Ena/VASP proteins regulate activated T-celltrafficking by promoting diapedesis duringtransendothelial migrationMiriam L. Estina,b, Scott B. Thompsona,b, Brianna Traxingera, Marlie H. Fishera,b, Rachel S. Friedmana,b,and Jordan Jacobellia,b,1

aDepartment of Biomedical Research, National Jewish Health, Denver, CO 80206; and bDepartment of Immunology and Microbiology, University ofColorado School of Medicine, Aurora, CO 80045

Edited by Philippa Marrack, Howard Hughes Medical Institute, National Jewish Health, Denver, CO, and approved February 24, 2017 (received for reviewFebruary 3, 2017)

Vasodilator-stimulated phosphoprotein (VASP) and Ena-VASP–like(EVL) are cytoskeletal effector proteins implicated in regulatingcell morphology, adhesion, and migration in various cell types.However, the role of these proteins in T-cell motility, adhesion,and in vivo trafficking remains poorly understood. This study iden-tifies a specific role for EVL and VASP in T-cell diapedesis andtrafficking. We demonstrate that EVL and VASP are selectively re-quired for activated T-cell trafficking but are not required for normalT-cell development or for naïve T-cell trafficking to lymph nodes andspleen. Using amodel of multiple sclerosis, we show an impairment intrafficking of EVL/VASP-deficient activated T cells to the inflamedcentral nervous system of mice with experimental autoimmuneencephalomyelitis. Additionally, we found a defect in traffickingof EVL/VASP double-knockout (dKO) T cells to the inflamed skinand secondary lymphoid organs. Deletion of EVL and VASP resulted inthe impairment in α4 integrin (CD49d) expression and function.Unexpectedly, EVL/VASP dKO T cells did not exhibit alterationsin shear-resistant adhesion to, or in crawling on, primary endothe-lial cells under physiologic shear forces. Instead, deletion of EVLand VASP impaired T-cell diapedesis. Furthermore, T-cell diapede-sis became equivalent between control and EVL/VASP dKO T cellsupon α4 integrin blockade. Overall, EVL and VASP selectively me-diate activated T-cell trafficking by promoting the diapedesis stepof transendothelial migration in a α4 integrin-dependent manner.

T cell | migration | cytoskeleton | extravasation

Activated T-cell trafficking across the vascular endothelium isessential for ongoing immune surveillance of tissues and for

effective immune responses to conditions such as infection andcancer. Conversely, in situations of immune dysregulation, in-hibition of self-reactive T-cell trafficking represents a promisingtarget for therapeutic immunomodulation. Disruption of thesepathways, such as by antibody blockade of α4 integrins, is a highlyeffective approach to immunomodulation (1, 2). However, themolecular mechanisms by which chemokine receptor and adhesionmolecule signaling induce the T-cell cytoskeletal machinery topromote extravasation are not yet fully elucidated.Transendothelial migration (TEM), the process by which

T cells extravasate from the blood into tissues, is characterized byfour distinct steps: rolling along the vascular wall, arrest or adhe-sion, intravascular crawling, and diapedesis across the endothelialbarrier (3). Surface adhesion molecules play well-characterizedroles in each step of the process. For example, the initial rollingstep of TEM is facilitated by interactions between T-cell andendothelial selectins, whereas the adhesion, intravascular crawling,and diapedesis steps of TEM are mainly regulated by chemokine-and shear force-stimulated modulation of lymphocyte function-associated antigen 1 (LFA-1, αLβ2 integrin, CD11a/CD18) and verylate antigen 4 (VLA-4, α4β1 integrin, CD49d/CD29) interactionswith intracellular adhesion molecule 1 (ICAM-1) and vascular cel-lular adhesion molecule 1 (VCAM-1), respectively. Dynamic cyto-

skeletal changes occur throughout the process of TEM (4, 5);however, the regulation of these cytoskeletal changes is not completelyunderstood.The lymphocyte actin-myosin cytoskeleton is composed of

networks of linear and branched actin filaments, cross-linked byclass-II nonmuscle myosin. We have previously shown that in-hibition of myosin-IIA, the main class-II myosin protein expressedin lymphocytes, alters T-cell trafficking, motility, and TEM (6–9).Although numerous studies have focused on the upstream regula-tory signaling cascades that control actin network remodeling dur-ing migration (5, 10), less is understood about how downstreameffectors of branched and linear actin polymerization might par-ticipate in lymphocyte TEM. Two major families of effector pro-teins of linear actin polymerization are expressed in lymphocytes.The Formin family, which is thought to nucleate new linear actinfilament production, has been implicated in T-cell activation andegress from the thymus (11–13). Less is known about the Ena/VASP (vasodilator-stimulated phosphoprotein) family of cyto-skeletal effectors in T cells.The Ena/VASP family is composed of three members:

mammalian-enabled (Mena), which is not typically expressed inhematopoietic cells; VASP; and Ena-VASP–like (EVL) (14, 15).These proteins coordinate monomeric actin recruitment to thebarbed end of the actin filament, prevent actin filament capping,and can play a role in actin filament bundling (16–20). Structurally,EVL and VASP share significant homology, containing anN-terminal EVH1 domain, which regulates cellular localization;

Significance

T-cell trafficking is essential for the function of the adaptiveimmune system, and regulation of T-cell entry into tissues can bean effective therapy in diseases such as autoimmunity. However,the mechanisms regulating T-cell migration and trafficking arepoorly understood. We have identified a key role for Ena/VASP(vasodilator-stimulated phosphoprotein) family cytoskeletal ef-fectors selectively in activated T-cell trafficking to secondarylymphoid organs and to peripheral sites of inflammation. Ena/VASP deficiency in T cells causes a defect in α4 integrin function,which impairs transendothelial migration. Our work suggeststhat further studies of the Ena/VASP pathway in T cells couldidentify therapeutically useful ways to more selectively modu-late α4 integrin activity and activated T-cell trafficking.

Author contributions: R.S.F. and J.J. designed research; M.L.E., S.B.T., B.T., M.H.F., and J.J.performed research; M.L.E., S.B.T., R.S.F., and J.J. analyzed data; and M.L.E. and J.J. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. Email: jacobellij@njhealth.org.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1701886114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1701886114 PNAS | Published online March 20, 2017 | E2901–E2910

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and a C-terminal EVH2 domain, which facilitates tetramerization,binds filamentous actin (F-actin), and is thought to be responsiblefor actin polymerization (21–24). Ena/VASP proteins are capableof compensating for deletion of one another, but there is someevidence that they are differentially regulated (15).EVL, and especially VASP, are implicated in the motility, ad-

hesion, and sensory capacity of many cell types. EVL and VASPlocalize to filopodia tips (25) as well as to adhesive sites, such asfibroblast focal adhesions (24). This localization pattern is consis-tent with a role in migration and adhesion. Fibroblasts lacking EVLand VASP produce shorter filopodia, and a slower-moving lamel-lipodium, which paradoxically leads to enhanced fibroblast motility(26, 27). Platelets from VASP-knockout mice demonstrate en-hanced vascular adhesion (28), whereas inside-out signaling throughβ2 integrins is impaired in VASP-deficient neutrophils (29).Only a few studies have examined the role of Ena/VASP proteins

in T cells, showing that Ena/VASP proteins can contribute to actinremodeling during T-cell receptor (TCR) signaling (24, 30). How-ever, previous studies did not focus on the role of EVL and VASPin T-cell development or T-cell motility. Therefore, we sought toelucidate the mechanisms by which EVL and VASPmight influenceT-cell adhesion, migration, and trafficking in vivo.

