PNAS PLUS
Ena/VASP proteins regulate activated T-cell 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 a Department 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)
T cell
| migration | cytoskeleton | extravasation
A
ctivated T-cell trafficking across the vascular endothelium is essential for ongoing immune surveillance of tissues and for 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 target for therapeutic immunomodulation. Disruption of these pathways, such as by antibody blockade of α4 integrins, is a highly effective approach to immunomodulation (1, 2). However, the molecular mechanisms by which chemokine receptor and adhesion molecule signaling induce the T-cell cytoskeletal machinery to promote extravasation are not yet fully elucidated. Transendothelial migration (TEM), the process by which T cells extravasate from the blood into tissues, is characterized by four distinct steps: rolling along the vascular wall, arrest or adhesion, intravascular crawling, and diapedesis across the endothelial barrier (3). Surface adhesion molecules play well-characterized roles in each step of the process. For example, the initial rolling step of TEM is facilitated by interactions between T-cell and endothelial selectins, whereas the adhesion, intravascular crawling, and diapedesis steps of TEM are mainly regulated by chemokineand shear force-stimulated modulation of lymphocyte functionassociated antigen 1 (LFA-1, αLβ2 integrin, CD11a/CD18) and very late antigen 4 (VLA-4, α4β1 integrin, CD49d/CD29) interactions with intracellular adhesion molecule 1 (ICAM-1) and vascular cellular adhesion molecule 1 (VCAM-1), respectively. Dynamic cyto-
www.pnas.org/cgi/doi/10.1073/pnas.1701886114
skeletal changes occur throughout the process of TEM (4, 5); however, the regulation of these cytoskeletal changes is not completely understood. The lymphocyte actin-myosin cytoskeleton is composed of networks of linear and branched actin filaments, cross-linked by class-II nonmuscle myosin. We have previously shown that inhibition of myosin-IIA, the main class-II myosin protein expressed in lymphocytes, alters T-cell trafficking, motility, and TEM (6–9). Although numerous studies have focused on the upstream regulatory signaling cascades that control actin network remodeling during migration (5, 10), less is understood about how downstream effectors of branched and linear actin polymerization might participate in lymphocyte TEM. Two major families of effector proteins of linear actin polymerization are expressed in lymphocytes. The Formin family, which is thought to nucleate new linear actin filament production, has been implicated in T-cell activation and egress from the thymus (11–13). Less is known about the Ena/ VASP (vasodilator-stimulated phosphoprotein) family of cytoskeletal effectors in T cells. The Ena/VASP family is composed of three members: mammalian-enabled (Mena), which is not typically expressed in hematopoietic cells; VASP; and Ena-VASP–like (EVL) (14, 15). These proteins coordinate monomeric actin recruitment to the barbed 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 an N-terminal EVH1 domain, which regulates cellular localization; Significance T-cell trafficking is essential for the function of the adaptive immune system, and regulation of T-cell entry into tissues can be an effective therapy in diseases such as autoimmunity. However, the mechanisms regulating T-cell migration and trafficking are poorly understood. We have identified a key role for Ena/VASP (vasodilator-stimulated phosphoprotein) family cytoskeletal effectors selectively in activated T-cell trafficking to secondary lymphoid 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 suggests that further studies of the Ena/VASP pathway in T cells could identify therapeutically useful ways to more selectively modulate 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. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1
To whom correspondence should be addressed. Email: [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1701886114/-/DCSupplemental.
PNAS | Published online March 20, 2017 | E2901–E2910
IMMUNOLOGY AND INFLAMMATION
Vasodilator-stimulated phosphoprotein (VASP) and Ena-VASP–like (EVL) are cytoskeletal effector proteins implicated in regulating cell 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 identifies a specific role for EVL and VASP in T-cell diapedesis and trafficking. We demonstrate that EVL and VASP are selectively required for activated T-cell trafficking but are not required for normal T-cell development or for naïve T-cell trafficking to lymph nodes and spleen. Using a model of multiple sclerosis, we show an impairment in trafficking of EVL/VASP-deficient activated T cells to the inflamed central nervous system of mice with experimental autoimmune encephalomyelitis. Additionally, we found a defect in trafficking of EVL/VASP double-knockout (dKO) T cells to the inflamed skin and secondary lymphoid organs. Deletion of EVL and VASP resulted in the impairment in α4 integrin (CD49d) expression and function. Unexpectedly, EVL/VASP dKO T cells did not exhibit alterations in shear-resistant adhesion to, or in crawling on, primary endothelial cells under physiologic shear forces. Instead, deletion of EVL and VASP impaired T-cell diapedesis. Furthermore, T-cell diapedesis became equivalent between control and EVL/VASP dKO T cells upon α4 integrin blockade. Overall, EVL and VASP selectively mediate activated T-cell trafficking by promoting the diapedesis step of transendothelial migration in a α4 integrin-dependent manner.
