetroxide, processed through dehydration for flat-embedding in
embed812/NMA, sectioned, stained with 1% uranylacetate, and examined
with a Philips CM10 Electron Microscope (Eindhoven, The Netherlands).
Determination of route of diapedesis
BEnd.3 cells were plated at 90% confluence on fibronectin-coated coverslips (50 ␮g/mL). After 24 hours, cells were activated by 500 U/mL TNF␣
overnight. T-cell blasts (5 ⫻ 106 in PBS⫹/⫹) were added to the SDF1␣bearing endothelial monolayer for 30 minutes at 37°C. Samples were fixed
in 4% PFA at RT for 15 minutes, and saturated in PBS 5% FCS for
20 minutes. T cells undergoing transendothelial migration were identified as
LFA-1–positive cells that extended down to the basal sections at the
endothelial cell substrate, as already described.27 Additional immunofluorescent stainings were performed to determine the distribution of T cells
relative to the endothelial cells in the X-Y and Z dimensions (for detailed
explanation see Figure S1, available on the Blood website; see the
Supplemental Materials link at the top of the online article). After
identifying the transmigrating cells, the route of diapedesis was essentially
determined as follows: cells were stained with LFA-1 to identify the T cells
and with PECAM-1 and ICAM-1 to identify endothelial cell perimeter and
the transmigration pore, respectively. Appropriate fluorescent-labeled secondary antibodies were subsequently used. Diapedesis not in close proximity to endothelial junctions was identified as transcellular. Diapedesis at
endothelial cell-cell contacts was scored as paracellular.7,9
Electron microscopy
BEnd.3 cells were plated at 90% confluence on fibronectin-coated (50 ␮g/
mL) Thermanox plastic coverslips (Nunc, Rochester, NY). After 24 hours,
cells were activated by 500 U/mL TNF␣ overnight. T-cell blasts (5 ⫻ 106 in
PBS⫹/⫹) were added to the SDF1␣-bearing endothelial monolayer for
30 minutes at 37°C. Cells were fixed in Karnovsky fixative and postfixed in
Chemotaxis assay
The inner and outer surface of transwells (5-␮m pore size; Costar,
Cambridge, MA) were coated with 0.5% ovalbumin (Ova) for 2 hours at
RT. Purified T cells (106 in 150 ␮L RPMI 0.1% Ova) were treated with
2 ␮M PKC␨ inhibitor for 1 hour when indicated and loaded in an
Ova-coated transwell, which was placed into a 24-well plate containing
250 ␮L RPMI supplemented with 0.1% Ova and SDF1␣, SLC (100 ng/
mL), or S1P (15 nM), as indicated. After 1 hour at 37°C, the cells that
migrated into the lower chamber were collected and counted.
Cell lysis and immunoblotting
Lysates were prepared in standard NP40 lysis buffer (10% glycerol, 50 mM
Tris-HCl [pH 7.4], 1% NP40, 150 mM NaCl, 20 mM NaF, 2 mM MgCl2,
1 mM Na3VO4, 1 mg/mL protease inhibitor) for 10 minutes at 4°C and
centrifuged at 16 000g for 10 minutes at 4°C. Lysates were denatured with
sodium dodecyl sulfate (SDS) and separated by SDS–polyacrylamide gel
electrophoresis (PAGE). For immunoblotting, membranes were blocked
with 5% BSA, probed with specific antibodies, and incubated with the
appropriate horseradish peroxidase–conjugated secondary antibodies. Immunoreactive bands were visualized by enhanced chemiluminescence
(Pierce, Rockford, IL).
Rac activity assay
Rac activity was determined as described previously.28 T cells (10 ⫻ 106)
were starved for 18 hours in IMDM medium with 0.5% BSA, stimulated as
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indicated, and lysed in standard NP40 buffer containing a biotinylated
Rac1/Cdc42-interactive binding motif peptide of PAK1. Active Rac was
precipitated with streptavidin-agarose beads.
Statistical analysis
Data were expressed as mean plus or minus SD. Comparisons between
groups were analyzed by using the Student t test with Microsoft Excel
Software (Redmond, WA). Data were considered significant when P values
were less than .05.
Results
Tiam1-mediated Rac activity is necessary for chemokine- and
S1P-induced T-cell migration
To investigate whether Tiam1 was required for Rac activation
induced by attractants, we used Tiam1-deficient (Tiam1⫺/⫺) T cells
and analyzed Rac activation in response to various extracellular
stimuli. Whereas basal Rac activity was not significantly different
between wild-type (WT) and Tiam1⫺/⫺ T cells (Figure 1A,C,E),
Tiam1⫺/⫺ cells were defective in SDF1␣-induced Rac activation
(Figure 1A) leading to impaired chemotaxis (Figure 1B). Tiam1
was also necessary for Rac activation and migration induced by the
chemokine SLC (Figure 1C,D). We wondered whether other types
of attractant for T cells could also signal via Tiam1. The lipid S1P is
a crucial component regulating T-cell egress from pLNs and
functions as an attractant for T cells (for review, see Cyster29).