ResultsDeletion of EVL and VASP Does Not Significantly Affect T-CellDevelopment. To determine if deletion of Ena/VASP proteinsaltered T-cell trafficking in vivo, we used EVL/VASP double-knockout (dKO) mice (generously provided by Frank Gertler,Massachusetts Institute of Technology, Cambridge, MA) (31, 32).We confirmed EVL and VASP deletion in T cells, and verified thatMena was not up-regulated as a compensatory mechanism in EVL/VASP dKO T cells by Western blot analysis (Fig. S1). We thenanalyzed if EVL/VASP deficiency caused defects in T-cell devel-opment. Flow cytometry analysis of lymphocyte populations in thethymus and secondary lymphoid organs showed no gross defects inT-cell development and normal proportions of mature CD4 andCD8 T cells in the periphery of EVL/VASP dKO mice (Fig. S2).

Deletion of Both EVL and VASP Selectively Impairs Activated CD4T-Cell Trafficking into Secondary Lymphoid Tissues. Next, we usedcoadoptive transfers of WT control and EVL/VASP dKO T cellsto study the T-cell–intrinsic effect of EVL and VASP deficiencyon trafficking in vivo. Consistent with the normal pattern of de-velopment and homeostatic trafficking, dKO and WT naïve CD4T cells had equivalent homing to the spleen and lymph nodes afterintravenous adoptive transfer in to WT recipient mice (Fig. 1A).Activated T cells have different requirements for migration

and trafficking than their naïve counterparts (33–36). Therefore,we investigated if Ena/VASP family proteins are specifically re-quired for activated T-cell trafficking. We first established that,upon ex vivo polyclonal activation with CD3 and CD28 stimu-lation, T-cell proliferation kinetics and activation profiles weresimilar for WT and EVL/VASP dKO T cells (Fig. S3). ActivatedT cells can become lodged in the vasculature (particularly in thelungs) (37, 38), potentially because of their increased size andadhesion properties. Therefore, we used an established tech-nique (39, 40) to distinguish extravasated T cells from thosestuck intravascularly by injecting intravenously a fluorophore-conjugated anti-CD4 antibody immediately before euthanasiaof the recipient mice (Fig. 1B). Only extravasated cells (T cellsnegative for intravascular anti-CD4 staining) were considered tohave infiltrated a tissue.In this setting, in vitro-activated CD4 T cells maintain expression

of CCR7 and can recirculate to secondary lymphoid organs. Todetermine if homeostatic trafficking of activated T cells was affectedby Ena/VASP deficiency, we cotransferred control and EVL/VASPdKO T cells into unimmunized recipient mice. Following in-travenous adoptive transfer, activated dKO CD4 T cells on average

exhibited a 2.2-fold reduction in spleen trafficking and a 3.3-fold re-duction in lymph node trafficking 2 h after adoptive transfer com-pared with WT controls (Fig. 1 C and D). This activated dKO T-celltrafficking defect still persisted 24 h posttransfer (Fig. S4 A and B).Furthermore, the intravascular staining used to quantify T cells

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Fig. 1. Deletion of both EVL and VASP selectively inhibits activated but notnaïve CD4 T-cell trafficking to secondary lymphoid organs. (A) Naïve T-celltrafficking is not affected by EVL and VASP deficiency. Differentially dye-labeled naïve WT and EVL/VASP dKO T cells were coadoptively transferredintravenously at a 1:1 ratio and T-cell trafficking to lymphoid tissues wasquantified by flow cytometry 2 h after adoptive transfer. The dKO:WT ratiowas normalized to the ratio in the injected sample to account for possibleminor variations in the injection mixture (typically < 10%). (B) Experimentalset-up for cotransfer of activated WT and KO cells, including intravascularstaining method to distinguish intravascular T cells from those that haveextravasated into the tissue of interest. (C–F) Dye-labeled CD4 WT andsingle-KO or dKO activated T cells were coadoptively transferred at a 1:1 ratioand T-cell trafficking to tissues 2 h after adoptive transfer was quantified byflow cytometry as above. A ratio below 1.0 (horizontal red line) indicates im-paired homing of KO T cells. (C) Quantification of activated T-cell trafficking.Ratio of extravasated WT and EVL/VASP dKO activated T cells, harvested andanalyzed as above. (D) Number of WT and dKO T cells recovered from theindicated tissues from data in C. (E and F) Trafficking of EVL (E) and VASP(F) single-KO activated T cells relative to WT controls. Data are the averagefrom a minimum of three independent experiments. Error bars are SEM. Sta-tistics are one-sample t test compared with a hypothetical value of 1.0 (A, C, E, F)or paired t test (D). LN, lymph node; ns, not significant.

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blood vessels also indicated that the defect in activated EVL/VASPdKO T-cell trafficking was not a result of selective trapping of thesecells in the lung microvasculature, the first capillary bed encoun-tered after intravenous adoptive transfer. In fact, significantly moreWT than dKO T cells were recovered from inside the lung micro-vasculature (Fig. S4C). In keeping with these data, there is a trendtoward more EVL/VASP dKO T cells than control cells in theblood compartment, suggesting that these cells may be unable togain full access to tissues from the blood (Fig. 1 C and D).We next analyzed if deletion of EVL or VASP alone was

sufficient to impair activated T-cell trafficking to secondarylymphoid organs. Consistent with the likelihood of redundant orcompensatory functions between Ena/VASP proteins, single-knockout of EVL or VASP did not give rise to activated T-celltrafficking defects (Fig. 1 E and F).

EVL and VASP Deletion Impairs Activated CD4 T-Cell Trafficking toSites of Inflammation. We next sought to determine if activatedCD4 T-cell trafficking to sites of inflammation was affected byEna/VASP protein deficiency. Because the vascular endothelialbarrier in the central nervous system (CNS) is particularly re-strictive, we first investigated EVL/VASP-deficient activatedT-cell trafficking to the CNS in the context of autoimmune in-flammation, using a mouse model of multiple sclerosis: experi-mental autoimmune encephalomyelitis (EAE). Twenty-fourhours after adoptive transfer of T cells into mice with ongoingEAE, we quantified the number of transferred T cells that hadextravasated into the CNS using the intravascular staining tech-nique described above. Activated dKO T cells exhibited on av-erage a 2.0-fold reduction in trafficking to the CNS relative tocontrol T cells (Fig. 2 A–C). Next, we analyzed T-cell trafficking

to the inflamed skin, using lipopolysaccharide (LPS) as the in-flammatory stimulus. Recipient mice were treated by sub-cutaneous LPS injections in the ear and 24 h later activatedcontrol and dKO T cells were intravenously transferred into therecipient mice. Quantification of transferred T cells that hadextravasated into the inflamed skin of the ear showed a 1.8-foldreduction in dKO T-cell trafficking to this site (Fig. 2 D–F).