altered T-cell trafficking in vivo, we used EVL/VASP doubleknockout (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 that Mena was not up-regulated as a compensatory mechanism in EVL/ VASP dKO T cells by Western blot analysis (Fig. S1). We then analyzed if EVL/VASP deficiency caused defects in T-cell development. Flow cytometry analysis of lymphocyte populations in the thymus and secondary lymphoid organs showed no gross defects in T-cell development and normal proportions of mature CD4 and CD8 T cells in the periphery of EVL/VASP dKO mice (Fig. S2). Deletion of Both EVL and VASP Selectively Impairs Activated CD4 T-Cell Trafficking into Secondary Lymphoid Tissues. Next, we used
coadoptive transfers of WT control and EVL/VASP dKO T cells to study the T-cell–intrinsic effect of EVL and VASP deficiency on trafficking in vivo. Consistent with the normal pattern of development and homeostatic trafficking, dKO and WT naïve CD4 T cells had equivalent homing to the spleen and lymph nodes after intravenous 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 required for activated T-cell trafficking. We first established that, upon ex vivo polyclonal activation with CD3 and CD28 stimulation, T-cell proliferation kinetics and activation profiles were similar for WT and EVL/VASP dKO T cells (Fig. S3). Activated T cells can become lodged in the vasculature (particularly in the lungs) (37, 38), potentially because of their increased size and adhesion properties. Therefore, we used an established technique (39, 40) to distinguish extravasated T cells from those stuck intravascularly by injecting intravenously a fluorophoreconjugated anti-CD4 antibody immediately before euthanasia 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 of CCR7 and can recirculate to secondary lymphoid organs. To determine if homeostatic trafficking of activated T cells was affected by Ena/VASP deficiency, we cotransferred control and EVL/VASP dKO T cells into unimmunized recipient mice. Following intravenous adoptive transfer, activated dKO CD4 T cells on average E2902 | www.pnas.org/cgi/doi/10.1073/pnas.1701886114
Naive dKO:WT Ratio Normalized to Injected
1.5
ns
ns
2 hours in vivo
Inject anti-CD4 PE Ab IV( ) Euthanize
1.0
Perfuse with saline
0.5
Harvest tissues and analyze by flow cytometry
0.0
Blood Spleen
Intravascular Cells (CD4 PE+)
LN
D
Activated 2.0 ns
p=0.004 p=0.005
1.5 1.0 0.5 0.0
Blood Spleen
LN
E
Transferred cells # (x104)
C
F 1.5
ns
ns
ns
1.0
0.5
0.0
Blood Spleen
LN
VASP sKO:WT Ratio Normalized to Injected
Deletion of EVL and VASP Does Not Significantly Affect T-Cell Development. To determine if deletion of Ena/VASP proteins
Naive 2.0 p=0.03
WT and KO CD4+ T cells
B
Extravasated Cells (CD4 PE-)
Activated dKO:WT Ratio Normalized to Injected
Results
A
EVL sKO:WT Ratio Normalized to Injected
and a C-terminal EVH2 domain, which facilitates tetramerization, binds filamentous actin (F-actin), and is thought to be responsible for actin polymerization (21–24). Ena/VASP proteins are capable of compensating for deletion of one another, but there is some evidence that they are differentially regulated (15). EVL, and especially VASP, are implicated in the motility, adhesion, and sensory capacity of many cell types. EVL and VASP localize to filopodia tips (25) as well as to adhesive sites, such as fibroblast focal adhesions (24). This localization pattern is consistent with a role in migration and adhesion. Fibroblasts lacking EVL and VASP produce shorter filopodia, and a slower-moving lamellipodium, which paradoxically leads to enhanced fibroblast motility (26, 27). Platelets from VASP-knockout mice demonstrate enhanced 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 in T cells, showing that Ena/VASP proteins can contribute to actin remodeling during T-cell receptor (TCR) signaling (24, 30). However, previous studies did not focus on the role of EVL and VASP in T-cell development or T-cell motility. Therefore, we sought to elucidate the mechanisms by which EVL and VASP might influence T-cell adhesion, migration, and trafficking in vivo.