Similarly as found for chemokines, S1P-mediated Rac activation
(Figure 1E) and migration (Figure 1F) appeared also impaired in
Tiam1⫺/⫺ T cells compared with WT T cells. From these results, we
conclude that Tiam1 is essential for chemokine- and S1P-mediated
Rac activation that controls chemotactic cell migration in T cells.
Tiam1 acts in conjunction with the PKC␨ to promote
S1P-induced Rac activation and migration
We previously found that Tiam1 regulates Rac activation and T-cell
migration induced by the chemokine SDF1␣ downstream of the
Par/PKC␨ polarity complex.30 As PKC␨ activation is required for
Tiam1-mediated Rac activation, we investigated whether PKC␨
could be activated in response to S1P by analyzing the phosphorylation status of PKC␨ (Thr410). S1P increased PKC␨ phosphorylation in both WT and Tiam1⫺/⫺ T cells (Figure 2A), demonstrating
that the PKC␨ is activated by S1P independently of Tiam1.
However, inhibition of PKC␨ downstream signaling by a myristoylated PKC␨ pseudosubstrate (PKC␨ inhibitor)31 abrogated S1Pinduced Rac activation in WT cells, indicating that PKC␨ functions
upstream of Tiam1 and is required for S1P-induced Rac activation
(Figure 2B,C). We finally determined whether PKC␨ activity was
necessary for S1P-mediated T-cell migration. Inhibition of PKC␨
downstream signaling reduced chemotactic migration toward S1P
of WT cells, whereas it had no effect on residual chemotactic
migration of Tiam1⫺/⫺ T cells (Figure 2D).
These data show that PKC␨ activity is required for Tiam1mediated Rac activation and that this pathway regulates S1Pmediated chemotactic migration of T cells.
Tiam1 is required for efficient T-cell trafficking
Chemokine signaling is required for T-cell adhesion and migration,32,33 processes that are essential for T cells to home to
secondary lymphoid organs (SLOs).34 By contrast, S1P-mediated
signaling has been implicated in the egress of T cells from pLNs.
Figure 2. Tiam1 cooperates with PKC␨ to regulate S1P-induced Rac activation
and chemotaxis. (A) WT and Tiam1⫺/⫺ T cells were either nonstimulated or
stimulated with 150 nM S1P for the indicated time periods. Subsequently PKC␨
phosphorylation status was determined. (Top panel) Phosphorylated PKC␨. (Middle
panel) Total PKC␨. (Bottom panel) Tiam1. Sizes are indicated in kilodaltons. (B) WT T
cells were either nontreated or treated with 2 ␮M PKC␨ inhibitor for 1 hour and
subsequently stimulated with 150 nM S1P for the indicated time periods. Subsequently, Rac activity status was determined. (Top panel) GTP-bound Rac. (Bottom
panel) Total Rac. Sizes are indicated in kilodaltons. (C) Quantification of the Rac
activation as shown in panel B. Rac activity value in nonstimulated WT T cells is set to
1. (D) Chemotaxis of WT and Tiam1⫺/⫺ lymphocytes either treated or untreated with
2 ␮M PKC␨ inhibitor was measured in response to 15 nM S1P after 1 hour. The
results are presented as the percentage of the input cells and are based on
3 independent experiments. Error bars indicate SD; p, P value.
Our in vitro studies showed that Tiam1 functions in both chemokine- and S1P-mediated T-cell migration and we investigated
therefore the function of Tiam1 in T-cell trafficking in vivo.
We first analyzed the requirement of Tiam1 for T-cell homing
by analyzing the capacity of adoptively transferred WT and
Tiam1⫺/⫺ T cells to reach SLOs in WT mice. Two hours after
adoptive transfer, approximately 2 times more WT T cells were
found in pLNs compared with Tiam1⫺/⫺ T cells (Figure 3A).
Moreover, we found that higher numbers of WT cells reached the
mesenteric lymph nodes and spleen compared with Tiam1⫺/⫺
T cells (Figure 3A). Consistent with these data, lower numbers of
WT cells remained in the circulation. The same difference in
homing was observed for CD4⫹ and CD8⫹ cells (Figure S2),
indicating that a lack of Tiam1 delays homing of both T-cell
subpopulations. Cryosections of pLNs from homing experiment
revealed that Tiam1⫺/⫺ cells were concentrated within or close to
HEVs, whereas WT T cells were already present within the pLNs
(Figure 3B). This supports our conclusion that Tiam1⫺/⫺ cells enter
pLNs less efficiently than WT T cells. From these data, we
conclude that Tiam1 deficiency delays short-term homing of
T cells, particularly to pLNs.