Deletion of EVL and VASP Reduces Chemokine-Triggered ActinPolymerization but Does Not Impair Activated T-Cell Chemotaxis inVitro. Chemokine signaling is instrumental in mediating leuko-cyte migration and trafficking (41, 42). Therefore, we analyzed ifthe trafficking defect of Ena/VASP-deficient activated T cellscould be because of an altered ability to respond to chemokinestimulation. First, we measured the expression of chemokinereceptors involved in both homeostatic and inflammatory traf-ficking. Our data showed no difference in the expression ofCCR7, CXCR3, CXCR4, and CCR5 between control and EVL/VASP dKO CD4 T cells (Fig. 3A).Ena/VASP proteins are cytoskeletal effectors that promote

actin filament polymerization (14). During migration, chemokinestimulation can trigger actin network remodeling and promotemotility (34). Therefore, we analyzed if Ena/VASP deficiencyimpaired actin polymerization in response to chemokine stimu-lation. To this end, we measured the F-actin content in controland EVL/VASP dKO T cells before and after chemokine stim-ulation in a time-course analysis. Our results showed that dKO-activated T cells had a small but significant defect in actin po-lymerization promoted by CCL21 stimulation and, to a lesserdegree, by CXCL10 (Fig. 3B). However, when we analyzed actinpolymerization in response to CCL21 in naïve T cells, we did notsee a defect in EVL/VASP dKO cells (Fig. S5A). This findingsuggests a different reliance on Ena/VASP proteins for actinpolymerization between naïve and activated T cells.Based on this result, we then measured chemokine-stimulated

migration using Transwell chambers. There were no significantdifferences in migration in the absence of chemokine, or inchemotaxis toward CCL21, CXCL10, CXCL12, or CCL5 in thelower chamber between control and EVL/VASP dKO-activatedT cells (Fig. 3C). Furthermore, chemokinetic migration in responseto CCL21 in both the upper and lower chambers was also un-affected (Fig. S5B). Although we detected slightly reducedchemokine-mediated actin polymerization in EVL/VASP-deficient activated T cells, overall these data showing normalchemotaxis suggest that the strong trafficking defect of EVL/VASP dKO T cells in vivo is not explained by an overall defect inmigratory capacity or chemokine sensing.

Activated CD4 EVL/VASP dKO T Cells Are Deficient in α4 IntegrinExpression and Function. Ena/VASP proteins are reported to af-fect adhesion and integrin function (28, 43). Therefore, havingruled-out impaired chemotaxis, we next examined the expressionand function of key integrins involved in extravasation to de-termine if the trafficking impairment we observed was because ofintegrin defects. Flow cytometry analysis revealed that activatedEVL/VASP dKO T cells expressed on average 28% less CD49d,but 28% more CD11a (Fig. 4 A and B).CD49d, the α4 subunit of the integrins α4β1 (VLA-4) and

α4β7 (LPAM-1), is primarily expressed on antigen-experiencedT cells, which may explain why only activated dKO T cellsexhibited a trafficking defect in vivo. Consistent with their normaltrafficking phenotype, activated EVL or VASP single-knockoutT cells did not exhibit reduced CD49d expression, nor did naïvedKO T cells (which only expressed negligible levels of CD49d) (Fig.S6 A–E). Additionally, expression of the α4 integrin binding part-ners β1 (CD29) and β7 in activated cells was also reduced in acti-vated dKO T cells compared with WT T cells (Fig. S6F).Furthermore, total expression versus surface expression of CD11a

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Fig. 2. EVL and VASP deletion inhibits activated CD4 T-cell trafficking to theCNS during EAE and to the inflamed skin. (A) Experimental set-up for cotransferof WT and EVL/VASP dKO activated T cells into mice with ongoing EAE. Acti-vated, dye-labeled polyclonal CD4 WT and dKO T cells were coadoptivelytransferred at a 1:1 ratio, and were harvested 24 h posttransfer from the bloodand CNS (brain and spinal cord). (B) Activated T-cell trafficking during EAE wasquantified by flow cytometry, shown as the ratio of dKO:WT T cells normalizedto the ratio in the injected sample. (C) Number of WT and dKO T cells recoveredfrom the indicated tissues from data in B. (D) Experimental set-up to quantifyactivated T-cell trafficking to the inflamed skin. Twenty-four hours after LPS-induced inflammation in the ears, WT and dKO activated T cells were coad-optively transferred at a 1:1 ratio, and were harvested 24 h posttransfer from theblood and ears of the recipient mice. (E) Ratio of dKO:WT T cells recovered fromblood and ears, normalized to the ratio in the injected sample. (F) Number ofWTand dKO T cells recovered from the indicated tissues from data in E. Data are theaverage of four independent experiments. Error bars are SEM. Statistics are one-sample t test compared with a hypothetical value of 1.0 (B, E) or paired t test (C,F). IP, intraperitoneal; ns, not significant; SC, subcutaneous.

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and CD49d were similarly affected in activated dKO T cells, in-dicating that the defect is not a result of altered integrin traffickingfrom intracellular stores to the cell surface (Fig. S6 H and I).Integrin activity can be regulated by conformation, which in-

fluences integrin affinity for ligands (34, 44). The main CD49dligands are fibronectin and VCAM-1, with the latter expressedon vascular endothelial cells. Therefore, to determine if CD49dfunction was compromised in EVL/VASP-deficient T cells, wemeasured WT and dKO T-cell binding to soluble VCAM-1. Inthe absence of stimulation, there was very low basal VCAM-1binding capacity and no difference between WT and dKO acti-vated T cells. However, in response to phorbol myristate acetate(PMA)/ionomycin stimulation or treatment with MnCl2 (whichexogenously forces integrins into a high-affinity conformation), alower percent of dKO cells bound VCAM-1 (Fig. 4C). BecausedKO T cells expressed less CD49d, we normalized for this dif-ference in expression to evaluate CD49d affinity for VCAM. Wecalculated a VCAM-1/CD49d affinity index for WT and dKOT cells upon stimulation with PMA/ionomycin by normalizingthe geometric mean fluorescence intensity (gMFI) of boundVCAM-1 to the surface expression of CD49d (also by gMFI)(Fig. 4D). This analysis showed significantly decreased CD49daffinity for VCAM-1 on a per receptor basis upon deletion ofEVL and VASP. Taken together, these data show that deletion

of EVL and VASP decreases CD49d expression on the T-cellsurface and impairs inside-out signaling activation of CD49d.

