Ena/VASP Proteins Regulate Activated T-Cell Trafficking by Promoting

Ena/VASP proteins regulate activated T-cell PNAS PLUS trafficking by promoting diapedesis during transendothelial migration

Miriam 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 of Colorado 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 review February 3, 2017) Vasodilator-stimulated phosphoprotein (VASP) and Ena-VASP–like skeletal changes occur throughout the process of TEM (4, 5); (EVL) are cytoskeletal effector proteins implicated in regulating however, the regulation of these cytoskeletal changes is not completely cell morphology, adhesion, and migration in various cell types. understood. However, the role of these proteins in T-cell motility, adhesion, The lymphocyte actin-myosin cytoskeleton is composed of and in vivo trafficking remains poorly understood. This study iden- networks of linear and branched actin filaments, cross-linked by tifies a specific role for EVL and VASP in T-cell diapedesis and class-II nonmuscle myosin. We have previously shown that in- trafficking. We demonstrate that EVL and VASP are selectively re- hibition of myosin-IIA, the main class-II myosin protein expressed quired for activated T-cell trafficking but are not required for normal in lymphocytes, alters T-cell trafficking, motility, and TEM (6–9). T-cell development or for naïve T-cell trafficking to lymph nodes and Although numerous studies have focused on the upstream regula- spleen. Using a model of multiple sclerosis, we show an impairment in tory signaling cascades that control actin network remodeling dur- trafficking of EVL/VASP-deficient activated T cells to the inflamed ing migration (5, 10), less is understood about how downstream central nervous system of mice with experimental autoimmune effectors of branched and linear actin polymerization might par- INFLAMMATION

encephalomyelitis. Additionally, we found a defect in trafficking ticipate in lymphocyte TEM. Two major families of effector pro- IMMUNOLOGY AND of EVL/VASP double-knockout (dKO) T cells to the inflamed skin teins of linear actin polymerization are expressed in lymphocytes. and secondary lymphoid organs. Deletion of EVL and VASP resulted in The Formin family, which is thought to nucleate new linear actin α the impairment in 4 integrin (CD49d) expression and function. filament production, has been implicated in T-cell activation and Unexpectedly, EVL/VASP dKO T cells did not exhibit alterations egress from the thymus (11–13). Less is known about the Ena/ in shear-resistant adhesion to, or in crawling on, primary endothe- VASP (vasodilator-stimulated phosphoprotein) family of cyto- lial cells under physiologic shear forces. Instead, deletion of EVL skeletal effectors in T cells. and VASP impaired T-cell diapedesis. Furthermore, T-cell diapede- The Ena/VASP family is composed of three members: sis became equivalent between control and EVL/VASP dKO T cells α mammalian-enabled (Mena), which is not typically expressed in upon 4 integrin blockade. Overall, EVL and VASP selectively me- hematopoietic cells; VASP; and Ena-VASP–like (EVL) (14, 15). diate activated T-cell trafficking by promoting the diapedesis step These proteins coordinate monomeric actin recruitment to the of transendothelial migration in a α4 integrin-dependent manner. barbed end of the actin filament, prevent actin filament capping, and can play a role in actin filament bundling (16–20). Structurally, Tcell| migration | cytoskeleton | extravasation EVL and VASP share significant homology, containing an N-terminal EVH1 domain, which regulates cellular localization; ctivated T-cell trafficking across the vascular endothelium is Aessential for ongoing immune surveillance of tissues and for Significance effective immune responses to conditions such as infection and cancer. Conversely, in situations of immune dysregulation, inhibition of self-reactive T-cell trafficking represents a promising T-cell trafficking is essential for the function of the adaptive target for therapeutic immunomodulation. Disruption of these immune system, and regulation of T-cell entry into tissues can be pathways, such as by antibody blockade of α4 integrins, is a highly an effective therapy in diseases such as autoimmunity. However, effective approach to immunomodulation (1, 2). However, the the mechanisms regulating T-cell migration and trafficking are molecular mechanisms by which chemokine receptor and adhesion poorly understood. We have identified a key role for Ena/VASP molecule signaling induce the T-cell cytoskeletal machinery to (vasodilator-stimulated phosphoprotein) family cytoskeletal ef- promote extravasation are not yet fully elucidated. fectors selectively in activated T-cell trafficking to secondary Transendothelial migration (TEM), the process by which lymphoid organs and to peripheral sites of inflammation. Ena/ α T cells extravasate from the blood into tissues, is characterized by VASP deficiency in T cells causes a defect in 4 integrin function, four distinct steps: rolling along the vascular wall, arrest or adhe- which impairs transendothelial migration. Our work suggests sion, intravascular crawling, and diapedesis across the endothelial that further studies of the Ena/VASP pathway in T cells could identify therapeutically useful ways to more selectively modu- barrier (3). Surface adhesion molecules play well-characterized late α4 integrin activity and activated T-cell trafficking. roles in each step of the process. For example, the initial rolling

step of TEM is facilitated by interactions between T-cell and Author contributions: R.S.F. and J.J. designed research; M.L.E., S.B.T., B.T., M.H.F., and J.J. endothelial selectins, whereas the adhesion, intravascular crawling, performed research; M.L.E., S.B.T., R.S.F., and J.J. analyzed data; and M.L.E. and J.J. wrote and diapedesis steps of TEM are mainly regulated by chemokine- the paper. and shear force-stimulated modulation of lymphocyte function- The authors declare no conflict of interest. associated antigen 1 (LFA-1, αLβ2 integrin, CD11a/CD18) and very This article is a PNAS Direct Submission. late antigen 4 (VLA-4, α4β1 integrin, CD49d/CD29) interactions 1To whom correspondence should be addressed. Email: [email protected]. with intracellular adhesion molecule 1 (ICAM-1) and vascular cel- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. lular adhesion molecule 1 (VCAM-1), respectively. Dynamic cyto- 1073/pnas.1701886114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1701886114 PNAS Early Edition | 1of10 Downloaded by guest on September 26, 2021 and a C-terminal EVH2 domain, which facilitates tetramerization, WT and KO ABCD4+ T cells binds filamentous actin (F-actin), and is thought to be responsible Naive for actin polymerization (21–24). Ena/VASP proteins are capable 2.0 2 hours of compensating for deletion of one another, but there is some in vivo p=0.03 ns ns evidence that they are differentially regulated (15). 1.5 Inject anti-CD4 PE Ab IV( ) EVL, and especially VASP, are implicated in the motility, ad- Euthanize hesion, and sensory capacity of many cell types. EVL and VASP 1.0 Perfuse with saline localize to filopodia tips (25) as well as to adhesive sites, such as Harvest tissues and fibroblast focal adhesions (24). This localization pattern is consis- 0.5 analyze by flow cytometry tent with a role in migration and adhesion. Fibroblasts lacking EVL

Naive dKO:WT Ratio Intravascular Cells and VASP produce shorter filopodia, and a slower-moving lamel- Normalized to Injected 0.0 (CD4 PE+) lipodium, which paradoxically leads to enhanced fibroblast motility Blood Spleen LN Extravasated Cells (26, 27). Platelets from VASP-knockout mice demonstrate en- (CD4 PE-) hanced vascular adhesion (28), whereas inside-out signaling through β2 integrins is impaired in VASP-deficient neutrophils (29). Only a few studies have examined the role of Ena/VASP proteins CDActivated WT dKO ) in T cells, showing that Ena/VASP proteins can contribute to actin 2.0 4 50 ns p=0.004 p=0.005 remodeling during T-cell receptor (TCR) signaling (24, 30). How- 40 ns p=0.01 ever, previous studies did not focus on the role of EVL and VASP 1.5 30 in T-cell development or T-cell motility. Therefore, we sought to 20 elucidate the mechanisms by which EVL and VASP might influence 1.0 10 T-cell adhesion, migration, and trafficking in vivo. p=0.04 0.5 2 Results 1