Subsequently, we analyzed whether the egress of T cells from
pLNs was also affected by Tiam1 deficiency. We performed egress
experiments in which further homing was blocked with anti–
L-selectin mAb injection 24 hours after adoptive transfer of WT
and Tiam1⫺/⫺ T cells. We determined the ratio between the
percentage of Tiam1⫺/⫺ and WT T cells retained in pLNs after
blocking homing. If the egress rate of Tiam1⫺/⫺ T cells is slower
than that of WT T cells, the retention ratio should increase
overtime. After 24 hours of homing (time point 0), we found a
retention ratio of approximately 0.7 (Figure 3C). When blocking
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Figure 3. Tiam1 is required for homing and egress of
T cells. (A) T-cell homing: CFDA-labeled WT and
CMTMR-labeled Tiam1⫺/⫺ naive T cells were mixed and
injected intravenously in a WT mouse. Mice were killed
2 hours after transfer and the percentage of labeled cells
in different organs was determined. Results are expressed as a homing ratio, corresponding to the ratio
between number of Tiam1⫺/⫺ and WT cells. Bars are
means. All data points are depicted by a dot; P values
between WT and Tiam1⫺/⫺ T cells are depicted for every
organ tested. (B) Representative cryosection of a pLN
after a homing experiment performed in panel A. Adoptively transferred WT T cells appear in green and Tiam1⫺/⫺
T cells, in red. HEVs (blue) are visualized using MECA-79
Ab. Scale bar represents 20 ␮m. (C-E) T-cell egress:
mixed adoptively transferred CMFDA-labeled WT and
CMTMR-labeled Tiam1⫺/⫺ T cells were allowed to home
to pLNs for 24 hours. Homing was then blocked by
Mel-14 Ab injection. Mice were killed 0, 12, and 24 hours
after blocking of homing. The relative number of adoptively transferred cells present in pLNs was calculated by
FACS analysis. (C) Ratio between the number of Tiam1⫺/⫺
and WT cells present in pLNs at different time points.
Bars are means. All data points are depicted by a dot.
Three independent experiments were performed; P values of 12- and 24-hour time points compared with
0 hours are depicted. (D) Kinetics of WT and Tiam1⫺/⫺
cell numbers present in pLNs during the egress assay.
Results are expressed as a percentage of the initial
population present at time point 0. Three independent
experiments were performed. Values indicate mean ⫾
SD; P values: comparison of the relative decrease of WT
and Tiam1⫺/⫺ T-cell numbers at the same time point.
(E) Representative cryosection of a pLN 24 hours after
adoptive transfer of WT (green) and Tiam1⫺/⫺ T cells
(red). HEVs (blue) are visualized using MECA-79 Ab.
homing, this retention ratio gradually increased to 1.8 at 24 hours
(Figure 3C). Analysis of the number of cells retained in pLNs at
different time points confirmed that Tiam1⫺/⫺ cells had a lower
egress rate than WT T cells (Figure 3D). The dwell time (time when
50% of the initial population had left pLNs) was 11.8 hours and
16 hours for WT and Tiam1⫺/⫺ T cells, respectively. The defect in
egress of Tiam1⫺/⫺ T cells was not caused by a retention of these
cells in the HEVs, as Tiam1⫺/⫺ cells freely migrated within the
pLNs 24 hours after adoptive transfer (Figure 3E).
From these data, we conclude that Tiam1-deficient T cells are
delayed in both homing and egress. Apparently, the established
defects in chemokine- and S1P-induced Rac activation and chemotaxis of Tiam1-deficient T cells in vitro lead to less efficient homing
and egress of these cells in vivo.