Activated EVL/VASP dKO CD4 T Cells Are Competent in Shear-Resistant Adhesion and Crawling. To determine how the pertur-bation in CD49d activity we observed could influence the im-paired trafficking phenotype of EVL/VASP-deficient T cells, wethen investigated if decreased expression and function of CD49din EVL/VASP-deficient T cells caused impaired adhesion ormotility on integrin ligands. Using flow chambers that recapitu-late the physiologic shear forces of the microvasculature (Fig.5A), we first quantified shear-resistant adhesion to integrin li-gands in the presence of chemokine. Compared with controls,EVL/VASP dKO T-cell adhesion to ICAM-1 was actuallymoderately improved (Fig. 5B), consistent with increased CD11aexpression, and no significant differences were observed inshear-resistant adhesion to VCAM-1 (Fig. 5C). We then ana-lyzed dKO T-cell 2D migration on ICAM-1– or VCAM-1–coated surfaces under flow. Time-lapse spinning-disk confocalmicroscopy was used to image T cells, and the mean crawlingspeed of dKO T cells compared with WT was found to be slightlyslower on ICAM-1 (13% less) (Fig. 5D) and equivalent onVCAM-1–coated surfaces (Fig. 5E), suggesting that adhesionand motility on integrins is not severely impaired in dKO T cells.Next, adhesion to primary endothelial cells was quantified in

vitro to determine if T-cell adhesion to the vascular wall waslikely to be inhibited in vivo. Primary brain microvascular en-dothelial cells were grown to confluence in flow chambers, ac-tivated with TNF-α 24 h before imaging (to up-regulate adhesionmolecule expression), and pretreated with CCL21 30 min beforeimaging (to stimulate T-cell adhesion). Fluorescently dyed T cellswere flowed through the chamber and imaged with fluorescent andphase-contrast spinning-disk microscopy to quantify the number ofadhered T cells. Surprisingly, deletion of EVL and VASP did notalter shear-resistant T-cell adhesion to primary brain endothelial

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Fig. 3. Deletion of both EVL and VASP in activated CD4 T cells reduceschemokine-triggered actin polymerization but does not impair chemotaxis.(A) Quantification of chemokine receptor expression in WT and EVL/VASPdKO activated T cells; data shown as gMFI. (B) Time-course analysis of actinpolymerization in WT and dKO activated T cells in response to 100 ng/mLCCL21 stimulation (WT vs. dKO curve comparison P < 0.0001) or 100 ng/mLCXCL10 stimulation (WT vs. dKO curve comparison P = 0.032), measured byflow cytometry quantification of fluorescent phalloidin staining. (C) Che-motactic migration across 5-μm pore Transwell chambers in the absence ofchemokine, or in the presence of CCL21 (100 ng/mL), CXCL10 (100 ng/mL),CXCL12 (1 μg/mL), or CCL5 (100 ng/mL) in the bottom wells as indicated. Dataare the average of at least three independent experiments; error bars areSEM; statistics are paired t tests in A and C, or two-way ANOVA in B. ns, notsignificant.

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Fig. 4. Activated EVL/VASP dKO CD4 T cells have a deficit in α4 integrin(CD49d) expression and function. (A) Examples of CD11a and CD49d ex-pression on WT and EVL/VASP dKO activated T cells by flow cytometry.(B) Quantification of CD11a and CD49d surface expression in activated WTand dKO T cells; data shown as gMFI. (C) CD49d function measured as sol-uble VCAM-1 binding to T cells in response to the indicated stimuli (MnCl2:manganese chloride; PMA/I: PMA and ionomycin). (D) Affinity for VCAM-1calculated as PMA/ionomycin-elicited VCAM-1 binding normalized to sur-face expression of CD49d by gMFI. Data in A are representative of 10 in-dependent experiments; data in B are the average of ten experiments; datain C and D are the average of three independent experiments. Error bars areSEM. All P values are paired t tests. ns, not significant.

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cells (Fig. 5F), and detachment from the endothelial monolayerunder flow conditions was also unchanged (Fig. 5G). Furthermore,no statistically significant change was noted in the percentage ofEVL/VASP dKO T cells that adhered to the endothelial monolayerbut never crawled (Fig. 5H). Similarly, T-cell crawling speed on theendothelial monolayer under flow conditions, an in vitro model forintravascular crawling in vivo, was unaffected by EVL and VASPdeletion (Fig. 5I). Taken together, these data indicate that despitedifferences in integrin expression and function, EVL/VASP dKOT cells are capable of normal adhesion and migration on endo-thelial cells in vitro, suggesting that a different mechanism causedthe trafficking impairment of EVL/VASP-deficient T cells.Furthermore, using the intravascular staining method de-

scribed above to quantify T cells that remained intravascular inour in vivo trafficking experiments, we did not see a significantdifference or defect in the frequency of EVL/VASP dKO acti-vated T cells remaining in the vasculature of isolated lymphnodes from recipient mice 2 h posttransfer (Fig. 5J). In fact,there might be a trend toward increased numbers of dKO T cellsadhered to the lymph node vasculature. We also did not observea significant difference in the frequency of EVL/VASP dKOT cells that are found in the CNS microvasculature of mice withEAE, 24 h after adoptive transfer (Fig. 5K). Although this is nota direct readout of adhesion to the microvasculature, it furtherindicates that the impairment in activated dKO T-cell traffickingis unlikely to be because of a deficiency in the adhesion stepof extravasation.

Deletion of EVL and VASP Inhibits the Diapedesis Step ofTransendothelial Migration in Vitro. In addition to playing a rolein adhesion and intravascular crawling, integrins are cruciallyimportant for the diapedesis step of TEM (33, 45). Therefore, weinvestigated whether the trafficking defect of EVL/VASP-deficientT cells could be caused by defects in migration through the endo-thelial barrier during extravasation. For these experiments, we im-aged T-cell TEM on primary brain endothelial cells in the flow-chamber system described above using phase contrast and spinningdisk confocal microscopy to determine the effects of EVL andVASP deletion on T-cell diapedesis in vitro. Using phase-contrastmicroscopy, with the plane of focus set to the endothelial mono-layer, T cells attached on the endothelium (above the plane of fo-cus) appear to have a white halo surrounding them. In contrast,T cells that have undergone diapedesis and the protrusions pro-duced during that process lose this halo (6, 46) (Fig. 6 A and B andMovies S1 and S2). Collected images were analyzed to determinethe frequency and timing of diapedesis attempts, defined as T-cellproduction of fluorescent protrusions lacking a phase halo, anddiapedesis completions, defined as events in which the T-cell bodyfollows after the protrusion and the entire T-cell loses its halo. Thisanalysis revealed that deletion of EVL and VASP significantly im-paired both T-cell diapedesis attempts and completions (Fig. 6 Cand D), indicating that the mechanism of reduced dKO T-celltrafficking in vivo lies at the stage of diapedesis.

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Fig. 5. EVL/VASP dKO activated CD4 T cells are competent in shear-resistantadhesion and migration. (A) Schematic of the experimental set-up formeasuring T-cell adhesion and motility under physiologic shear forces.(B and C) WT and EVL/VASP dKO T-cell shear-resistant adhesion to ICAM-1(B), or to VCAM-1 (C). (D and E) Mean WT and dKO T-cell crawling speedunder flow on ICAM-1 in the presence of CCL21 (D) or on VCAM-1 in thepresence of CCL21 (E). (F) Quantification of WT and dKO T-cell shear-resistant

adhesion to TNF-α–activated primary microvascular brain endothelial cells inthe presence of CCL21. (G) Detachment of initially adhered T cells from ac-tivated endothelial monolayers after shear flow increase from 0.2 to 2 dyne/cm2.(H) Hyperadhesiveness of WT and dKO T cells to primary brain endothe-lial cells, measured as the frequency of adhered T cells that failed to crawl.(I) Mean T-cell crawling speed under shear forces on activated brain endo-thelial monolayers. (J) Percentage of in vivo adoptively transferred WTand dKO T cells recovered from lymph nodes that remained in the vascula-ture 2 h posttransfer (identified by intravascular staining). (K) Percentageof transferred T cells recovered from the CNS that remained intravascular,24 h posttransfer. Data represent the average of a minimum of three in-dependent experiments; error bars are SEM; P values are paired t tests. ns,not significant.