WT 50 40 30 20 10
ns
dKO
p=0.01
p=0.04
2 1 0
Blood Spleen
LN
1.5 ns
ns
ns
1.0
0.5
0.0
Blood Spleen
LN
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 trafficking is not affected by EVL and VASP deficiency. Differentially dyelabeled naïve WT and EVL/VASP dKO T cells were coadoptively transferred intravenously at a 1:1 ratio and T-cell trafficking to lymphoid tissues was quantified by flow cytometry 2 h after adoptive transfer. The dKO:WT ratio was normalized to the ratio in the injected sample to account for possible minor variations in the injection mixture (typically < 10%). (B) Experimental set-up for cotransfer of activated WT and KO cells, including intravascular staining method to distinguish intravascular T cells from those that have 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 and T-cell trafficking to tissues 2 h after adoptive transfer was quantified by flow cytometry as above. A ratio below 1.0 (horizontal red line) indicates impaired homing of KO T cells. (C) Quantification of activated T-cell trafficking. Ratio of extravasated WT and EVL/VASP dKO activated T cells, harvested and analyzed as above. (D) Number of WT and dKO T cells recovered from the indicated 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 average from a minimum of three independent experiments. Error bars are SEM. Statistics 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.
exhibited a 2.2-fold reduction in spleen trafficking and a 3.3-fold reduction in lymph node trafficking 2 h after adoptive transfer compared with WT controls (Fig. 1 C and D). This activated dKO T-cell trafficking defect still persisted 24 h posttransfer (Fig. S4 A and B). Furthermore, the intravascular staining used to quantify T cells that had entered tissues versus cells that remained adhered within Estin et al.
EVL and VASP Deletion Impairs Activated CD4 T-Cell Trafficking to Sites of Inflammation. We next sought to determine if activated
CD4 T-cell trafficking to sites of inflammation was affected by Ena/VASP protein deficiency. Because the vascular endothelial barrier in the central nervous system (CNS) is particularly restrictive, we first investigated EVL/VASP-deficient activated T-cell trafficking to the CNS in the context of autoimmune inflammation, using a mouse model of multiple sclerosis: experimental autoimmune encephalomyelitis (EAE). Twenty-four hours after adoptive transfer of T cells into mice with ongoing EAE, we quantified the number of transferred T cells that had extravasated into the CNS using the intravascular staining technique described above. Activated dKO T cells exhibited on average a 2.0-fold reduction in trafficking to the CNS relative to control T cells (Fig. 2 A–C). Next, we analyzed T-cell trafficking
Transfer activated T cells into EAE mice, 24h later euthanize and harvest tissues
E 0h
Inject LPS SC in ear
24h
Transfer T cells
48h
Euthanize and harvest tissues
dKO:WT Ratio Normalized to Injected
D
ns
p=0.01
1.0
0.5
0.0
Blood
CNS
ns
p=0.02
60 50 40 30 20 10
1.0
0.5
0.0
Blood
Skin
WT dKO
p=0.02 5 0
Blood
F 1500
1.5
ns
CNS
ns WT dKO
1000 500 10 8 6 4 2 0
p=0.01
cyte migration and trafficking (41, 42). Therefore, we analyzed if the trafficking defect of Ena/VASP-deficient activated T cells could be because of an altered ability to respond to chemokine stimulation. First, we measured the expression of chemokine receptors involved in both homeostatic and inflammatory trafficking. Our data showed no difference in the expression of CCR7, 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, chemokine stimulation can trigger actin network remodeling and promote motility (34). Therefore, we analyzed if Ena/VASP deficiency impaired actin polymerization in response to chemokine stimulation. To this end, we measured the F-actin content in control and EVL/VASP dKO T cells before and after chemokine stimulation in a time-course analysis. Our results showed that dKOactivated T cells had a small but significant defect in actin polymerization promoted by CCL21 stimulation and, to a lesser degree, by CXCL10 (Fig. 3B). However, when we analyzed actin 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. Based on this result, we then measured chemokine-stimulated migration using Transwell chambers. There were no significant differences in migration in the absence of chemokine, or in chemotaxis toward CCL21, CXCL10, CXCL12, or CCL5 in the lower chamber between control and EVL/VASP dKO-activated T cells (Fig. 3C). Furthermore, chemokinetic migration in response to CCL21 in both the upper and lower chambers was also unaffected (Fig. S5B). Although we detected slightly reduced chemokine-mediated actin polymerization in EVL/VASPdeficient activated T cells, overall these data showing normal chemotaxis suggest that the strong trafficking defect of EVL/ VASP dKO T cells in vivo is not explained by an overall defect in migratory capacity or chemokine sensing. Activated CD4 EVL/VASP dKO T Cells Are Deficient in α4 Integrin Expression and Function. Ena/VASP proteins are reported to af-