Tiam1 is required for contact hypersensitivity
As migration of T cells is required not only for steady-state
trafficking but also for reaching sites of inflammation during
immune responses, we suspected a function of Tiam1 during
T-cell–mediated immune responses. To test this hypothesis, we
performed contact hypersensitivity experiments on WT and
Tiam1⫺/⫺ mice and compared ear swelling induced by FITC
sensitization. As shown in Figure 4A, 1 day after challenge,
Tiam1⫺/⫺ mice showed significantly decreased ear swelling compared with WT mice (Figure 4A), which was accompanied by a
reduced T-cell infiltration in the ear (Figure 4B). Interestingly,
4 days after challenge, whereas WT mice showed a decrease in ear
swelling, Tiam1⫺/⫺ mice mounted up a response, and showed also
significant ear swelling (Figure 4A). This delay in contact hypersensitivity response suggests that T-cell recruitment, rather than
activation, is affected by Tiam1 deficiency. Indeed, hapten uptake
and recruitment of antigen-presenting cells to the draining LN was
not impaired since the percentage of CD11c- and FITC-positive
cells in the inguinal LN was identical between WT and Tiam1⫺/⫺
mice 3 days after sensitization (Figure 4C). Furthermore, LN
amplification after hapten sensitization was not impaired in
Tiam1⫺/⫺ mice (Figure 4D), arguing for a normal initiation of the
immune response in those mice. Together, these data suggest that
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Tiam1 REGULATES T-CELL TRAFFICKING
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Figure 4. Tiam1 functions during contact hypersensitivity. (A) WT and Tiam1⫺/⫺ mice were sensitized with
0.5% FITC solution. After 6 days, 10 ␮L 0.5% FITC
solution or control solution was applied to both ears and
the thickness of each ear was measured on days 1 and 4
after challenge. Values are means ⫾ SD (n ⫽ 10);
p indicates P value. (B) Infiltrating T cells in ears of WT
and Tiam1⫺/⫺ mice 1 day after FITC challenge. T-cell
infiltrates are presented as the number of CD3⫹ T cells
per unit surface area in the FITC-challenged ear. Values
are means ⫾ SD (n ⫽ 6). (C,D) WT and Tiam1⫺/⫺ mice
were sensitized with 0.5% FITC solution. On day 3,
draining LNs were isolated and single cell suspensions
were stained for CD11c and counted. (C) Representative
FACS analysis showing Cd11c/FITC double-positive cells
in draining LNs. (D) Increased LN cell number after
hapten sensitization. Results are expressed as fold
increase (LN cell number of sensitized mouse/LN cell
number of control mouse). Values are means ⫾ SD
(n ⫽ 4).
the ear swelling defect observed in Tiam1⫺/⫺ mice is due to a
reduced capacity of T cells to leave the LN and/or to reach the
inflammation site as a result of impaired Tiam1-mediated Rac
activation.
Tiam1 is optional for T-cell arrest but essential for T-cell
migration
During processes of homing to pLNs and recruitment to inflammation site, T cells have to cross a natural barrier constituted by
specialized endothelial cells. Chemokines are important in attracting T cells, but are mainly required during transendothelial
migration for arrest, migration, and diapedesis.5,35 Therefore, we
wondered in which chemokine-dependent steps necessary for
transendothelial migration Tiam1 was required.
We first determined the ability of naive WT and Tiam1⫺/⫺
T cells to arrest and adhere to a TNF␣-activated endothelial
monolayer under physiologic shear flow. No significant differences
in adhesion capacity to the endothelial monolayer were found
between WT and Tiam1⫺/⫺ T cells (Figure 5A). We also analyzed
the ability of activated T cells to adhere to an ICAM-1 substrate
under shear flow condition in the presence of Ca2⫹/Mg2⫹. We could
not detect significant differences between adhesion capacity of WT
and Tiam1⫺/⫺ T cells (Figure 5B). Intriguingly, although Tiam1⫺/⫺
T cells adhered similarly as WT cells, they were unable to activate
Rac upon adhesion to an ICAM-1 substrate (Figure 5C), suggesting
that disturbed Tiam1-mediated Rac activation is not essential for
T-cell arrest on ICAM-1 under shear flow condition.
To further understand what Tiam1-mediated Rac activation was
responsible for, we analyzed the migratory capacity of WT and
Tiam1⫺/⫺ T cells under shear flow condition. Shortly after their
arrest, most of the control WT T cells (⬎ 65%) started to migrate
on ICAM-1, whereas only a small portion of the Tiam1⫺/⫺ T cells
(⬃ 20% after 15 minutes) were able to do so (Figure 5D).
Time-lapse imaging revealed that after arrest most of the WT
T cells became stably front-rear polarized (Figure 5E) and started
to migrate in one direction (Video S1). This was confirmed by
real-time localization of GFP-actin, showing stable lamellipodium
formation at the leading edge of migrating WT cells (Figure S3A).
In contrast, the polarization of Tiam1⫺/⫺ T cells was much more
transient and unstable. At the arrest site, Tiam1⫺/⫺ T cells
frequently changed their polarity and position of lamellipodium, as
seen in phase-contrast images (Figure 5E) and images of GFP-actin–
expressing cells (Figure S3A). This defect resulted in less efficient
directional migration (Figure S3B, Video S2) and in a decrease in
overall migration speed (Figure S3C). Consistent with this finding,
the percentage of polarized cells on ICAM-1 after 15 minutes under
shear flow conditions was 2 times higher in WT cells compared
with Tiam1⫺/⫺ T cells (Figure 5F).