Deletion of EVL and VASP Does Not Significantly Affect T-Cell Normalized to Injected

Activated dKO:WT Ratio 0.0 0 Development. To determine if deletion of Ena/VASP proteins Blood Spleen LN # (x10 cells Transferred Blood Spleen LN altered 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). EF 1.5 1.5 WeconfirmedEVLandVASPdeletioninTcells,andverifiedthat ns ns ns ns ns ns Mena was not up-regulated as a compensatory mechanism in EVL/ VASP dKO T cells by Western blot analysis (Fig. S1). We then 1.0 analyzed if EVL/VASP deficiency caused defects in T-cell devel- 1.0 opment. Flow cytometry analysis of lymphocyte populations in the thymus and secondary lymphoid organs showed no gross defects in 0.5 0.5 T-cell development and normal proportions of mature CD4 and

CD8 T cells in the periphery of EVL/VASP dKO mice (Fig. S2). EVL sKO:WT Ratio VASP sKO:WT Ratio Normalized to Injected Normalized to Injected 0.0 0.0 Deletion of Both EVL and VASP Selectively Impairs Activated CD4 Blood Spleen LN Blood Spleen LN T-Cell Trafficking into Secondary Lymphoid Tissues. Next, we used coadoptive transfers of WT control and EVL/VASP dKO T cells Fig. 1. Deletion of both EVL and VASP selectively inhibits activated but not – naïve CD4 T-cell trafficking to secondary lymphoid organs. (A) Naïve T-cell to study the T-cell intrinsic effect of EVL and VASP deficiency trafficking is not affected by EVL and VASP deficiency. Differentially dye- on trafficking in vivo. Consistent with the normal pattern of de- labeled naïve WT and EVL/VASP dKO T cells were coadoptively transferred velopment and homeostatic trafficking, dKO and WT naïve CD4 intravenously at a 1:1 ratio and T-cell trafficking to lymphoid tissues was T cells had equivalent homing to the spleen and lymph nodes after quantified by flow cytometry 2 h after adoptive transfer. The dKO:WT ratio intravenous adoptive transfer in to WT recipient mice (Fig. 1A). was normalized to the ratio in the injected sample to account for possible Activated T cells have different requirements for migration minor variations in the injection mixture (typically < 10%). (B) Experimental and trafficking than their naïve counterparts (33–36). Therefore, set-up for cotransfer of activated WT and KO cells, including intravascular we investigated if Ena/VASP family proteins are specifically re- staining method to distinguish intravascular T cells from those that have – quired for activated T-cell trafficking. We first established that, extravasated into the tissue of interest. (C F) Dye-labeled CD4 WT and single-KO or dKO activated T cells were coadoptively transferred at a 1:1 ratio upon ex vivo polyclonal activation with CD3 and CD28 stimu- and T-cell trafficking to tissues 2 h after adoptive transfer was quantified by lation, T-cell proliferation kinetics and activation profiles were flow cytometry as above. A ratio below 1.0 (horizontal red line) indicates im- similar for WT and EVL/VASP dKO T cells (Fig. S3). Activated paired homing of KO T cells. (C) Quantification of activated T-cell trafficking. T cells can become lodged in the vasculature (particularly in the Ratio of extravasated WT and EVL/VASP dKO activated T cells, harvested and lungs) (37, 38), potentially because of their increased size and analyzed as above. (D) Number of WT and dKO T cells recovered from the adhesion properties. Therefore, we used an established tech- indicated tissues from data in C.(E and F) Trafficking of EVL (E)andVASP nique (39, 40) to distinguish extravasated T cells from those (F) single-KO activated T cells relative to WT controls. Data are the average stuck intravascularly by injecting intravenously a fluorophore- from a minimum of three independent experiments. Error bars are SEM. Sta- conjugated anti-CD4 antibody immediately before euthanasia 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. of the recipient mice (Fig. 1B). Only extravasated cells (T cells negative for intravascular anti-CD4 staining) were considered to have infiltrated a tissue. In this setting, in vitro-activated CD4 T cells maintain expression exhibited a 2.2-fold reduction in spleen trafficking and a 3.3-fold re- of CCR7 and can recirculate to secondary lymphoid organs. To duction in lymph node trafficking 2 h after adoptive transfer com- determine if homeostatic trafficking of activated T cells was affected paredwithWTcontrols(Fig.1C and D). This activated dKO T-cell by Ena/VASP deficiency, we cotransferred control and EVL/VASP trafficking defect still persisted 24 h posttransfer (Fig. S4 A and B). dKO T cells into unimmunized recipient mice. Following in- Furthermore, the intravascular staining used to quantify T cells travenous adoptive transfer, activated dKO CD4 T cells on average that had entered tissues versus cells that remained adhered within

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1701886114 Estin et al. Downloaded by guest on September 26, 2021 blood vessels also indicated that the defect in activated EVL/VASP to the inflamed skin, using lipopolysaccharide (LPS) as the in- PNAS PLUS dKO T-cell trafficking was not a result of selective trapping of these flammatory stimulus. Recipient mice were treated by sub- cells in the lung microvasculature, the first capillary bed encoun- cutaneous LPS injections in the ear and 24 h later activated tered after intravenous adoptive transfer. In fact, significantly more control and dKO T cells were intravenously transferred into the WT than dKO T cells were recovered from inside the lung micro- recipient mice. Quantification of transferred T cells that had vasculature (Fig. S4C). In keeping with these data, there is a trend extravasated into the inflamed skin of the ear showed a 1.8-fold toward more EVL/VASP dKO T cells than control cells in the reduction in dKO T-cell trafficking to this site (Fig. 2 D–F). blood compartment, suggesting that these cells may be unable to gain full access to tissues from the blood (Fig. 1 C and D). Deletion of EVL and VASP Reduces Chemokine-Triggered Actin We next analyzed if deletion of EVL or VASP alone was Polymerization but Does Not Impair Activated T-Cell Chemotaxis in sufficient to impair activated T-cell trafficking to secondary Vitro. Chemokine signaling is instrumental in mediating leuko- lymphoid organs. Consistent with the likelihood of redundant or cyte migration and trafficking (41, 42). Therefore, we analyzed if compensatory functions between Ena/VASP proteins, single- the trafficking defect of Ena/VASP-deficient activated T cells knockout of EVL or VASP did not give rise to activated T-cell could be because of an altered ability to respond to chemokine trafficking defects (Fig. 1 E and F). stimulation. First, we measured the expression of chemokine receptors involved in both homeostatic and inflammatory traf- EVL and VASP Deletion Impairs Activated CD4 T-Cell Trafficking to ficking. Our data showed no difference in the expression of Sites of Inflammation. We next sought to determine if activated CCR7, CXCR3, CXCR4, and CCR5 between control and EVL/ CD4 T-cell trafficking to sites of inflammation was affected by VASP dKO CD4 T cells (Fig. 3A). Ena/VASP protein deficiency. Because the vascular endothelial Ena/VASP proteins are cytoskeletal effectors that promote barrier in the central nervous system (CNS) is particularly re- actin filament polymerization (14). During migration, chemokine strictive, we first investigated EVL/VASP-deficient activated stimulation can trigger actin network remodeling and promote T-cell trafficking to the CNS in the context of autoimmune in- motility (34). Therefore, we analyzed if Ena/VASP deficiency flammation, using a mouse model of multiple sclerosis: experi- impaired actin polymerization in response to chemokine stimu- mental autoimmune encephalomyelitis (EAE). Twenty-four lation. To this end, we measured the F-actin content in control hours after adoptive transfer of T cells into mice with ongoing and EVL/VASP dKO T cells before and after chemokine stim- EAE, we quantified the number of transferred T cells that had ulation in a time-course analysis. Our results showed that dKO-