Blood
Skin
Fig. 2. EVL and VASP deletion inhibits activated CD4 T-cell trafficking to the CNS during EAE and to the inflamed skin. (A) Experimental set-up for cotransfer of WT and EVL/VASP dKO activated T cells into mice with ongoing EAE. Activated, dye-labeled polyclonal CD4 WT and dKO T cells were coadoptively transferred at a 1:1 ratio, and were harvested 24 h posttransfer from the blood and CNS (brain and spinal cord). (B) Activated T-cell trafficking during EAE was quantified by flow cytometry, shown as the ratio of dKO:WT T cells normalized to the ratio in the injected sample. (C) Number of WT and dKO T cells recovered from the indicated tissues from data in B. (D) Experimental set-up to quantify activated T-cell trafficking to the inflamed skin. Twenty-four hours after LPSinduced inflammation in the ears, WT and dKO activated T cells were coadoptively transferred at a 1:1 ratio, and were harvested 24 h posttransfer from the blood and ears of the recipient mice. (E) Ratio of dKO:WT T cells recovered from blood and ears, normalized to the ratio in the injected sample. (F) Number of WT and dKO T cells recovered from the indicated tissues from data in E. Data are the average of four independent experiments. Error bars are SEM. Statistics are onesample 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.
Estin et al.
Deletion of EVL and VASP Reduces Chemokine-Triggered Actin Polymerization but Does Not Impair Activated T-Cell Chemotaxis in Vitro. Chemokine signaling is instrumental in mediating leuko-
fect adhesion and integrin function (28, 43). Therefore, having ruled-out impaired chemotaxis, we next examined the expression and function of key integrins involved in extravasation to determine if the trafficking impairment we observed was because of integrin defects. Flow cytometry analysis revealed that activated EVL/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-experienced T cells, which may explain why only activated dKO T cells exhibited a trafficking defect in vivo. Consistent with their normal trafficking phenotype, activated EVL or VASP single-knockout T cells did not exhibit reduced CD49d expression, nor did naïve dKO T cells (which only expressed negligible levels of CD49d) (Fig. S6 A–E). Additionally, expression of the α4 integrin binding partners β1 (CD29) and β7 in activated cells was also reduced in activated dKO T cells compared with WT T cells (Fig. S6F). Furthermore, total expression versus surface expression of CD11a PNAS | Published online March 20, 2017 | E2903
IMMUNOLOGY AND INFLAMMATION
Day 14-20
Mice begin to develop symptoms
C
1.5
Transferred cells # (x104)
Start scoring mice
B
Transferred cells # (x102)
Day 7 Day 10-14
EAE Scoring
Day 2
Inject MOG35-55 SC & PTX IP PTX IP
dKO:WT Ratio Normalized to Injected
A Day 0
to the inflamed skin, using lipopolysaccharide (LPS) as the inflammatory stimulus. Recipient mice were treated by subcutaneous LPS injections in the ear and 24 h later activated control and dKO T cells were intravenously transferred into the recipient mice. Quantification of transferred T cells that had extravasated into the inflamed skin of the ear showed a 1.8-fold reduction in dKO T-cell trafficking to this site (Fig. 2 D–F).
PNAS PLUS
blood vessels also indicated that the defect in activated EVL/VASP dKO T-cell trafficking was not a result of selective trapping of these cells in the lung microvasculature, the first capillary bed encountered after intravenous adoptive transfer. In fact, significantly more WT than dKO T cells were recovered from inside the lung microvasculature (Fig. S4C). In keeping with these data, there is a trend toward more EVL/VASP dKO T cells than control cells in the blood compartment, suggesting that these cells may be unable to gain 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 secondary lymphoid organs. Consistent with the likelihood of redundant or compensatory functions between Ena/VASP proteins, singleknockout of EVL or VASP did not give rise to activated T-cell trafficking defects (Fig. 1 E and F).