Together, these data indicate that the impaired Rac activation
observed in Tiam1⫺/⫺ T cells upon adhesion is required for stable
polarization and subsequent efficient migration rather than adhesion.
Tiam1 and PKC␨ activity are essential for crawling during T-cell
transendothelial migration
To further investigate the function of Tiam1 during transendothelial
migration, we used an approach allowing us to study diapedesis
independently from crawling.26 The behavior of WT and Tiam1⫺/⫺
T-cell blasts on TNF␣-activated endothelial monolayers was analyzed under shear flow condition. Approximately 16% of the
adherent WT T cells remained nonmotile (stationary), 42% of the
cells migrated on top of the monolayer without transmigrating
(crawling), whereas 40% of the cells succeeded in transmigration
(diapedesis; Figure 6A). In contrast, only a small proportion (20%)
of Tiam1⫺/⫺ T cells were able to crawl on top of endothelial
monolayers, consistent with the finding that Tiam1 is required for
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results were found with naive T cells (Figure S4), indicating that
Tiam1 deficiency affected both naive and activated T cells.
As PKC␨, a component of the Par polarity complex, acts
upstream of Tiam1 and Rac1 (Figure 2), we also inhibited PKC␨
signaling in WT cells. Similarly as found for Tiam1⫺/⫺ cells, WT
T cells treated with the PKC␨ inhibitor showed impaired crawling
on the endothelial monolayer but showed normal transmigration
(Figure 6B), suggesting that Tiam1 and PKC␨ are acting in the
same signaling pathway to control T-cell polarization and crawling
on endothelial monolayers. Indeed, we found also reduced percentage of polarized Tiam1⫺/⫺ cells compared with WT cells on top of
the endothelial monolayer (Figure 6C). Although the percentage of
diapedesis over time was slightly higher for WT T cells, we could
not find significant differences between WT and Tiam1⫺/⫺ T cells
(Figure 6D). Moreover, the average time for one single cell to cross
the endothelial monolayer was not different between the 2 genotypes (Figure S5). Together, we conclude that Tiam1 and PKC␨ are
specifically required for stable polarization and crawling of T cells
on top of an endothelial monolayer.
Inhibition of PKC␨/Tiam1 signaling leads to a switch from
paracellular to transcellular T-cell diapedesis
Figure 5. Tiam1 is dispensable for T-cell arrest but essential for subsequent
T-cell migration. (A) WT and Tiam1⫺/⫺ T cells were subjected to shear flow for
15 minutes after an accumulation phase on activated bEnd.3 monolayer. The
average number of adherent cells per field (⫻20 objective) at the end time point was
determined from 4 independent experiments (2 fields per experiment). Error bars
indicate SD; p, P value. No significant difference was observed between WT and
Tiam1⫺/⫺ T cells. (B) WT and Tiam1⫺/⫺ T-cell blasts were subjected to shear flow on
ICAM-1 substrate. At the indicated time points, average number of adherent cells per
field (⫻10 objective) was calculated from 3 independent experiments (2 fields per
experiment). Error bars indicate SD; p, P value between number of adherent WT and
Tiam1⫺/⫺ T cells for the same time point. No significant difference was observed.
(C) Blasts from WT and Tiam1⫺/⫺ mice were either nonstimulated or stimulated by
adhesion to ICAM-1 for 5 minutes. Subsequently, Rac activity was determined. (Top
panel) GTP-bound Rac. (Middle panel) Total Rac. (Bottom panel) Tiam1. (D) Locomotion of adherent WT and Tiam1⫺/⫺ blasts on an ICAM-1 substrate under shear flow
was recorded for 15 minutes, and the percentage of migrating cells was determined
at the indicated time points. At least 3 independent experiments were performed and
2 fields for each time point per experiment were recorded. Values indicate mean plus
or minus SD; p, P value between percentage of migrating WT and Tiam1⫺/⫺ T cells,
for each time point. (E) Representative confocal images of adherent WT and
Tiam1⫺/⫺ T-cell blasts on an ICAM-1 substrate under shear flow. Images are recorded
every 20 seconds. Scale bar represents 5 ␮m. Arrows indicate the direction of
polarization. Note the switches in direction of polarization of Tiam1⫺/⫺ T cells.