extravasated into the CNS using the intravascular staining tech- activated T cells had a small but significant defect in actin po- INFLAMMATION nique described above. Activated dKO T cells exhibited on av- lymerization promoted by CCL21 stimulation and, to a lesser IMMUNOLOGY AND erage a 2.0-fold reduction in trafficking to the CNS relative to degree, by CXCL10 (Fig. 3B). However, when we analyzed actin control T cells (Fig. 2 A–C). Next, we analyzed T-cell trafficking polymerization in response to CCL21 in naïve T cells, we did not see a defect in EVL/VASP dKO cells (Fig. S5A). This finding suggests a different reliance on Ena/VASP proteins for actin polymerization between naïve and activated T cells. A Inject MOG35-55 SC B C ) ns Based on this result, we then measured chemokine-stimulated Day 0 1.5 4 60 & PTX IP 50 WT ns p=0.01 dKO Day 2 PTX IP 40 migration using Transwell chambers. There were no significant 30 Day 7 Start scoring mice 1.0 differences in migration in the absence of chemokine, or in 20 Mice begin to chemotaxis toward CCL21, CXCL10, CXCL12, or CCL5 in the Day 10-14 10 develop symptoms p=0.02 0.5 lower chamber between control and EVL/VASP dKO-activated Transfer activated 5 C dKO:WT Ratio T cells (Fig. 3 ). Furthermore, chemokinetic migration in response

EAE Scoring T cells into EAE mice, Day 14-20

24h later euthanize Normalized to Injected 0.0 0 to CCL21 in both the upper and lower chambers was also un- and harvest tissues Blood CNS # (x10 cells Transferred Blood CNS affected (Fig. S5B). Although we detected slightly reduced chemokine-mediated actin polymerization in EVL/VASP- ns

DFE ) deficient activated T cells, overall these data showing normal

1.5 2 1500 WT ns p=0.02 dKO chemotaxis suggest that the strong trafficking defect of EVL/ 0h Inject LPS SC in ear 1000 1.0 500 VASP dKO T cells in vivo is not explained by an overall defect in 24h Transfer T cells migratory capacity or chemokine sensing. 10 Euthanize and p=0.01 48h 8 harvest tissues 0.5 6 Activated CD4 EVL/VASP dKO T Cells Are Deficient in α4 Integrin

dKO:WT Ratio 4 2 Expression and Function. Ena/VASP proteins are reported to af- Normalized to Injected 0.0 0 Blood Skin # (x10 cells Transferred Blood Skin fect adhesion and integrin function (28, 43). Therefore, having ruled-out impaired chemotaxis, we next examined the expression Fig. 2. EVL and VASP deletion inhibits activated CD4 T-cell trafficking to the and function of key integrins involved in extravasation to de- CNS during EAE and to the inflamed skin. (A) Experimental set-up for cotransfer termine if the trafficking impairment we observed was because of of WT and EVL/VASP dKO activated T cells into mice with ongoing EAE. Acti- integrin defects. Flow cytometry analysis revealed that activated vated, dye-labeled polyclonal CD4 WT and dKO T cells were coadoptively EVL/VASP dKO T cells expressed on average 28% less CD49d, transferred at a 1:1 ratio, and were harvested 24 h posttransfer from the blood A B and CNS (brain and spinal cord). (B) Activated T-cell trafficking during EAE was but 28% more CD11a (Fig. 4 and ). quantified by flow cytometry, shown as the ratio of dKO:WT T cells normalized CD49d, the α4 subunit of the integrins α4β1 (VLA-4) and to the ratio in the injected sample. (C) Number of WT and dKO T cells recovered α4β7 (LPAM-1), is primarily expressed on antigen-experienced from the indicated tissues from data in B.(D) Experimental set-up to quantify T cells, which may explain why only activated dKO T cells activated T-cell trafficking to the inflamed skin. Twenty-four hours after LPS- exhibited a trafficking defect in vivo. Consistent with their normal induced inflammation in the ears, WT and dKO activated T cells were coad- trafficking phenotype, activated EVL or VASP single-knockout optively transferred at a 1:1 ratio, and were harvested 24 h posttransfer from the T cells did not exhibit reduced CD49d expression, nor did naïve blood and ears of the recipient mice. (E) Ratio of dKO:WT T cells recovered from dKO T cells (which only expressed negligible levels of CD49d) (Fig. blood and ears, normalized to the ratio in the injected sample. (F) Number of WT S6 A–E). Additionally, expression of the α4 integrin binding part- and dKO T cells recovered from the indicated tissues from data in E. Data are the β β average of four independent experiments. Error bars are SEM. Statistics are one- ners 1(CD29)and 7 in activated cells was also reduced in acti- sample t test compared with a hypothetical value of 1.0 (B, E)orpairedt test (C, vated dKO T cells compared with WT T cells (Fig. S6F). F). IP, intraperitoneal; ns, not significant; SC, subcutaneous. Furthermore, total expression versus surface expression of CD11a

Estin et al. PNAS Early Edition | 3of10 Downloaded by guest on September 26, 2021 A CCR7 CXCR3 CXCR4 CCR5 of EVL and VASP decreases CD49d expression on the T-cell 100 600 250 150 ns ns ns ns surface and impairs inside-out signaling activation of CD49d. 80 200 400 100 Activated EVL/VASP dKO CD4 T Cells Are Competent in Shear- 60 150 Resistant Adhesion and Crawling. To determine how the pertur- 40 100 200 50 bation in CD49d activity we observed could influence the im- CCR7 gMFI CCR7 CCR5 gMFI 20 CXCR3 gMFI CXCR4 gMFI 50 paired trafficking phenotype of EVL/VASP-deficient T cells, we 0 0 0 0 then investigated if decreased expression and function of CD49d WT dKO WT dKO WT dKO WT dKO in EVL/VASP-deficient T cells caused impaired adhesion or motility on integrin ligands. Using flow chambers that recapitu- B CCL21 CXCL10 1.8 2.0 late the physiologic shear forces of the microvasculature (Fig. WT WT } p<0.0001 } p=0.032 5A), we first quantified shear-resistant adhesion to integrin li- dKO 1.8 dKO 1.6 gands in the presence of chemokine. Compared with controls, 1.6 1.4 EVL/VASP dKO T-cell adhesion to ICAM-1 was actually 1.4 moderately improved (Fig. 5B), consistent with increased CD11a 1.2 Phalloidin MFI Phalloidin MFI 1.2 expression, and no significant differences were observed in (fold over no stim.) (fold over no stim.) C 1.0 1.0 shear-resistant adhesion to VCAM-1 (Fig. 5 ). We then ana- 0 15 30 45 60 0 15 30 45 60 – – Time (sec.) Time (sec.) lyzed dKO T-cell 2D migration on ICAM-1 or VCAM-1 coated surfaces under flow. Time-lapse spinning-disk confocal C No Chem. CCL21 CXCL10 CXCL12 CCL5 microscopy was used to image T cells, and the mean crawling 100 100 100 ns 100 100 speed of dKO T cells compared with WT was found to be slightly D 80 80 80 80 80 slower on ICAM-1 (13% less) (Fig. 5 ) and equivalent on ns – E 60 60 60 60 60 VCAM-1 coated surfaces (Fig. 5 ), suggesting that adhesion ns and motility on integrins is not severely impaired in dKO T cells. 40 40 40 40 40 ns Next, adhesion to primary endothelial cells was quantified in 20 20 ns 20 20 20 vitro to determine if T-cell adhesion to the vascular wall was