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CD49d-Independent Diapedesis Does Not Require EVL and VASP. Fi-nally, having identified a defect in CD49d function in the EVL/VASP dKO T cells, we examined how CD49d affected the di-apedesis of control and EVL/VASP-deficient T cells. To thisend, we preincubated T cells with CD49d-blocking or isotypecontrol antibodies before introducing the T cells into flowchambers, and then imaged for 30 min and analyzed the imagingdata, as described above. As expected, shear-resistant adhesionof both control and dKO T cells to the endothelial monolayerwas dramatically impaired by CD49d blockade. However, therewas a trend toward more EVL/VASP dKO than WT T cellsadhering to the endothelial monolayer with CD49d blockade,

suggesting that they may use alternate adhesion molecules moreeffectively (Fig. 6E). As opposed to control antibody treatment,which confirmed a significant reduction in TEM of the dKOT cells, CD49d blockade greatly reduced the number of adheredT cells on the endothelial monolayer and resulted in a similarability of the adhered WT and dKO T cells to complete di-apedesis (Fig. 6F). These data support the idea that EVL andVASP promote T-cell diapedesis via a CD49d-using mecha-nism, and that impaired CD49d function in EVL/VASP-deficient T cells is a mechanism for their in vivo defect inT-cell trafficking.

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Fig. 6. EVL/VASP-deficient activated CD4 T cells are impaired in the diapedesis step of transendothelial migration in vitro through a CD49d-dependentmechanism. Fluorescently labeled WT and EVL/VASP dKO activated T cells were flowed on primary brain microvascular endothelial monolayers and imaged bytime-lapse fluorescent and phase-contrast microscopy. (A and B) Representative examples of diapedesis completion (A) and diapedesis attempts withoutcompletion (B), as visualized by phase microscopy. (Upper) Fluorescence overlay on phase channel; (Lower) phase channel alone. WT T cells are in green, dKOT cells in red. Red arrows indicate diapedesis attempts (extension of protrusions), green arrows indicate diapedesis completion. (Scale bars, 5 μm.) Timestampsare minutes:seconds. (C and D) Quantification of the percentage of adhered WT and dKO T cells that attempted (C) or completed (D) diapedesis. (E) Shear-resistant adhesion of WT and dKO T cells to a monolayer of primary endothelial cells with or without CD49d blockade (overall ANOVA P = 0.0002).(F) Frequency of adhered T cells that completed diapedesis with or without CD49d-blockade (overall ANOVA P = 0.005). Images in A and B are representativeof six independent experiments; data are the average of six (C and D) or four (E and F) independent experiments. Error bars are SEM. P values are pairedt tests (C and D) or one-way ANOVA with post hoc Tukey test (E and F). ns, not significant.

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DiscussionThis report on the role of EVL and VASP in T-cell migrationand trafficking identifies a key role for the actin effector proteinsEVL and VASP in activated, but not naïve T-cell trafficking, anddetermines that the trafficking defect in EVL/VASP dKO T cellsoccurs at the diapedesis step of TEM. EVL/VASP deficiencyresulted in impaired activated T-cell trafficking to the CNS duringautoimmune neuroinflammation, to the inflamed skin and to sec-ondary lymphoid organs. Nonetheless, based on the varying endo-thelial barrier properties and adhesion molecules used forextravasation into different tissues, it is possible that the Ena/VASP family may have a more or less profound impact ontrafficking to different anatomical sites.The observation that deletion of EVL and VASP impaired

activated T-cell trafficking in vivo is consistent with the literatureimplicating Ena/VASP proteins in cellular motility (27, 47–51).Although we identified reduced actin polymerization in responseto chemokine triggering in activated EVL/VASP dKO T cells,surprisingly we found no major effect on activated T-cell che-motaxis or crawling in vitro. The finding that shear-resistantT-cell adhesion to primary brain endothelial cells was not af-fected by deletion of EVL and VASP was even more surprising,as Ena/VASP proteins are established negative regulators ofplatelet adhesion to the vascular wall (28).To determine the mechanism of the trafficking defect of EVL/

VASP-deficient T cells, we analyzed adhesion molecule functionand observed alterations in expression and activity of T-cellintegrins upon deletion of EVL and VASP. Surface and totalexpression of CD49d was decreased in EVL/VASP dKO T cells,whereas CD11a expression was slightly but significantly increased.Shear-resistant adhesion to purified ICAM-1 was enhanced upondeletion of EVL and VASP, as expected based on CD11a levels.However, although CD49d expression was reduced in EVL/VASPdKO T cells, there was no corresponding decrease in shear-resistantadhesion to VCAM-1. Because surface integrins need to be acti-vated to achieve a high-affinity conformation, differences in ex-pression do not necessarily correspond to alterations in function.However, when we quantified CD49d affinity for VCAM-1, wefound that EVL/VASP dKO T cells not only bound less solubleVCAM-1 than WT control cells, but were also less capable of in-creasing their VCAM-1 binding capacity in response to PMA/ion-omycin stimulation. This finding suggests that EVL and VASPpositively regulate both expression and activation of CD49d.These data suggest that differential regulation of CD49d and

CD11a upon deletion of EVL/VASP could explain the preser-vation of endothelial adhesion we observed, with increased CD11acompensating for decreased CD49d. Indeed, upon CD49d block-ade, dKO T cells trended toward increased endothelial adhesionrelative to WT controls, suggesting that they may have developedenhanced CD49d-independent adhesion mechanisms in response topoor CD49d function. However, because adhesion to and crawlingon endothelial cells is unaffected in EVL/VASP dKO T cells, simplealterations in the adhesive functions of these integrins do not pro-vide a sufficient explanation for the trafficking defects we observedin vivo. In contrast, our in vitro TEM data point to a critical non-redundant role for EVL and VASP in diapedesis, suggesting thatthe requirement for Ena/VASP family proteins in activated T-celltrafficking occurs specifically at the diapedesis step of TEM.Our finding that EVL and VASP are required for diapedesis

but not adhesion, motility, or chemotaxis is currently uniqueamong actin cytoskeletal regulators. For example, our previouswork (6) has indicated that the cytoskeletal effector myosin-IIAaffects both T-cell motility and diapedesis, and deletion ofmDia1, another actin effector protein, both impairs thymocytedevelopment and produces defects in chemotactic migration andin vivo homing of naïve T cells (11). Similarly, deletion of thecytoskeletal regulators Rap1, RIAM, talin, or RAPL impair

chemokine-stimulated ICAM-1 adhesion and naïve T-cell traf-ficking (52–54), whereas CRK proteins regulate T-cell adhesion,chemotaxis, and diapedesis, leading to reduced T-cell traffickingselectively to inflamed tissues (55). In contrast, recent reportsshow that Kindlin-3 is not required for diapedesis, although ithas been shown to play an important role in adhesion and CNStrafficking more generally (56, 57).We propose that the unique and selective role of EVL and