(F) Polarization of WT or Tiam1⫺/⫺ T-cell blasts was monitored after 10 minutes on
ICAM-1–coated coverslips under shear flow. At least 100 cells were monitored in
each group. Two independent experiments were performed and 2 fields per condition
were analyzed. Values indicate mean ⫾ SD; p, P value between percentage of
polarized WT and Tiam1⫺/⫺ T cells.
efficient migration on ICAM-1 (Figure 5D). As a consequence, we
found an increased percentage (42%) of Tiam1⫺/⫺ T cells in the
stationary phase. Tiam1⫺/⫺ T cells showed no obvious difference in
diapedesis capacity compared with WT cells (Figure 6A). Similar
Leukocytes can use 2 different routes to cross the endothelial
barrier (ie, the paracellular and the transcellular route).5,36 Paracellular migration is the most frequently route used for transendothelial migration, and implies that leukocytes transmigrate between
endothelial cells at cell junctions. When using the transcellular
route, leukocytes cross the endothelial barrier by transmigrating
directly through individual endothelial cells. As we found no major
differences in percentage of WT and Tiam1⫺/⫺ T cells undergoing
diapedesis, we investigated whether disturbed PKC␨/Tiam1/Rac
signaling affected the route of transendothelial migration. By
fixed-cell imaging, we blindly quantified the percentage of cells
transmigrating through the endothelial cytoplasm or at endothelial
junctions. Endothelial cell junctions and the transmigration pore
were visualized by PECAM-1 and ICAM-1 staining, whereas
T cells were visualized by LFA-1 staining as depicted in Figure 7A.
Using this method of quantification, most of WT T cells used the
paracellular route (Figure 7B). Intriguingly, more than 30% of the
Tiam1⫺/⫺ T cells crossed the endothelial monolayer via the
transcellular route (Figure 7B), suggesting that impaired Tiam1dependent polarization caused this shift in transmigration route.
Recent studies have demonstrated that transcellular, but not
paracellular, migration is dependent on Src-mediated formation of
invasive podosomes on T cells.7 Indeed, electron microscopic
studies revealed the presence of podosomes particularly in Tiam1⫺/⫺
cells before diapedesis (Figure 7C), suggesting that transcellular
migration in Tiam1⫺/⫺ T cells is dependent on podosome formation. We treated therefore T cells with the Src inhibitor PP2 and
quantified the percentage of adherent T cells undergoing diapedesis
under shear flow. Treatment of WT T cells with the Src inhibitor
PP2 only slightly decreased the percentage of transmigrating cells
(Figure 7D), consistent with the low percentage of WT cells
(⬃ 5%) using the transcellular route. In contrast, diapedesis was
approximately 40% reduced in Src-inhibited Tiam1⫺/⫺ T cells,
consistent with the higher percentage of Tiam1⫺/⫺ cells using the
transcellular route. These results further support the conclusion
drawn from visual analysis (Figure 7B) that transcellular migration
is enhanced in Tiam1⫺/⫺ T cells.
Similarly as the effects of PKC␨ inhibition on stabilization of
polarization and T-cell crawling, inhibition of PKC␨ also led to a
strong reduction in diapedesis upon Src inhibition in WT but not in
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Tiam1 REGULATES T-CELL TRAFFICKING
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Figure 6. Tiam1 is required for T-cell crawling. T-cell blasts were
subjected to shear flow for 15 minutes after accumulation on activated
bEnd.3 monolayer. Four independent experiments were performed and
2 fields per condition for every experiment were recorded. (A,B) The
behavior of single adherent T-cell blasts was analyzed and classified as
stationary, crawling (without transendothelial migration), or transmigrating
(diapedesis condition). Values indicate mean ⫾ SD. (A) Analysis of WT
and Tiam1⫺/⫺ T cells; p indicates P value between the percentage of WT
and Tiam1⫺/⫺ T cells. (B) Analysis of WT T cells treated with or without a
PKC␨ inhibitor (2 ␮M); p indicates P value between percentage of treated
and nontreated WT T cells. (C) Polarization of WT or Tiam1⫺/⫺ T-cell
blasts was monitored after 15 minutes. At least 50 cells were monitored in
each group. Values indicate mean ⫾ SD; p, P value between percentage
of polarized WT and Tiam1⫺/⫺ T cells. (D) The percentage of WT and
Tiam1⫺/⫺ T cells undergoing diapedesis was analyzed over time (every
3 minutes). Values indicate mean ⫾ SD; p, P value between percentage of
WT and Tiam1⫺/⫺ T cells undergoing diapedesis at the same time point.
No significant difference in diapedesis was observed between WT and
Tiam1⫺/⫺ T cells.
Tiam1⫺/⫺ T cells (Figure 7E). This indicates that upon PKC␨
inhibition WT cells preferentially use the transcellular route during
diapedesis similarly as Tiam1-deficient T cells. Together we
conclude that T cells with impaired PKC␨/Tiam1/Rac signaling
show unstable polarization and impaired crawling, and shift from
paracellular to transcellular migration during the process of transendothelial migration (Figure 7F).