Transwell Migration (%) 0 0 0 0 0 likely to be inhibited in vivo. Primary brain microvascular en- WT dKO WT dKO WT dKO WT dKO WT dKO dothelial cells were grown to confluence in flow chambers, ac- α Fig. 3. Deletion of both EVL and VASP in activated CD4 T cells reduces tivated with TNF- 24 h before imaging (to up-regulate adhesion chemokine-triggered actin polymerization but does not impair chemotaxis. molecule expression), and pretreated with CCL21 30 min before (A) Quantification of chemokine receptor expression in WT and EVL/VASP imaging (to stimulate T-cell adhesion). Fluorescently dyed T cells dKO activated T cells; data shown as gMFI. (B) Time-course analysis of actin were flowed through the chamber and imaged with fluorescent and polymerization in WT and dKO activated T cells in response to 100 ng/mL phase-contrast spinning-disk microscopy to quantify the number of CCL21 stimulation (WT vs. dKO curve comparison P < 0.0001) or 100 ng/mL adhered T cells. Surprisingly, deletion of EVL and VASP did not = CXCL10 stimulation (WT vs. dKO curve comparison P 0.032), measured by alter shear-resistant T-cell adhesion to primary brain endothelial flow cytometry quantification of fluorescent phalloidin staining. (C) Che- motactic migration across 5-μm pore Transwell chambers in the absence of chemokine, 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. Data WT dKO 400 125 are the average of at least three independent experiments; error bars are ABunstained p=0.002 p=0.004 I

SEM; statistics are paired t tests in A and C, or two-way ANOVA in B. ns, not F 300 100 significant. 75 200 50 100 25 CD49d gMFI and CD49d were similarly affected in activated dKO T cells, in- CD11a gM %of Mode 0 0 dicating that the defect is not a result of altered integrin trafficking CD11a CD49d WT dKO WT dKO from intracellular stores to the cell surface (Fig. S6 H and I). Integrin activity can be regulated by conformation, which in- CDWT dKO fluences integrin affinity for ligands (34, 44). The main CD49d 30 p=0.02 30 ligands are fibronectin and VCAM-1, with the latter expressed p=0.02 on vascular endothelial cells. Therefore, to determine if CD49d p=0.02 20 20 function was compromised in EVL/VASP-deficient T cells, we measured WT and dKO T-cell binding to soluble VCAM-1. In 10 10

the absence of stimulation, there was very low basal VCAM-1 Affinity Index ns (VCAM-Fc gMFI:

binding capacity and no difference between WT and dKO acti- CD49d gMFI ratio) vated T cells. However, in response to phorbol myristate acetate VCAM-1 binding (%) 0 0 No Stim PMA/I MnCl dKO (PMA)/ionomycin stimulation or treatment with MnCl2 (which 2 WT exogenously forces integrins into a high-affinity conformation), a α lower percent of dKO cells bound VCAM-1 (Fig. 4C). Because Fig. 4. Activated EVL/VASP dKO CD4 T cells have a deficit in 4 integrin (CD49d) expression and function. (A) Examples of CD11a and CD49d ex- dKO T cells expressed less CD49d, we normalized for this dif- pression on WT and EVL/VASP dKO activated T cells by flow cytometry. ference in expression to evaluate CD49d affinity for VCAM. We (B) Quantification of CD11a and CD49d surface expression in activated WT calculated a VCAM-1/CD49d affinity index for WT and dKO and dKO T cells; data shown as gMFI. (C) CD49d function measured as sol- T cells upon stimulation with PMA/ionomycin by normalizing uble VCAM-1 binding to T cells in response to the indicated stimuli (MnCl2: the geometric mean fluorescence intensity (gMFI) of bound manganese chloride; PMA/I: PMA and ionomycin). (D) Affinity for VCAM-1 VCAM-1 to the surface expression of CD49d (also by gMFI) calculated as PMA/ionomycin-elicited VCAM-1 binding normalized to sur- D face expression of CD49d by gMFI. Data in A are representative of 10 in- (Fig. 4 ). This analysis showed significantly decreased CD49d dependent experiments; data in B are the average of ten experiments; data affinity for VCAM-1 on a per receptor basis upon deletion of in C and D are the average of three independent experiments. Error bars are EVL and VASP. Taken together, these data show that deletion SEM. All P values are paired t tests. ns, not significant.

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1701886114 Estin et al. Downloaded by guest on September 26, 2021 F PNAS PLUS Flow cells (Fig. 5 ), and detachment from the endothelial monolayer A under flow conditions was also unchanged (Fig. 5G). Furthermore, T cells no statistically significant change was noted in the percentage of EVL/VASP dKO T cells that adhered to the endothelial monolayer but never crawled (Fig. 5H). Similarly, T-cell crawling speed on the endothelial monolayer under flow conditions, an in vitro model for Phase contrast intravascular crawling in vivo, was unaffected by EVL and VASP objective ICAM-1, VCAM-1, deletion (Fig. 5I). Taken together, these data indicate that despite or endothelial cells differences in integrin expression and function, EVL/VASP dKO T cells are capable of normal adhesion and migration on endo- B C thelial cells in vitro, suggesting that a different mechanism caused 50 50 p=0.02 ns the trafficking impairment of EVL/VASP-deficient T cells. 40 40 Furthermore, using the intravascular staining method de- 30 30 scribed above to quantify T cells that remained intravascular in our in vivo trafficking experiments, we did not see a significant 20 20 difference or defect in the frequency of EVL/VASP dKO acti- 10 10 vated T cells remaining in the vasculature of isolated lymph ICAM-1 Adhesion VCAM-1 Adhesion nodes from recipient mice 2 h posttransfer (Fig. 5J). In fact, (Mean T cells per FOV) 0 (Mean T cells per FOV) 0 WT dKO WT dKO there might be a trend toward increased numbers of dKO T cells D adhered to the lymph node vasculature. We also did not observe p=0.04 a significant difference in the frequency of EVL/VASP dKO E 15 15 T cells that are found in the CNS microvasculature of mice with ns EAE, 24 h after adoptive transfer (Fig. 5K). Although this is not 10 10 a direct readout of adhesion to the microvasculature, it further indicates that the impairment in activated dKO T-cell trafficking 5 5 is unlikely to be because of a deficiency in the adhesion step of extravasation. INFLAMMATION IMMUNOLOGY AND Mean T cell Crawling Mean T cell Crawling 0 0 Deletion of EVL and VASP Inhibits the Diapedesis Step of Speed on ICAM-1 (μm/min)

WT dKO Speed on VCAM-1 (μm/min) WT dKO Transendothelial Migration in Vitro. In addition to playing a role FG in adhesion and intravascular crawling, integrins are crucially 80 30 ns ns important for the diapedesis step of TEM (33, 45). Therefore, we

60 investigated whether the trafficking defect of EVL/VASP-deficient 20 T cells could be caused by defects in migration through the endo- 40 thelial barrier during extravasation. For these experiments, we im- 10 aged T-cell TEM on primary brain endothelial cells in the flow- 20 chamber system described above using phase contrast and spinning that Detach (%) Adhered T cells disk confocal microscopy to determine the effects of EVL and Endothelial Adhesion