50
WT dKO
} p
Ena/VASP proteins regulate activated T-cell 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 a Department 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)
T cell
| migration | cytoskeleton | extravasation
A
ctivated T-cell trafficking across the vascular endothelium is essential for ongoing immune surveillance of tissues and for 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 target for therapeutic immunomodulation. Disruption of these pathways, such as by antibody blockade of α4 integrins, is a highly effective approach to immunomodulation (1, 2). However, the molecular mechanisms by which chemokine receptor and adhesion molecule signaling induce the T-cell cytoskeletal machinery to promote extravasation are not yet fully elucidated. Transendothelial migration (TEM), the process by which T cells extravasate from the blood into tissues, is characterized by four distinct steps: rolling along the vascular wall, arrest or adhesion, intravascular crawling, and diapedesis across the endothelial barrier (3). Surface adhesion molecules play well-characterized roles in each step of the process. For example, the initial rolling step of TEM is facilitated by interactions between T-cell and endothelial selectins, whereas the adhesion, intravascular crawling, and diapedesis steps of TEM are mainly regulated by chemokineand shear force-stimulated modulation of lymphocyte functionassociated antigen 1 (LFA-1, αLβ2 integrin, CD11a/CD18) and very late antigen 4 (VLA-4, α4β1 integrin, CD49d/CD29) interactions with intracellular adhesion molecule 1 (ICAM-1) and vascular cellular adhesion molecule 1 (VCAM-1), respectively. Dynamic cyto-
www.pnas.org/cgi/doi/10.1073/pnas.1701886114
skeletal changes occur throughout the process of TEM (4, 5); however, the regulation of these cytoskeletal changes is not completely understood. The lymphocyte actin-myosin cytoskeleton is composed of networks of linear and branched actin filaments, cross-linked by class-II nonmuscle myosin. We have previously shown that inhibition of myosin-IIA, the main class-II myosin protein expressed in lymphocytes, alters T-cell trafficking, motility, and TEM (6–9). Although numerous studies have focused on the upstream regulatory signaling cascades that control actin network remodeling during migration (5, 10), less is understood about how downstream effectors of branched and linear actin polymerization might participate in lymphocyte TEM. Two major families of effector proteins of linear actin polymerization are expressed in lymphocytes. The Formin family, which is thought to nucleate new linear actin filament production, has been implicated in T-cell activation and egress from the thymus (11–13). Less is known about the Ena/ VASP (vasodilator-stimulated phosphoprotein) family of cytoskeletal effectors in T cells. The Ena/VASP family is composed of three members: mammalian-enabled (Mena), which is not typically expressed in hematopoietic cells; VASP; and Ena-VASP–like (EVL) (14, 15). These proteins coordinate monomeric actin recruitment to the barbed 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 an N-terminal EVH1 domain, which regulates cellular localization; Significance T-cell trafficking is essential for the function of the adaptive immune system, and regulation of T-cell entry into tissues can be an effective therapy in diseases such as autoimmunity. However, the mechanisms regulating T-cell migration and trafficking are poorly understood. We have identified a key role for Ena/VASP (vasodilator-stimulated phosphoprotein) family cytoskeletal effectors selectively in activated T-cell trafficking to secondary lymphoid 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 suggests that further studies of the Ena/VASP pathway in T cells could identify therapeutically useful ways to more selectively modulate 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. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1
To whom correspondence should be addressed. Email: [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1701886114/-/DCSupplemental.
PNAS | Published online March 20, 2017 | E2901–E2910
IMMUNOLOGY AND INFLAMMATION
Vasodilator-stimulated phosphoprotein (VASP) and Ena-VASP–like (EVL) are cytoskeletal effector proteins implicated in regulating cell 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 identifies a specific role for EVL and VASP in T-cell diapedesis and trafficking. We demonstrate that EVL and VASP are selectively required for activated T-cell trafficking but are not required for normal T-cell development or for naïve T-cell trafficking to lymph nodes and spleen. Using a model of multiple sclerosis, we show an impairment in trafficking of EVL/VASP-deficient activated T cells to the inflamed central nervous system of mice with experimental autoimmune encephalomyelitis. Additionally, we found a defect in trafficking of EVL/VASP double-knockout (dKO) T cells to the inflamed skin and secondary lymphoid organs. Deletion of EVL and VASP resulted in the impairment in α4 integrin (CD49d) expression and function. Unexpectedly, EVL/VASP dKO T cells did not exhibit alterations in shear-resistant adhesion to, or in crawling on, primary endothelial cells under physiologic shear forces. Instead, deletion of EVL and VASP impaired T-cell diapedesis. Furthermore, T-cell diapedesis became equivalent between control and EVL/VASP dKO T cells upon α4 integrin blockade. Overall, EVL and VASP selectively mediate activated T-cell trafficking by promoting the diapedesis step of transendothelial migration in a α4 integrin-dependent manner.