Discussion
Trafficking of immune cells relies on their capacity to migrate
toward a chemotactic gradient, or in other terms, to undergo
chemotaxis.37 Here, we found that the Rac activator Tiam1 in
conjunction with PKC␨ are required for efficient chemokine- and
S1P-induced Rac activation and migration and thereby are essential
for efficient T-cell trafficking. Indeed, Tiam1-deficient T cells show
impaired homing and egress while the recruitment to sites of
inflammation is strongly delayed. During T-cell trafficking, Tiam1mediated Rac activation is not required for T-cell arrest but is
essential for stable polarization and crawling of T cells on top of
endothelial monolayers. Intriguingly, a large percentage of T cells
with impaired PKC␨/Tiam1/Rac signaling switch from paracellular
to transcellular migration (Figure 7F), suggesting that stable
polarization is a prerequisite for transmigration at endothelial
junctions.
We and others have shown that Tiam1 associates with Par3 of
the Par polarity complex (consisting of Par3, Par6, and atypical
PKC␨) and thereby regulates various polarization processes such as
axon specification in neuronal cells,38 epithelial apical-basal cell
polarization,28,39,40 and front-rear polarization of keratinocytes and
T cells.30,41 Here we found that T cells lacking Tiam1 or having
impaired PKC␨ signaling show short-lived polarization whereby
cells frequently change their direction of polarity, preventing
efficient migration on ICAM1 or endothelial monolayers. These
data strongly suggest that Tiam1 and the Par complex regulate the
maintenance and stability of polarization in T cells. As a conse-
quence of the polarization and migration defects, Tiam1-deficient
T cells switch the route of transendothelial migration and show a
delay in homing, egress, and recruitment to inflammatory sites.
It is likely that other Rac activators contribute to the different
steps of T-cell trafficking. In vitro studies indicate that Dock2 is
required for crawling endothelial monolayers but is not essential
for T-cell arrest and diapedesis.26 The striking similarities between
the function of Dock2 and Tiam1 suggest that they act in the same
signaling pathway. However, Tiam1 activation is controlled by
Rap1 activity independently of Dock2,30 whereas Dock2 deletion
does not influence chemokine-induced Rap activation.19 This
suggests that Tiam1 and Dock2 initiate 2 separate signaling
pathways that possibly converge and that are both essential for
T-cell migration.42 The residual migration seen in both Dock2⫺/⫺
and Tiam1⫺/⫺ T cells could be controlled by the redundant function
of these different Rac activators.
Tiam1 deficiency did not affect the adhesion of T cells under
shear flow. This is consistent with the findings that PKC␨ is not
involved in LFA-1 high-affinity state and T-cell arrest on
HEVs.43 Apparently PKC␨/Tiam1 signaling is dispensable for
T-cell adhesion, suggesting that other Rac activators may
control integrin-mediated adhesion of T cells.44 Likely candidates are Rac activators of the Vav family. Vav1 activates Rac
upon LFA-1 stimulation45 and has been implicated in T-cell
adhesion mediated by the integrin ␣4␤1.16 Together these data
suggest that different activators of Rac have specific functions in
the various steps of T-cell trafficking.
Crawling of leukocytes seems to be important for seeking
appropriate sites for transendothelial migration. Mac1-deficient
neutrophils are impaired in crawling and show delayed transmigration across HEVs, preferentially through the transcellular
pathway.46 These observations are consistent with our conclusions that delayed homing, impaired ear swelling, and the switch
to transcellular migration are the result of the inability of
Tiam1-deficient cells to respond properly to chemokine stimuli
required for stable polarization and crawling.
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BLOOD, 11 JUNE 2009 䡠 VOLUME 113, NUMBER 24
Figure 7. Impaired PKC␨/Tiam1/Rac signaling induces a switch from paracellular to transcellular
migration. (A,B) Activated WT and Tiam1⫺/⫺ T cells were
incubated on top of a monolayer of TNF␣-activated
bEnd.3 cells overlaid with SDF1␣ for 30 minutes. Fixed
samples were subjected to LFA-1, ICAM-1, and PECAM-1
staining. (A) Representative examples of immunofluorescence images showing paracellular and transcellular
migration of a T cell. Scale bars represent 5 ␮m. (B) Percentage of WT and Tiam1⫺/⫺ T cells showing diapedesis
via the transcellular route. Three independent experiments were performed. Values are means ⫾ SD; p indicates P value between WT and Tiam1⫺/⫺ T cells.