(Mean T cells per FOV) 0 0 WT dKO WT dKO VASP deletion on T-cell diapedesis in vitro. Using phase-contrast microscopy, with the plane of focus set to the endothelial mono- H I layer, T cells attached on the endothelium (above the plane of fo- 20 15 ns ns cus) appear to have a white halo surrounding them. In contrast, T cells that have undergone diapedesis and the protrusions pro- 15 10 duced during that process lose this halo (6, 46) (Fig. 6 A and B and Movies S1 and S2). Collected images were analyzed to determine 10 the frequency and timing of diapedesis attempts, defined as T-cell 5 5 production of fluorescent protrusions lacking a phase halo, and

Attached T cells diapedesis completions, defined as events in which the T-cell body that Never Crawl (%)

0 on Endothelium (μm/min) 0 follows after the protrusion and the entire T-cell loses its halo. This WT dKO Mean T cell Crawling Speed WT dKO analysis revealed that deletion of EVL and VASP significantly im- J K paired both T-cell diapedesis attempts and completions (Fig. 6 C 6 ns 8 D ns and ), indicating that the mechanism of reduced dKO T-cell 5 trafficking in vivo lies at the stage of diapedesis. 6 4 3 4 adhesion to TNF-α–activated primary microvascular brain endothelial cells in 2 2 the presence of CCL21. (G) Detachment of initially adhered T cells from ac- 1 tivated endothelial monolayers after shear flow increase from 0.2 to 2 dyne/cm2. T cells in LNs (%) T cells in CNS (%) (H) Hyperadhesiveness of WT and dKO T cells to primary brain endothe- 0 0 Transferred intravascular Transferred intravascular WT dKO WT dKO 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- Fig. 5. EVL/VASP dKO activated CD4 T cells are competent in shear-resistant thelial monolayers. (J) Percentage of in vivo adoptively transferred WT adhesion and migration. (A) Schematic of the experimental set-up for and dKO T cells recovered from lymph nodes that remained in the vascula- measuring T-cell adhesion and motility under physiologic shear forces. ture 2 h posttransfer (identified by intravascular staining). (K) Percentage (B and C) WT and EVL/VASP dKO T-cell shear-resistant adhesion to ICAM-1 of transferred T cells recovered from the CNS that remained intravascular, (B), or to VCAM-1 (C). (D and E) Mean WT and dKO T-cell crawling speed 24 h posttransfer. Data represent the average of a minimum of three in- under flow on ICAM-1 in the presence of CCL21 (D) or on VCAM-1 in the dependent experiments; error bars are SEM; P values are paired t tests. ns, presence of CCL21 (E). (F) Quantification of WT and dKO T-cell shear-resistant not significant.

Estin et al. PNAS Early Edition | 5of10 Downloaded by guest on September 26, 2021 A 0:00 5:20 6:20 20:00

B 0:00 5:20 14:00 25:00

CDEFWT dKO WT dKO p<0.05 100 p=0.0006 20 p=0.04 50 ns 30 40 75 15 ns 20 30 50 10 ns 20 10 25 5 10 Adhered T cells that Adhered T cells that 0 Adhered T cells that 0 0 0 attempt diapedesis (%) Adhered T cells per FOV complete diapedesis (%) WT dKO complete diapedesis (%) WT dKO Isotype CD49d Isotype CD49d control block control block

Fig. 6. EVL/VASP-deficient activated CD4 T cells are impaired in the diapedesis step of transendothelial migration in vitro through a CD49d-dependent mechanism. Fluorescently labeled WT and EVL/VASP dKO activated T cells were flowed on primary brain microvascular endothelial monolayers and imagedby time-lapse fluorescent and phase-contrast microscopy. (A and B) Representative examples of diapedesis completion (A) and diapedesis attempts without completion (B), as visualized by phase microscopy. (Upper) Fluorescence overlay on phase channel; (Lower) phase channel alone. WT T cells are in green, dKO T cells in red. Red arrows indicate diapedesis attempts (extension of protrusions), green arrows indicate diapedesis completion. (Scale bars, 5 μm.) Timestamps are 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 representative of 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 paired t tests (C and D) or one-way ANOVA with post hoc Tukey test (E and F). ns, not significant.

CD49d-Independent Diapedesis Does Not Require EVL and VASP. Fi- suggesting that they may use alternate adhesion molecules more nally, having identified a defect in CD49d function in the EVL/ effectively (Fig. 6E). As opposed to control antibody treatment, VASP dKO T cells, we examined how CD49d affected the di- which confirmed a significant reduction in TEM of the dKO apedesis of control and EVL/VASP-deficient T cells. To this T cells, CD49d blockade greatly reduced the number of adhered end, we preincubated T cells with CD49d-blocking or isotype T cells on the endothelial monolayer and resulted in a similar control antibodies before introducing the T cells into flow ability of the adhered WT and dKO T cells to complete di- chambers, and then imaged for 30 min and analyzed the imaging F data, as described above. As expected, shear-resistant adhesion apedesis (Fig. 6 ). These data support the idea that EVL and of both control and dKO T cells to the endothelial monolayer VASP promote T-cell diapedesis via a CD49d-using mecha- was dramatically impaired by CD49d blockade. However, there nism, and that impaired CD49d function in EVL/VASP- was a trend toward more EVL/VASP dKO than WT T cells deficient T cells is a mechanism for their in vivo defect in adhering to the endothelial monolayer with CD49d blockade, T-cell trafficking.

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1701886114 Estin et al. Downloaded by guest on September 26, 2021 Discussion chemokine-stimulated ICAM-1 adhesion and naïve T-cell traf- PNAS PLUS This report on the role of EVL and VASP in T-cell migration ficking (52–54), whereas CRK proteins regulate T-cell adhesion, and trafficking identifies a key role for the actin effector proteins chemotaxis, and diapedesis, leading to reduced T-cell trafficking EVL and VASP in activated, but not naïve T-cell trafficking, and selectively to inflamed tissues (55). In contrast, recent reports determines that the trafficking defect in EVL/VASP dKO T cells show that Kindlin-3 is not required for diapedesis, although it occurs at the diapedesis step of TEM. EVL/VASP deficiency has been shown to play an important role in adhesion and CNS resulted in impaired activated T-cell trafficking to the CNS during trafficking more generally (56, 57). autoimmune neuroinflammation, to the inflamed skin and to sec- We propose that the unique and selective role of EVL and ondary lymphoid organs. Nonetheless, based on the varying endo- VASP in activated T-cell diapedesis is related to alterations in thelial barrier properties and adhesion molecules used for CD49d activity. Poor trafficking of activated EVL/VASP dKO extravasation into different tissues, it is possible that the Ena/ T cells correlated with decreased CD49d expression and function. VASP family may have a more or less profound impact on Conversely, naïve EVL/VASP dKO T cells trafficked normally and trafficking to different anatomical sites. their CD49d expression, although very low, was not significantly The observation that deletion of EVL and VASP impaired different from the low level expressed in control T cells. Further- activated T-cell trafficking in vivo is consistent with the literature more, activated EVL or VASP single-knockout T cells expressed implicating Ena/VASP proteins in cellular motility (27, 47–51). normal CD49d levels, possibly explaining the normal trafficking Although we identified reduced actin polymerization in response pattern they exhibit. Taken together, these data identify impaired to chemokine triggering in activated EVL/VASP dKO T cells, CD49d expression and function, which are known to regulate ac- surprisingly we found no major effect on activated T-cell che- tivated T-cell homing to peripheral lymph nodes and sites of in- motaxis or crawling in vitro. The finding that shear-resistant flammation (58), as a mechanism mediating the impairment in T-cell adhesion to primary brain endothelial cells was not af- EVL/VASP-deficient T-cell diapedesis and trafficking. fected by deletion of EVL and VASP was even more surprising, In line with this notion, control and EVL/VASP dKO cells that adhered to endothelial monolayers despite CD49d blockade as Ena/VASP proteins are established negative regulators of were equally likely to undergo diapedesis in vitro. This finding platelet adhesion to the vascular wall (28). suggests that EVL and VASP are specifically required for CD49d- To determine the mechanism of the trafficking defect of EVL/ dependent diapedesis, and opens up new questions about how such VASP-deficient T cells, we analyzed adhesion molecule function a specific requirement might be regulated. and observed alterations in expression and activity of T-cell