altered T-cell trafficking in vivo, we used EVL/VASP doubleknockout (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 that Mena was not up-regulated as a compensatory mechanism in EVL/ VASP dKO T cells by Western blot analysis (Fig. S1). We then analyzed if EVL/VASP deficiency caused defects in T-cell development. Flow cytometry analysis of lymphocyte populations in the thymus and secondary lymphoid organs showed no gross defects in T-cell development and normal proportions of mature CD4 and CD8 T cells in the periphery of EVL/VASP dKO mice (Fig. S2). Deletion of Both EVL and VASP Selectively Impairs Activated CD4 T-Cell Trafficking into Secondary Lymphoid Tissues. Next, we used
coadoptive transfers of WT control and EVL/VASP dKO T cells to study the T-cell–intrinsic effect of EVL and VASP deficiency on trafficking in vivo. Consistent with the normal pattern of development and homeostatic trafficking, dKO and WT naïve CD4 T cells had equivalent homing to the spleen and lymph nodes after intravenous 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 required for activated T-cell trafficking. We first established that, upon ex vivo polyclonal activation with CD3 and CD28 stimulation, T-cell proliferation kinetics and activation profiles were similar for WT and EVL/VASP dKO T cells (Fig. S3). Activated T cells can become lodged in the vasculature (particularly in the lungs) (37, 38), potentially because of their increased size and adhesion properties. Therefore, we used an established technique (39, 40) to distinguish extravasated T cells from those stuck intravascularly by injecting intravenously a fluorophoreconjugated anti-CD4 antibody immediately before euthanasia 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 of CCR7 and can recirculate to secondary lymphoid organs. To determine if homeostatic trafficking of activated T cells was affected by Ena/VASP deficiency, we cotransferred control and EVL/VASP dKO T cells into unimmunized recipient mice. Following intravenous adoptive transfer, activated dKO CD4 T cells on average E2902 | www.pnas.org/cgi/doi/10.1073/pnas.1701886114
Naive dKO:WT Ratio Normalized to Injected
1.5
ns
ns
2 hours in vivo
Inject anti-CD4 PE Ab IV( ) Euthanize
1.0
Perfuse with saline
0.5
Harvest tissues and analyze by flow cytometry
0.0
Blood Spleen
Intravascular Cells (CD4 PE+)
LN
D
Activated 2.0 ns
p=0.004 p=0.005
1.5 1.0 0.5 0.0
Blood Spleen
LN
E
Transferred cells # (x104)
C
F 1.5
ns
ns
ns
1.0
0.5
0.0
Blood Spleen
LN
VASP sKO:WT Ratio Normalized to Injected
Deletion of EVL and VASP Does Not Significantly Affect T-Cell Development. To determine if deletion of Ena/VASP proteins
Naive 2.0 p=0.03
WT and KO CD4+ T cells
B
Extravasated Cells (CD4 PE-)
Activated dKO:WT Ratio Normalized to Injected
Results
A
EVL sKO:WT Ratio Normalized to Injected
and a C-terminal EVH2 domain, which facilitates tetramerization, binds filamentous actin (F-actin), and is thought to be responsible for actin polymerization (21–24). Ena/VASP proteins are capable of compensating for deletion of one another, but there is some evidence that they are differentially regulated (15). EVL, and especially VASP, are implicated in the motility, adhesion, and sensory capacity of many cell types. EVL and VASP localize to filopodia tips (25) as well as to adhesive sites, such as fibroblast focal adhesions (24). This localization pattern is consistent with a role in migration and adhesion. Fibroblasts lacking EVL and VASP produce shorter filopodia, and a slower-moving lamellipodium, which paradoxically leads to enhanced fibroblast motility (26, 27). Platelets from VASP-knockout mice demonstrate enhanced 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 in T cells, showing that Ena/VASP proteins can contribute to actin remodeling during T-cell receptor (TCR) signaling (24, 30). However, previous studies did not focus on the role of EVL and VASP in T-cell development or T-cell motility. Therefore, we sought to elucidate the mechanisms by which EVL and VASP might influence T-cell adhesion, migration, and trafficking in vivo.