VASP in activated T-cell diapedesis is related to alterations inCD49d activity. Poor trafficking of activated EVL/VASP dKOT cells correlated with decreased CD49d expression and function.Conversely, naïve EVL/VASP dKO T cells trafficked normally andtheir CD49d expression, although very low, was not significantlydifferent from the low level expressed in control T cells. Further-more, activated EVL or VASP single-knockout T cells expressednormal CD49d levels, possibly explaining the normal traffickingpattern they exhibit. Taken together, these data identify impairedCD49d expression and function, which are known to regulate ac-tivated T-cell homing to peripheral lymph nodes and sites of in-flammation (58), as a mechanism mediating the impairment inEVL/VASP-deficient T-cell diapedesis and trafficking.In line with this notion, control and EVL/VASP dKO cells that

adhered to endothelial monolayers despite CD49d blockadewere equally likely to undergo diapedesis in vitro. This findingsuggests that EVL and VASP are specifically required for CD49d-dependent diapedesis, and opens up new questions about how sucha specific requirement might be regulated.The role of CD49d and VCAM-1 in T-cell extravasation is well

established. Various studies have reported that ligation of VCAM-1stimulates changes in endothelial permeability, triggering disso-ciation of vascular endothelial (VE) protein tyrosine-phosphatase(VE-PTP) from VE-cadherin, phosphorylation of VE-cadherin attyrosine 731, and ultimately, down-regulation of VE-cadherin fromendothelial cell junctions (59–61). T-cell protrusions that are gen-erated during the “intravascular crawling” stage of extravasationhave previously been proposed to trigger chemokine depot-initiateddiapedesis (35). It has also been suggested that filopodia generatedby T cells can promote the sensing of permissive sites for diapedesis(62). Ena/VASP family proteins are involved in generating mem-brane protrusions such as filopodia (20, 63, 64) and localize to themicrospikes generated upon ligation of the TCR (24). Consistentwith this finding, our data showed reduced protrusions initiatingdiapedesis by EVL/VASP-deficient T cells. Furthermore, activatedCD49d clusters at the leading edge of migrating T cells (65), andthe microvilli that form at the leukocyte-endothelial cell interfacein migrating leukocytes are rich in CD49d but not CD18 integrins(66). Further study may allow determination of whether EVL andVASP coordinate the presentation of CD49d on T-cell protru-sions, which are able to trigger the opening of endothelial junctionsvia signaling through VCAM-1.Similar to our results using EVL/VASP dKO T cells, condi-

tional knockout of CD49d in mice results in impaired T-celltrafficking to an inflamed CNS (67). Interference with CD49d-mediated T-cell trafficking via monoclonal antibody blockadehas made natalizumab a very effective treatment for multiplesclerosis (1, 2) and Crohn’s disease (68). This therapy targetsactivated or memory T cells without significantly impairing naïveT-cell surveillance. However, use of natalizumab is limited inpatients who are infected with the JC Virus, as they are at risk forprogressive multifocal leukoencephalopathy, a fatal complication(69, 70). Natalizumab-treated patients can also experience in-creased susceptibility to urinary and respiratory tract infections,which can trigger relapses (71).Our work supports the idea that CD49d has a specific, EVL/

VASP-dependent function during the diapedesis step of TEM,which is distinct from its roles in adhesion and intravascularcrawling. Although EVL/VASP-directed therapies would needto be targeted to lymphocytes to avoid effects on other cells, our

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work suggests that further studies of EVL and VASP in T cellscould identify therapeutically useful ways to more selectivelymodulate CD49d activity and activated T-cell trafficking.

Materials and MethodsEthics Statement. All experiments involving mice were conducted in compliancewith the NIH’s Guide for the Care and Use of Laboratory Animals (72) and withthe approval by the Institutional Animal Care and Use Committee of NationalJewish Health (Protocol #AS2811-02-17). All efforts were made to minimizemouse suffering.

Mice. KO mice lacking EVL and VASP were generated, respectively, byKwiatkowski et al. (31) and Aszódi et al. (32) (EVL/VASP dKO mice weregenerously provided by Frank Gertler, Massachusetts Institute of Technol-ogy, Cambridge, MA). These mice were on a 129 × C57BL/6 mixed back-ground and bred in-house at National Jewish Health. Single EVL or VASP KOand WT 129 × C57BL/6 mice were derived from the double-KO mice andwere maintained in parallel. Recipient mice used for homeostatic traffickingassays were 129S1 × C57BL/6 F1 hybrid mice (Stock #101043, The JacksonLaboratory). For EAE induction and short-term CNS trafficking, as well asskin trafficking experiments, CD45.1 congenically marked C57BL/6 recipientmice (Strain #564) were purchased from Charles River. CD45.1 C57BL/6 micewere also used to provide third-party WT splenocytes for T-cell activations.

T-Cell Isolation. Naïve CD4+ T cells were isolated and purified by negativeselection. Briefly, spleens, as well as mesenteric, brachial, axial, and inguinallymph nodes, were harvested and single-cell suspensions were made bypassing tissues through 100-μm sterile filters. T cells were purified usingStemCell EasySep magnetic isolation kits. Naive CD4 T-cell selection kits wereused for naïve cells; whereas total CD4 T-cell selection kits were used whenT cells were then subsequently activated in vitro.

T-Cell Activation and Culture. T cells were cultured using R10: RPMI 1640 withthe addition of L-glutamine, penicillin, streptomycin, and β-mercaptoethanol(all purchased from Invitrogen) and 10% (vol/vol) FCS (Fisher Scientific).Purified CD4+ T cells were activated in vitro with plate-bound anti-CD3 andsoluble anti-CD28 antibodies (BioXcell), in the presence of third-party WTfeeder splenocytes. Feeder cells that could be identified by a congenicmarker (CD45.1) were harvested from third-party mouse spleens as above,and red blood cells were lysed in 175 mM ammonium chloride. Splenocyteswere then irradiated at 1,500 rads, and mixed in a 2:1 ratio with purifiedT cells. Next, 2 μg/mL soluble anti-CD28 (clone PV-1) was added to the mix-ture, and cells were plated at 2 × 106/mL in a 24-well plate that had beencoated with 2 μg/mL anti-CD3 (clone 2C11) for 1–2 h at 37 °C. On day2 postactivation, cells were resuspended at 1 × 106/mL in fresh R10 + 10 U/mLrecombinant human interleukin 2 (rIL2) (obtained through the AIDS Re-search and Reference Reagent Program, Division of AIDS, National Instituteof Allergy and Infectious Diseases, NIH from Maurice Gately, Hoffmann-LaRoche Inc., Basel, Switzerland). On day 4 postactivation, cells were resus-pended at 2 × 106/mL in fresh R10 + 10 U/mL rIL2. Before use on day 5, deadcells were removed by Histopacque-1119 (Sigma-Aldrich) density gradient,and cells were resuspended at 2 × 106/mL in R10 + 10 units/mL rIL2.