The role of CD49d and VCAM-1 in T-cell extravasation is well INFLAMMATION

integrins upon deletion of EVL and VASP. Surface and total IMMUNOLOGY AND established. Various studies have reported that ligation of VCAM-1 expression of CD49d was decreased in EVL/VASP dKO T cells, stimulates changes in endothelial permeability, triggering disso- whereas CD11a expression was slightly but significantly increased. ciation of vascular endothelial (VE) protein tyrosine-phosphatase Shear-resistant adhesion to purified ICAM-1 was enhanced upon (VE-PTP) from VE-cadherin, phosphorylation of VE-cadherin at deletion of EVL and VASP, as expected based on CD11a levels. tyrosine 731, and ultimately, down-regulation of VE-cadherin from However, although CD49d expression was reduced in EVL/VASP endothelial cell junctions (59–61). T-cell protrusions that are gen- dKO T cells, there was no corresponding decrease in shear-resistant erated during the “intravascular crawling” stage of extravasation adhesion to VCAM-1. Because surface integrins need to be acti- have previously been proposed to trigger chemokine depot-initiated vated to achieve a high-affinity conformation, differences in ex- diapedesis (35). It has also been suggested that filopodia generated pression do not necessarily correspond to alterations in function. by T cells can promote the sensing of permissive sites for diapedesis However, when we quantified CD49d affinity for VCAM-1, we (62). Ena/VASP family proteins are involved in generating mem- found that EVL/VASP dKO T cells not only bound less soluble brane protrusions such as filopodia (20, 63, 64) and localize to the VCAM-1 than WT control cells, but were also less capable of in- microspikes generated upon ligation of the TCR (24). Consistent creasing their VCAM-1 binding capacity in response to PMA/ion- with this finding, our data showed reduced protrusions initiating omycin stimulation. This finding suggests that EVL and VASP diapedesis by EVL/VASP-deficient T cells. Furthermore, activated positively regulate both expression and activation of CD49d. CD49d clusters at the leading edge of migrating T cells (65), and These data suggest that differential regulation of CD49d and the microvilli that form at the leukocyte-endothelial cell interface CD11a upon deletion of EVL/VASP could explain the preser- in migrating leukocytes are rich in CD49d but not CD18 integrins vation of endothelial adhesion we observed, with increased CD11a (66). Further study may allow determination of whether EVL and compensating for decreased CD49d. Indeed, upon CD49d block- VASP coordinate the presentation of CD49d on T-cell protru- ade, dKO T cells trended toward increased endothelial adhesion sions, which are able to trigger the opening of endothelial junctions relative to WT controls, suggesting that they may have developed via signaling through VCAM-1. enhanced CD49d-independent adhesion mechanisms in response to Similar to our results using EVL/VASP dKO T cells, condi- poor CD49d function. However, because adhesion to and crawling tional knockout of CD49d in mice results in impaired T-cell on endothelial cells is unaffected in EVL/VASP dKO T cells, simple trafficking to an inflamed CNS (67). Interference with CD49d- alterations in the adhesive functions of these integrins do not pro- mediated T-cell trafficking via monoclonal antibody blockade vide a sufficient explanation for the trafficking defects we observed has made natalizumab a very effective treatment for multiple in vivo. In contrast, our in vitro TEM data point to a critical non- sclerosis (1, 2) and Crohn’s disease (68). This therapy targets redundant role for EVL and VASP in diapedesis, suggesting that activated or memory T cells without significantly impairing naïve the requirement for Ena/VASP family proteins in activated T-cell T-cell surveillance. However, use of natalizumab is limited in trafficking occurs specifically at the diapedesis step of TEM. patients who are infected with the JC Virus, as they are at risk for Our finding that EVL and VASP are required for diapedesis progressive multifocal leukoencephalopathy, a fatal complication but not adhesion, motility, or chemotaxis is currently unique (69, 70). Natalizumab-treated patients can also experience in- among actin cytoskeletal regulators. For example, our previous creased susceptibility to urinary and respiratory tract infections, work (6) has indicated that the cytoskeletal effector myosin-IIA which can trigger relapses (71). affects both T-cell motility and diapedesis, and deletion of Our work supports the idea that CD49d has a specific, EVL/ mDia1, another actin effector protein, both impairs thymocyte VASP-dependent function during the diapedesis step of TEM, development and produces defects in chemotactic migration and which is distinct from its roles in adhesion and intravascular in vivo homing of naïve T cells (11). Similarly, deletion of the crawling. Although EVL/VASP-directed therapies would need cytoskeletal regulators Rap1, RIAM, talin, or RAPL impair to be targeted to lymphocytes to avoid effects on other cells, our