(C) Tiam1⫺/⫺ T cells migrating on activated bEnd.3 were
fixed after 30 minutes and processed for transmission
electron microscopy (EM). Image shows typical podosome structures invading the endothelial cell (¡).
(D,E) Naive T cells were either untreated or treated 2 ␮m PP2
for 1 hour. After accumulation on activated bEnd.3 monolayer, T cells were subjected to shear flow for 15 minutes.
Percentages of adherent cells undergoing diapedesis
during this period were evaluated. Diapedesis of PP2treated T cells was expressed as a percentage of control
cells (untreated cells ⫽ 100%). Two fields for each condition were recorded in 4 independent experiments. Values
indicate mean ⫾ SD. (D) Comparison of diapedesis route
used by WT and Tiam1⫺/⫺ T cells upon PP2 treatment;
p indicates P value between percentage WT and Tiam1⫺/⫺
T cells undergoing diapedesis. (E) WT T cells were
untreated or treated for 1 hour at 37°C with a PKC␨
inhibitor (2 ␮M) before shear flow experiments; p indicates P value between percentage of untreated WT cells
and PKC␨ inhibitor–treated WT cells undergoing diapedesis. (F) Model illustrating that crawling and the route of
transendothelial migration depends on PKC␨/Tiam1dependent polarization and crawling. Tiam1 is dispensable for T-cell adhesion to endothelial monolayers but is
required for polarization and subsequent T-cell crawling
on top of endothelial monolayers. T cells with intact
PKC␨/Tiam1 signaling preferentially transmigrate through
endothelial junctions (paracellular migration). Loss of
Tiam1 and disturbed PKC␨ signaling in T cells prevents
chemokine-induced Rac activation leading to unstable
polarization and thereby impaired T-cell crawling on top of
endothelial monolayers. As a result, these T cells preferentially cross the endothelial monolayer through individual endothelial cells (transcellular migration).
Diapedesis is a critical component of T-cell trafficking
necessary for immune function. For transcellular migration,
ICAM1, PECAM1, caveolae, and vimentin are required on the
endothelial cells as well as Src-dependent formation of invasive
podosomes on the leukocytes.7,9,10,47 The type of vascular bed
may determine the conditions and the type of leukocytes that use
transcellular migration.7 Transcellular migration has been observed in the microvasculature, notably at the blood-brain
barrier11 and at HEVs.11,48 Electron microscopic studies revealed
the formation of podosomes in Tiam1⫺/⫺ T cells in vitro before
diapedesis. These data are consistent with the recently observed
requirement of podosome formation for transcellular migration.
Podosome formation is dependent on Src7 and indeed inhibition
of Src strongly reduced diapedesis of Tiam1⫺/⫺ but not WT
T cells, indicating Tiam1⫺/⫺ T cells preferentially use the
transcellular route of diapedesis. The shift from paracellular to
transcellular migration likely contributes to the delayed homing
and egress observed in Tiam1-deficient T cells. We cannot rule
out the possibility that Tiam1⫺/⫺ T cells are more predisposed to
make podosomes than WT cells while contacting endothelial
cells, thereby enhancing transcellular migration. However, it
is more conceivable that the reduced polarization and crawling capacity of Tiam1-deficient cells makes it more difficult to
find suitable transmigration sites at endothelial junctions, as
suggested earlier.46 In that scenario Tiam1-deficient T cells
could specifically initiate alternatives by the formation of
podosomes allowing their transmigration through individual
endothelial cells.
Acknowledgments
We thank coworkers from The Netherlands Cancer Institute
(NKI) mouse facility for their assistance with animal experiments, and P. Hordijk and F. van Alphen (Sanquin, Amsterdam,
The Netherlands) for their help with the shear flow experiments.
Furthermore, we thank S. Iden and S. Mertens for critical advice
on the paper.
This work is supported by grants from the Dutch Cancer Society
(Amsterdam, The Netherlands; J.G.C.).
BLOOD, 11 JUNE 2009 䡠 VOLUME 113, NUMBER 24
Tiam1 REGULATES T-CELL TRAFFICKING
Authorship
Contribution: A.G. conceptualized the study, performed all experiments (unless otherwise specified), analyzed the data, drew
the conclusions, and wrote the article; R.A.v.d.K. helped with
mice breeding and contact hypersensitivity experiments; H.J.
6147
performed the EM experiments; S.I.E. quantified the T-cell migration data from the videos; and J.G.C. supervised the work and
contributed to the writing of this article.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: John G. Collard, The Netherlands Cancer
Institute, Division of Cell Biology, Plesmanlaan 121, 1066 CX
Amsterdam, The Netherlands; e-mail: j.collard@nki.nl.
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