Antibody Clones Used for Flow Cytometry. Antibody clones used for flowcytometry were: CD4 (GK1.5), CD5 (53-7.3), CD8a (53-6.7), CD11a (M17/4),CD19 (6D5), CD25 (PC61), CD29 (HMB1-1), CD44 (IM7), CD45.1 (A20), CD49d(R1.2), CD62L (Mel14), CD69 (H1.2F3), CD197 (4B12), CCR5 (7A4), CCR7 (4B12),CXCR3 (CXCR3-173), CXCR4 (L276F12). All purchased from Biolegend oreBioscience.

Antibodies Used for Western Blot. Antibodies used for Western blot were:VASP (9A2, CellSignaling); EVL (ab108406, Abcam); Mena (sc-135988, SantaCruz); mouse anti-Tubulin (Sigma-Aldrich); secondary antibodies (Licordonkey anti-mouse IR680 and IR800, donkey anti-rabbit IR800).

Dye-Labeling T Cells. Naïve or activated CD4 EVL/VASP dKO andWT T cells weredifferentially labeled with either 2 μM carboxy-fluorescein diacetate succini-midyl ester (CFSE; Invitrogen) or 1 μM Violet Proliferation Dye (VPD; eBio-sciences), mixed at a 1:1 ratio, and used for in vitro adhesion and migrationassays under flow, as well as in vivo trafficking experiments. Between experi-mental repeats, dyes were swapped to control for potential effects of the dyes.

In Vivo Adoptive Transfer and Intravascular Staining. For homeostatic traf-ficking in untreated recipient mice, 5 × 106 dKO and WT dye-labeled cells

were transferred intravenously into recipient mice which were then eutha-nized 2 or 24 h after adoptive transfer and peripheral lymph nodes, spleen,blood, and lungs were harvested. Intravascular staining of cells remaining inblood vessels was performed via intravenous injection of 3 μg PE- or APC-conjugated anti-CD4 antibody (GK1.5) 3 min before euthanasia (39). Aftereuthanizing with CO2, blood was then harvested by cardiocentesis, and theperipheral vasculature and lungs were fully perfused through the heart withsaline. Single-cell suspensions were generated from lymph nodes and spleenby passing organs through a 100-μm filter. Blood and splenic red blood cellswere lysed in 175 mM ammonium chloride. Lungs were minced, digested inDNase and Collagenase D for 30 min, passed through a 100-μM filter, andspun through a Histopacque-1119 density gradient to isolate leukocytes.One to three recipient mice were typically used per experiment, dependingon cell numbers available.

Induction of EAE and Scoring. EAE was induced using MOG induction kits fromHooke Laboratories according to their protocol. Briefly, WT female CD45.1C57BL/6 mice of at least 8 wk of age were immunized with 200 μg ofMOG35–55

peptide emulsified in complete Freund’s adjuvant injected subcutane-ously, followed by intraperitoneal injection of 200 ng pertussis toxin on theday of induction and the following day. Typical EAE onset was within 10–15 dpostimmunization. Mice were monitored and scored daily for developmentof EAE based on the following 0–5 scoring criteria: 0, no disease; 1 limp tail;2, weakness or partial paralysis of hind limbs; 3, full paralysis of hind limbs;4, complete hind limb paralysis and partial front limb paralysis; 5, completeparalysis of front and hind limbs or moribund state. Mice with a score ≥ 4 wereeuthanized immediately. Mice with scores of 2 or greater were used for T-celltrafficking experiments. These procedures were approved and carried out inaccordance to the regulations of the Institutional Animal Care and Use Com-mittees of National Jewish Health, and all efforts were made to minimizemouse suffering. Five mice were induced per group.

CNS Trafficking. For CNS trafficking, 5 × 106 WT and dKO activated T cellswere transferred IV into acutely ill EAE mice (score ≥ 2.0, see above), andrecipients were euthanized 24 h after adoptive transfer following in-travascular staining of cells as above. Intravascular staining and saline per-fusion after cardiocentesis was performed as above. Blood, brain, and spinalcord were isolated and single-cell suspensions were generated from brainand spinal cord by passing tissues through a 100-μm filter. A 70%/30% (vol/vol)percoll gradient was used to isolate leukocytes. Total leukocytes in all sampleswere manually enumerated on a hemocytometer, and adoptively transferredcells were quantified by flow cytometry (CyAN, Beckman Coulter). One tothree recipient mice were typically used per experiment, depending onavailability of sufficiently ill recipient mice.

Skin Trafficking. Inflammation was induced in the ears of CD45.1 recipientmice via subcutaneous injection of 20 μg LPS. Dye-labeled control or dKOactivated T cells were transferred intravenously 24 h after the induction ofinflammation. Then, 24 h after transfer of T cells, mice were injected in-travenously with 3 μg of CD4-APC and then euthanized 3 min later. Bloodwas collected via cardiocentesis and the mouse was gravity-perfused withsaline. Ears were removed, peeled apart, cut into small pieces, and placedinto 10 mL digestion media [RPMI with 10% (vol/vol) FBS, 0.25 mg/mL DNase,Roche, 0.786 Wunsch U/mL collagenase; Roche]. Ears were digested for45 min at 37 °C with occasional mixing. After digestion, the remaining eartissue was further mechanically dissociated and then filtered out on a100-μM nylon mesh strainer followed by rinsing with RPMI with 10% (vol/vol)FBS. Lymphocytes were then separated from debris using Histopaque-1119,washed and resuspended in FACS buffer for staining. Blood and nondraininglymph nodes and spleen were also harvested and processed as describedabove. All samples were stained with anti–CD45.1-BUV-395 before analysisby flow cytometry using a LSR Fortessa (Beckton Dickinson). TransferredT cells that fully extravasated were identified as dye-positive (CFSE or VPD),CD4-APC–negative, and CD45.1-BUV395–negative. Two recipient mice wereused per experiment.

Actin Polymerization Assay.Activated T cellswere stimulatedwith either 1 μg/mLor 0.1 μg/mL of chemokine (Peprotech) for 5, 15, or 60 s at 37 °C in 2% (wt/vol)BSA in RPMI. The reaction was stopped using 4% (wt/vol) paraformaldehyde(Electron Microscopy Sciences) in PBS and the cells were fixed for 10 min. T cellswere then permeabilized with saponin (Sigma-Aldrich) buffer [0.5% (wt/vol)saponin, 2% (vol/vol) FBS, and 0.05% sodium azide in PBS] for 30 min at roomtemperature. Cells were stained with a 1:50 dilution of Phalloidin-Alexa Fluor-647 (Life Technologies) in saponin buffer for 30 min and then washed twice.Analysis was done using a Beckman-Coulter Cyan flow cytometer.

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Transwell Migration. Wells of a 24-well plate were prepared containing RPMIwith 2% (wt/vol) BSA and 10 mM Hepes, with or without indicated che-mokines (CCL21, CXCL10, CXCL12, and CCL5, all from Peprotech). For che-motaxis assays, 1 × 106 control or dKO T cells were added to the topchambers of 5-μm Transwell plates (Corning) and allowed to migrate for 1 hat 37 °C in the presence or absence of chemokines in the lower well. Forchemokinesis, chemokine was present in both upper and lower wells. As astandard, 2 × 105 cells (20% of input cells added to Transwells) were placeddirectly into bottom wells with no Transwell to calculate the percentage ofmigrated cells. Each condition was set up in duplicate Transwells. MigratedT cells were collected from the bottom wells and 25 μL of cell counting beads(123count eBeads, eBioscience) were added. Each sample was quantified fora fixed period (30 s) using a flow cytometer (CyAn ADP Beckman Coulter).The number of cells counted during this time was normalized to the numberof beads counted to adjust for any variations in flow rate during the run.