Estin et al. PNAS Early Edition | 7of10 Downloaded by guest on September 26, 2021 work suggests that further studies of EVL and VASP in T cells were transferred intravenously into recipient mice which were then eutha- could identify therapeutically useful ways to more selectively nized 2 or 24 h after adoptive transfer and peripheral lymph nodes, spleen, modulate CD49d activity and activated T-cell trafficking. blood, and lungs were harvested. Intravascular staining of cells remaining in blood vessels was performed via intravenous injection of 3 μg PE- or APC- Materials and Methods conjugated anti-CD4 antibody (GK1.5) 3 min before euthanasia (39). After euthanizing with CO2, blood was then harvested by cardiocentesis, and the Ethics Statement. All experiments involving mice were conducted in compliance peripheral vasculature and lungs were fully perfused through the heart with with the NIH’s Guide for the Care and Use of Laboratory Animals (72) and with saline. Single-cell suspensions were generated from lymph nodes and spleen the approval by the Institutional Animal Care and Use Committee of National by passing organs through a 100-μm filter. Blood and splenic red blood cells Jewish Health (Protocol #AS2811-02-17). All efforts were made to minimize were lysed in 175 mM ammonium chloride. Lungs were minced, digested in mouse suffering. DNase and Collagenase D for 30 min, passed through a 100-μM filter, and spun through a Histopacque-1119 density gradient to isolate leukocytes. Mice. KO mice lacking EVL and VASP were generated, respectively, by One to three recipient mice were typically used per experiment, depending Kwiatkowski et al. (31) and Aszódi et al. (32) (EVL/VASP dKO mice were on cell numbers available. generously provided by Frank Gertler, Massachusetts Institute of Technol- ogy, Cambridge, MA). These mice were on a 129 × C57BL/6 mixed back- Induction of EAE and Scoring. EAE was induced using MOG induction kits from ground and bred in-house at National Jewish Health. Single EVL or VASP KO Hooke Laboratories according to their protocol. Briefly, WT female CD45.1 and WT 129 × C57BL/6 mice were derived from the double-KO mice and C57BL/6 mice of at least 8 wk of age were immunized with 200 μgofMOG35–55 were maintained in parallel. Recipient mice used for homeostatic trafficking peptide emulsified in complete Freund’s adjuvant injected subcutane- assays were 129S1 × C57BL/6 F1 hybrid mice (Stock #101043, The Jackson ously, followed by intraperitoneal injection of 200 ng pertussis toxin on the Laboratory). For EAE induction and short-term CNS trafficking, as well as day of induction and the following day. Typical EAE onset was within 10–15 d skin trafficking experiments, CD45.1 congenically marked C57BL/6 recipient postimmunization. Mice were monitored and scored daily for development mice (Strain #564) were purchased from Charles River. CD45.1 C57BL/6 mice of EAE based on the following 0–5 scoring criteria: 0, no disease; 1 limp tail; were also used to provide third-party WT splenocytes for T-cell activations. 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, complete T-Cell Isolation. Naïve CD4 T cells were isolated and purified by negative paralysis of front and hind limbs or moribund state. Mice with a score ≥ 4were selection. Briefly, spleens, as well as mesenteric, brachial, axial, and inguinal euthanized immediately. Mice with scores of 2 or greater were used for T-cell lymph nodes, were harvested and single-cell suspensions were made by trafficking experiments. These procedures were approved and carried out in μ passing tissues through 100- m sterile filters. T cells were purified using accordance to the regulations of the Institutional Animal Care and Use Com- StemCell EasySep magnetic isolation kits. Naive CD4 T-cell selection kits were mittees of National Jewish Health, and all efforts were made to minimize used for naïve cells; whereas total CD4 T-cell selection kits were used when mouse suffering. Five mice were induced per group. T cells were then subsequently activated in vitro. CNS Trafficking. For CNS trafficking, 5 × 106 WT and dKO activated T cells T-Cell Activation and Culture. T cells were cultured using R10: RPMI 1640 with were transferred IV into acutely ill EAE mice (score ≥ 2.0, see above), and β the addition of L-glutamine, penicillin, streptomycin, and -mercaptoethanol recipients were euthanized 24 h after adoptive transfer following in- (all purchased from Invitrogen) and 10% (vol/vol) FCS (Fisher Scientific). + travascular staining of cells as above. Intravascular staining and saline per- Purified CD4 T cells were activated in vitro with plate-bound anti-CD3 and fusion after cardiocentesis was performed as above. Blood, brain, and spinal soluble anti-CD28 antibodies (BioXcell), in the presence of third-party WT cord were isolated and single-cell suspensions were generated from brain feeder splenocytes. Feeder cells that could be identified by a congenic and spinal cord by passing tissues through a 100-μm filter. A 70%/30% (vol/vol) marker (CD45.1) were harvested from third-party mouse spleens as above, percoll gradient was used to isolate leukocytes. Total leukocytes in all samples and red blood cells were lysed in 175 mM ammonium chloride. Splenocytes were manually enumerated on a hemocytometer, and adoptively transferred were then irradiated at 1,500 rads, and mixed in a 2:1 ratio with purified cells were quantified by flow cytometry (CyAN, Beckman Coulter). One to μ T cells. Next, 2 g/mL soluble anti-CD28 (clone PV-1) was added to the mix- three recipient mice were typically used per experiment, depending on × 6 ture, and cells were plated at 2 10 /mL in a 24-well plate that had been availability of sufficiently ill recipient mice. coated with 2 μg/mL anti-CD3 (clone 2C11) for 1–2 h at 37 °C. On day × 6 + 2 postactivation, cells were resuspended at 1 10 /mL in fresh R10 10 U/mL Skin Trafficking. Inflammation was induced in the ears of CD45.1 recipient recombinant human interleukin 2 (rIL2) (obtained through the AIDS Re- mice via subcutaneous injection of 20 μg LPS. Dye-labeled control or dKO search and Reference Reagent Program, Division of AIDS, National Institute activated T cells were transferred intravenously 24 h after the induction of of Allergy and Infectious Diseases, NIH from Maurice Gately, Hoffmann-La inflammation. Then, 24 h after transfer of T cells, mice were injected in- Roche Inc., Basel, Switzerland). On day 4 postactivation, cells were resus- travenously with 3 μg of CD4-APC and then euthanized 3 min later. Blood × 6 + pended at 2 10 /mL in fresh R10 10 U/mL rIL2. Before use on day 5, dead was collected via cardiocentesis and the mouse was gravity-perfused with cells were removed by Histopacque-1119 (Sigma-Aldrich) density gradient, saline. Ears were removed, peeled apart, cut into small pieces, and placed × 6 + and cells were resuspended at 2 10 /mL in R10 10 units/mL rIL2. into 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 for Antibody Clones Used for Flow Cytometry. Antibody clones used for flow 45 min at 37 °C with occasional mixing. After digestion, the remaining ear cytometry were: CD4 (GK1.5), CD5 (53-7.3), CD8a (53-6.7), CD11a (M17/4), tissue was further mechanically dissociated and then filtered out on a CD19 (6D5), CD25 (PC61), CD29 (HMB1-1), CD44 (IM7), CD45.1 (A20), CD49d 100-μM nylon mesh strainer followed by rinsing with RPMI with 10% (vol/vol) (R1.2), CD62L (Mel14), CD69 (H1.2F3), CD197 (4B12), CCR5 (7A4), CCR7 (4B12), FBS. Lymphocytes were then separated from debris using Histopaque-1119, CXCR3 (CXCR3-173), CXCR4 (L276F12). All purchased from Biolegend or washed and resuspended in FACS buffer for staining. Blood and nondraining eBioscience. lymph nodes and spleen were also harvested and processed as described above. All samples were stained with anti–CD45.1-BUV-395 before analysis Antibodies Used for Western Blot. Antibodies used for Western blot were: by flow cytometry using a LSR Fortessa (Beckton Dickinson). Transferred VASP (9A2, CellSignaling); EVL (ab108406, Abcam); Mena (sc-135988, Santa T cells that fully extravasated were identified as dye-positive (CFSE or VPD), Cruz); mouse anti-Tubulin (Sigma-Aldrich); secondary antibodies (Licor CD4-APC–negative, and CD45.1-BUV395–negative. Two recipient mice were donkey anti-mouse IR680 and IR800, donkey anti-rabbit IR800). used per experiment.

Dye-Labeling T Cells. NaïveoractivatedCD4EVL/VASPdKOandWTTcellswere Actin Polymerization Assay. Activated T cells were stimulated with either 1 μg/mL differentially labeled with either 2 μM carboxy-fluorescein diacetate succini- or 0.1 μg/mL of chemokine (Peprotech) for 5, 15, or 60 s at 37 °C in 2% (wt/vol) midyl ester (CFSE; Invitrogen) or 1 μM Violet Proliferation Dye (VPD; eBio- BSA in RPMI. The reaction was stopped using 4% (wt/vol) paraformaldehyde sciences), mixed at a 1:1 ratio, and used for in vitro adhesion and migration (Electron Microscopy Sciences) in PBS and the cells were fixed for 10 min. T cells assays under flow, as well as in vivo trafficking experiments. Between experi- were then permeabilized with saponin (Sigma-Aldrich) buffer [0.5% (wt/vol) mental repeats, dyes were swapped to control for potential effects of the dyes. saponin, 2% (vol/vol) FBS, and 0.05% sodium azide in PBS] for 30 min at room temperature. Cells were stained with a 1:50 dilution of Phalloidin-Alexa Fluor- In Vivo Adoptive Transfer and Intravascular Staining. For homeostatic traf- 647 (Life Technologies) in saponin buffer for 30 min and then washed twice. ficking in untreated recipient mice, 5 × 106 dKO and WT dye-labeled cells Analysis was done using a Beckman-Coulter Cyan flow cytometer.