WT 50 40 30 20 10
ns
dKO
p=0.01
p=0.04
2 1 0
Blood Spleen
LN
1.5 ns
ns
ns
1.0
0.5
0.0
Blood Spleen
LN
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 trafficking is not affected by EVL and VASP deficiency. Differentially dyelabeled naïve WT and EVL/VASP dKO T cells were coadoptively transferred intravenously at a 1:1 ratio and T-cell trafficking to lymphoid tissues was quantified by flow cytometry 2 h after adoptive transfer. The dKO:WT ratio was normalized to the ratio in the injected sample to account for possible minor variations in the injection mixture (typically < 10%). (B) Experimental set-up for cotransfer of activated WT and KO cells, including intravascular staining method to distinguish intravascular T cells from those that have 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 and T-cell trafficking to tissues 2 h after adoptive transfer was quantified by flow cytometry as above. A ratio below 1.0 (horizontal red line) indicates impaired homing of KO T cells. (C) Quantification of activated T-cell trafficking. Ratio of extravasated WT and EVL/VASP dKO activated T cells, harvested and analyzed as above. (D) Number of WT and dKO T cells recovered from the indicated 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 average from a minimum of three independent experiments. Error bars are SEM. Statistics 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.
exhibited a 2.2-fold reduction in spleen trafficking and a 3.3-fold reduction in lymph node trafficking 2 h after adoptive transfer compared with WT controls (Fig. 1 C and D). This activated dKO T-cell trafficking defect still persisted 24 h posttransfer (Fig. S4 A and B). Furthermore, the intravascular staining used to quantify T cells that had entered tissues versus cells that remained adhered within Estin et al.
EVL and VASP Deletion Impairs Activated CD4 T-Cell Trafficking to Sites of Inflammation. We next sought to determine if activated
CD4 T-cell trafficking to sites of inflammation was affected by Ena/VASP protein deficiency. Because the vascular endothelial barrier in the central nervous system (CNS) is particularly restrictive, we first investigated EVL/VASP-deficient activated T-cell trafficking to the CNS in the context of autoimmune inflammation, using a mouse model of multiple sclerosis: experimental autoimmune encephalomyelitis (EAE). Twenty-four hours after adoptive transfer of T cells into mice with ongoing EAE, we quantified the number of transferred T cells that had extravasated into the CNS using the intravascular staining technique described above. Activated dKO T cells exhibited on average a 2.0-fold reduction in trafficking to the CNS relative to control T cells (Fig. 2 A–C). Next, we analyzed T-cell trafficking
Transfer activated T cells into EAE mice, 24h later euthanize and harvest tissues
E 0h
Inject LPS SC in ear
24h
Transfer T cells
48h
Euthanize and harvest tissues
dKO:WT Ratio Normalized to Injected
D
ns
p=0.01
1.0
0.5
0.0
Blood
CNS
ns
p=0.02
60 50 40 30 20 10
1.0
0.5
0.0
Blood
Skin
WT dKO
p=0.02 5 0
Blood
F 1500
1.5
ns
CNS
ns WT dKO
1000 500 10 8 6 4 2 0
p=0.01
cyte migration and trafficking (41, 42). Therefore, we analyzed if the trafficking defect of Ena/VASP-deficient activated T cells could be because of an altered ability to respond to chemokine stimulation. First, we measured the expression of chemokine receptors involved in both homeostatic and inflammatory trafficking. Our data showed no difference in the expression of CCR7, 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, chemokine stimulation can trigger actin network remodeling and promote motility (34). Therefore, we analyzed if Ena/VASP deficiency impaired actin polymerization in response to chemokine stimulation. To this end, we measured the F-actin content in control and EVL/VASP dKO T cells before and after chemokine stimulation in a time-course analysis. Our results showed that dKOactivated T cells had a small but significant defect in actin polymerization promoted by CCL21 stimulation and, to a lesser degree, by CXCL10 (Fig. 3B). However, when we analyzed actin 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. Based on this result, we then measured chemokine-stimulated migration using Transwell chambers. There were no significant differences in migration in the absence of chemokine, or in chemotaxis toward CCL21, CXCL10, CXCL12, or CCL5 in the lower chamber between control and EVL/VASP dKO-activated T cells (Fig. 3C). Furthermore, chemokinetic migration in response to CCL21 in both the upper and lower chambers was also unaffected (Fig. S5B). Although we detected slightly reduced chemokine-mediated actin polymerization in EVL/VASPdeficient activated T cells, overall these data showing normal chemotaxis suggest that the strong trafficking defect of EVL/ VASP dKO T cells in vivo is not explained by an overall defect in migratory capacity or chemokine sensing. Activated CD4 EVL/VASP dKO T Cells Are Deficient in α4 Integrin Expression and Function. Ena/VASP proteins are reported to af-