Soluble VCAM-1 Binding Assay. T cells were resuspended in RPMI withoutphenol red, supplemented with 5% (wt/vol) BSA (Sigma-Aldrich). Experi-mental wells were set up in duplicate, each with 0.5 × 106 cells in 25 μL RPMI +BSA medium in a round-bottom 96-well plate. Twenty-five microliters ofR10 medium, MnCl2 treatment medium (R10 with 4 nM MnCl2), or PMA/ionomycin treatment medium (R10 with 50 nm/mL PMA and 1 μg/mL ion-omycin) were added to each experimental well. The plate was gently vor-texed and then incubated for 5 min at 37 °C. Next, 50 μL VCAM-Fc (1 mg/mL;R&D Systems) was added to each well and incubated for 10 min at 37 °C.Samples were then transferred to ice and 100 μL ice-cold RPMI + BSA me-dium was added to each well. Samples were washed once in 200 μL ice-coldRPMI + BSA medium, and were then resuspended in 100 μL APC-conjugatedanti–human-Fc antibody (clone HP6017) in RPMI + BSA (1 μL per test).Samples were incubated on ice for 1 h and then washed three times. Cellswere then filtered through Nytex filters, and samples were analyzed by flowcytometry (CyAN, Beckman Coulter).

Microscopy. We used a 3i (Intelligent Imaging Innovations) Marianasspinning-disk confocal microscope system equipped with a Zeiss invertedstand and a Yokogawa spinning disk unit. We imaged through a 20× phase-contrast objective. CFSE- and VPD-labeled lymphocytes were excited withthe 488 and 445 laser lines, respectively. Endothelial cells stained with A647-conjugated CD31 antibody were excited with the 640 laser line. Appropriateemission wavelengths were acquired for each channel. Time-lapse imageswere acquired every 20 s for 30 min, at three or four separate stage positionsper run. Three XY planes (with 3-μm z-spacing) were acquired to accom-modate potential z-drift because of alterations in shear-flow speed. Acqui-sition was managed using Slidebook software (3i, v6.0).

Adhesion and Crawling on ICAM-1 and VCAM-1. A 1:1 mixture of differentlydyed T cells was resuspended in RPMIwithout phenol red, supplementedwith2% (wt/vol) BSA (R&D) 10 mM Hepes at 4 × 106/mL. T cells were perfused intoa flow chamber (μ-slide VI, IBIDI) that had been coated with ICAM-1 orVCAM-1 (1 mg/mL) in PBS for 1 h at 37 °C. Cells were initially perfused intothe chamber at 0.2 dyne/cm2 shear-flow for 5 min, and then the shear-flowwas raised to 2 dyne/cm2. Spinning-disk confocal fluorescence images wereacquired every 20 s for 30 min.

Endothelial Adhesion, Crawling, and Diapedesis Under Flow. A 1:1 mixture ofdifferentially dyed WT and dKO T cells was resuspended in RPMI withoutphenol red, supplementedwith 2% (wt/vol) BSA, and 10mMHepes at 2× 106/mL.T cells were perfused into a flow chamber (μ-slide VI, IBIDI) coated with a

monolayer of mouse primary brain microvascular endothelial cells (Cell Bi-ologics) that had been activated with TNF-α 24 h before imaging, andtreated with CCL21 30 min before imaging. The endothelial cells were alsolabeled with APC-conjugated anti-CD31 antibody (clone 390) to visualize theendothelial cell membrane and monolayer integrity; this staining does notperturb the T-cell TEM process (73, 74). T cells were initially perfused into thechamber at 0.2 dyne/cm2 shear-flow for 5 min, and then the shear-flow wasraised to 2 dyne/cm2. Phase contrast and fluorescence images were acquiredevery 20 s for 30 min using a spinning-disk confocal microscope.

In CD49d (α4 integrin) blockade experiments, a 1:1 mixture of control andEVL/VASP dKO T cells at 10 × 106 cells/mL was pretreated with a mixture oftwo CD49d-blocking antibodies (clones 9C10 and R1-2) at 2 μg/mL each, for30 min on ice before imaging. IgG2b antibody was used as an isotype con-trol. Cells were then perfused into a flow chamber at 10 × 106 cells/mLas above.

Quantification of Adhesion, Crawling, and Diapedesis Under Flow. Manualscoring and automated quantification were completed on blinded images,using Imaris software (Bitplane). Adhesion (cells per field of view 1 min afterthe increase in flow) was quantified using the Imaris “spot” algorithm. Insome cases the number of cells per field-of-view was normalized betweenexperiments to account for a different number of T-cell input. Meancrawling speed was quantified by following fluorescent cells using the Imaris“track” algorithm. In endothelial TEM experiments, cells that never crawled,cell detachment, and diapedesis attempts and completions were also scoredmanually. Briefly, T cells that contained partial regions that lost and thenregained the white phase contrast ring in a stepwise manner were scored ashaving attempted diapedesis, whereas T cells that underwent a stepwisedarkening and completely lost the white phase contrast ring were scored ashaving completed diapedesis. Diapedesis data were filtered to exclude allcells that were not present in the field of view for at least 13 min (the meantime required to complete diapedesis).

Statistical Analysis. Prism software (GraphPad) was used to graph the dataand calculate statistical significance. The statistical significance of singlecomparison data were determined by performing paired or unpaired Stu-dent’s t tests, or one-sample t tests versus a hypothetical ratio of 1.0, asappropriate. One-way ANOVA with Tukey posttests was used for multiplecomparisons. Two-way ANOVA was used for comparison of data with twoindependent variables.

ACKNOWLEDGMENTS. We thank F. Gertler and R. Fassler for the gift of thevasodilator-stimulated phosphoprotein (VASP) and Ena-VASP–like knockoutmice; M. Gebert, R. Long, and D. Tracy for help with mouse genotyping andcolony maintenance; R. Lindsay for technical help with some experiments;J. Loomis and S. Sobus for expert technical assistance with cell sorting;J. Loomis for microscope maintenance; and P. Henson, R. Kedl, and R. Torresfor reagents and comments on the manuscript. This work was funded in partby National Institute of Allergy and Infectious Diseases/NIH Awards R01AI125553and R56AI105111 (to J.J.); awards from the Dana Foundation (Brain-Immuno Imaging grant), National Multiple Sclerosis Society (Pilot grant),and American Society of Hematology (Bridge grant) (all to J.J.); NIH train-ing Grant T32AI007405 (to M.L.E.); and NIH/National Center for AdvancingTranslational Sciences Colorado CTSI TL1 Grant 8TL1TR00155 (to M.L.E.);NIH Training Grant T32AI007405 (to S.B.T.). The spinning-disk confocal mi-croscope used was acquired thanks to Shared Instrumentation Grant AwardS10RR029218. The content of this work is solely the responsibility of theauthors and does not necessarily represent the official views of the NIH orother funding agencies.

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