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1701886114 Estin et al. Downloaded by guest on September 26, 2021 Transwell Migration. Wells of a 24-well plate were prepared containing RPMI monolayer of mouse primary brain microvascular endothelial cells (Cell Bi- PNAS PLUS with 2% (wt/vol) BSA and 10 mM Hepes, with or without indicated che- ologics) that had been activated with TNF-α 24 h before imaging, and mokines (CCL21, CXCL10, CXCL12, and CCL5, all from Peprotech). For che- treated with CCL21 30 min before imaging. The endothelial cells were also motaxis assays, 1 × 106 control or dKO T cells were added to the top labeled with APC-conjugated anti-CD31 antibody (clone 390) to visualize the chambers of 5-μm Transwell plates (Corning) and allowed to migrate for 1 h endothelial cell membrane and monolayer integrity; this staining does not at 37 °C in the presence or absence of chemokines in the lower well. For perturb the T-cell TEM process (73, 74). T cells were initially perfused into the chemokinesis, chemokine was present in both upper and lower wells. As a chamber at 0.2 dyne/cm2 shear-flow for 5 min, and then the shear-flow was 5 standard, 2 × 10 cells (20% of input cells added to Transwells) were placed raised to 2 dyne/cm2. Phase contrast and fluorescence images were acquired directly into bottom wells with no Transwell to calculate the percentage of every 20 s for 30 min using a spinning-disk confocal microscope. migrated cells. Each condition was set up in duplicate Transwells. Migrated In CD49d (α4 integrin) blockade experiments, a 1:1 mixture of control and μ T cells were collected from the bottom wells and 25 L of cell counting beads EVL/VASP dKO T cells at 10 × 106 cells/mL was pretreated with a mixture of (123count eBeads, eBioscience) were added. Each sample was quantified for two CD49d-blocking antibodies (clones 9C10 and R1-2) at 2 μg/mL each, for a fixed period (30 s) using a flow cytometer (CyAn ADP Beckman Coulter). 30 min on ice before imaging. IgG2b antibody was used as an isotype con- The number of cells counted during this time was normalized to the number trol. Cells were then perfused into a flow chamber at 10 × 106 cells/mL of beads counted to adjust for any variations in flow rate during the run. as above.

Soluble VCAM-1 Binding Assay. T cells were resuspended in RPMI without Quantification of Adhesion, Crawling, and Diapedesis Under Flow. Manual phenol red, supplemented with 5% (wt/vol) BSA (Sigma-Aldrich). Experi- scoring and automated quantification were completed on blinded images, × 6 μ + mental wells were set up in duplicate, each with 0.5 10 cells in 25 L RPMI using Imaris software (Bitplane). Adhesion (cells per field of view 1 min after BSA medium in a round-bottom 96-well plate. Twenty-five microliters of the increase in flow) was quantified using the Imaris “spot” algorithm. In R10 medium, MnCl treatment medium (R10 with 4 nM MnCl ), or PMA/ 2 2 some cases the number of cells per field-of-view was normalized between ionomycin treatment medium (R10 with 50 nm/mL PMA and 1 μg/mL ion- experiments to account for a different number of T-cell input. Mean omycin) were added to each experimental well. The plate was gently vor- crawling speed was quantified by following fluorescent cells using the Imaris texed and then incubated for 5 min at 37 °C. Next, 50 μL VCAM-Fc (1 mg/mL; “track” algorithm. In endothelial TEM experiments, cells that never crawled, R&D Systems) was added to each well and incubated for 10 min at 37 °C. cell detachment, and diapedesis attempts and completions were also scored Samples were then transferred to ice and 100 μL ice-cold RPMI + BSA me- manually. Briefly, T cells that contained partial regions that lost and then dium was added to each well. Samples were washed once in 200 μL ice-cold regained the white phase contrast ring in a stepwise manner were scored as RPMI + BSA medium, and were then resuspended in 100 μL APC-conjugated having attempted diapedesis, whereas T cells that underwent a stepwise anti–human-Fc antibody (clone HP6017) in RPMI + BSA (1 μL per test). darkening and completely lost the white phase contrast ring were scored as Samples were incubated on ice for 1 h and then washed three times. Cells having completed diapedesis. Diapedesis data were filtered to exclude all

were then filtered through Nytex filters, and samples were analyzed by flow INFLAMMATION

cells that were not present in the field of view for at least 13 min (the mean IMMUNOLOGY AND cytometry (CyAN, Beckman Coulter). time required to complete diapedesis). Microscopy. We used a 3i (Intelligent Imaging Innovations) Marianas Statistical Analysis. Prism software (GraphPad) was used to graph the data spinning-disk confocal microscope system equipped with a Zeiss inverted stand and a Yokogawa spinning disk unit. We imaged through a 20× phase- and calculate statistical significance. The statistical significance of single contrast objective. CFSE- and VPD-labeled lymphocytes were excited with comparison data were determined by performing paired or unpaired Stu- ’ the 488 and 445 laser lines, respectively. Endothelial cells stained with A647- dent s t tests, or one-sample t tests versus a hypothetical ratio of 1.0, as conjugated CD31 antibody were excited with the 640 laser line. Appropriate appropriate. One-way ANOVA with Tukey posttests was used for multiple emission wavelengths were acquired for each channel. Time-lapse images comparisons. Two-way ANOVA was used for comparison of data with two were acquired every 20 s for 30 min, at three or four separate stage positions independent variables. per 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- ACKNOWLEDGMENTS. We thank F. Gertler and R. Fassler for the gift of the – sition was managed using Slidebook software (3i, v6.0). vasodilator-stimulated phosphoprotein (VASP) and Ena-VASP like knockout mice; M. Gebert, R. Long, and D. Tracy for help with mouse genotyping and colony maintenance; R. Lindsay for technical help with some experiments; Adhesion and Crawling on ICAM-1 and VCAM-1. A 1:1 mixture of differently J. Loomis and S. Sobus for expert technical assistance with cell sorting; dyed T cells was resuspended in RPMI without phenol red, supplemented with J. Loomis for microscope maintenance; and P. Henson, R. Kedl, and R. Torres 6 2% (wt/vol) BSA (R&D) 10 mM Hepes at 4 × 10 /mL. T cells were perfused into for reagents and comments on the manuscript. This work was funded in part a flow chamber (μ-slide VI, IBIDI) that had been coated with ICAM-1 or by National Institute of Allergy and Infectious Diseases/NIH Awards R01AI125553 VCAM-1 (1 mg/mL) in PBS for 1 h at 37 °C. Cells were initially perfused into and R56AI105111 (to J.J.); awards from the Dana Foundation (Brain- the chamber at 0.2 dyne/cm2 shear-flow for 5 min, and then the shear-flow Immuno Imaging grant), National Multiple Sclerosis Society (Pilot grant), was raised to 2 dyne/cm2. Spinning-disk confocal fluorescence images were and American Society of Hematology (Bridge grant) (all to J.J.); NIH train- acquired every 20 s for 30 min. ing Grant T32AI007405 (to M.L.E.); and NIH/National Center for Advancing Translational Sciences Colorado CTSI TL1 Grant 8TL1TR00155 (to M.L.E.); NIH Training Grant T32AI007405 (to S.B.T.). The spinning-disk confocal mi- Endothelial Adhesion, Crawling, and Diapedesis Under Flow. A 1:1 mixture of croscope used was acquired thanks to Shared Instrumentation Grant Award differentially dyed WT and dKO T cells was resuspended in RPMI without S10RR029218. The content of this work is solely the responsibility of the phenol red, supplemented with 2% (wt/vol) BSA, and 10 mM Hepes at 2 × 106/mL. authors and does not necessarily represent the official views of the NIH or T cells were perfused into a flow chamber (μ-slide VI, IBIDI) coated with a other funding agencies.

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