Blood
Skin
Fig. 2. EVL and VASP deletion inhibits activated CD4 T-cell trafficking to the CNS during EAE and to the inflamed skin. (A) Experimental set-up for cotransfer of WT and EVL/VASP dKO activated T cells into mice with ongoing EAE. Activated, dye-labeled polyclonal CD4 WT and dKO T cells were coadoptively transferred at a 1:1 ratio, and were harvested 24 h posttransfer from the blood and CNS (brain and spinal cord). (B) Activated T-cell trafficking during EAE was quantified by flow cytometry, shown as the ratio of dKO:WT T cells normalized to the ratio in the injected sample. (C) Number of WT and dKO T cells recovered from the indicated tissues from data in B. (D) Experimental set-up to quantify activated T-cell trafficking to the inflamed skin. Twenty-four hours after LPSinduced inflammation in the ears, WT and dKO activated T cells were coadoptively transferred at a 1:1 ratio, and were harvested 24 h posttransfer from the blood and ears of the recipient mice. (E) Ratio of dKO:WT T cells recovered from blood and ears, normalized to the ratio in the injected sample. (F) Number of WT and dKO T cells recovered from the indicated tissues from data in E. Data are the average of four independent experiments. Error bars are SEM. Statistics are onesample 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.
Estin et al.
Deletion of EVL and VASP Reduces Chemokine-Triggered Actin Polymerization but Does Not Impair Activated T-Cell Chemotaxis in Vitro. Chemokine signaling is instrumental in mediating leuko-
fect adhesion and integrin function (28, 43). Therefore, having ruled-out impaired chemotaxis, we next examined the expression and function of key integrins involved in extravasation to determine if the trafficking impairment we observed was because of integrin defects. Flow cytometry analysis revealed that activated EVL/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-experienced T cells, which may explain why only activated dKO T cells exhibited a trafficking defect in vivo. Consistent with their normal trafficking phenotype, activated EVL or VASP single-knockout T cells did not exhibit reduced CD49d expression, nor did naïve dKO T cells (which only expressed negligible levels of CD49d) (Fig. S6 A–E). Additionally, expression of the α4 integrin binding partners β1 (CD29) and β7 in activated cells was also reduced in activated dKO T cells compared with WT T cells (Fig. S6F). Furthermore, total expression versus surface expression of CD11a PNAS | Published online March 20, 2017 | E2903
IMMUNOLOGY AND INFLAMMATION
Day 14-20
Mice begin to develop symptoms
C
1.5
Transferred cells # (x104)
Start scoring mice
B
Transferred cells # (x102)
Day 7 Day 10-14
EAE Scoring
Day 2
Inject MOG35-55 SC & PTX IP PTX IP
dKO:WT Ratio Normalized to Injected
A Day 0
to the inflamed skin, using lipopolysaccharide (LPS) as the inflammatory stimulus. Recipient mice were treated by subcutaneous LPS injections in the ear and 24 h later activated control and dKO T cells were intravenously transferred into the recipient mice. Quantification of transferred T cells that had extravasated into the inflamed skin of the ear showed a 1.8-fold reduction in dKO T-cell trafficking to this site (Fig. 2 D–F).
PNAS PLUS
blood vessels also indicated that the defect in activated EVL/VASP dKO T-cell trafficking was not a result of selective trapping of these cells in the lung microvasculature, the first capillary bed encountered after intravenous adoptive transfer. In fact, significantly more WT than dKO T cells were recovered from inside the lung microvasculature (Fig. S4C). In keeping with these data, there is a trend toward more EVL/VASP dKO T cells than control cells in the blood compartment, suggesting that these cells may be unable to gain 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 secondary lymphoid organs. Consistent with the likelihood of redundant or compensatory functions between Ena/VASP proteins, singleknockout of EVL or VASP did not give rise to activated T-cell trafficking defects (Fig. 1 E and F).
50
WT dKO
} p
