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

Abstract

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.

Keywords: T cell; cytoskeleton; extravasation; migration.

Fig. S1.

EVL/VASP dKO T cells do not express EVL, VASP, or Mena proteins. T-cell lysates were obtained from activated T cells 5–6 d postactivation. (A) EVL protein is expressed in WT T cells, but is not expressed in EVL/VASP dKO T cells. (B) VASP protein is expressed in WT T cells, but is not expressed in EVL/VASP dKO T cells. (C) Compared with WT T cells, Mena protein levels do not increase in EVL/VASP dKO cells to compensate for deletion of EVL and VASP. Brain lysates and PC12 cells were used as positive controls for Mena expression. Data are representative of at least two independent experiments.

Fig. S2.

T-cell development and lymphocyte populations in peripheral lymphoid organs are not altered in EVL/VASP dKO mice. (A and B) Thymus CD4, CD8, and double-positive T-cell fractions are maintained in EVL/VASP dKO mice both by frequency and by number. (C and D) Spleen CD4, CD8, and B-cell populations are equivalent in EVL/VASP dKO and WT mice, both by frequency and by number. (E and F) Peripheral lymph node CD4, CD8, and B-cell populations are not reduced in EVL/VASP dKO mice, both by frequency and by number. In C and E the red squares highlight the gates used for the Lower panels. Data in A, C, and E are representative of five independent experiments; data in B, D, and F are the mean of five independent experiments. Error bars are SEM; statistics are t tests. ns, not significant.

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 dye-labeled 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.

Fig. S3.

Polyclonal CD3-induced T-cell activation is not affected by deletion of EVL and VASP. (A) Time-course of the fold-increase in cell numbers after T-cell activation by anti-CD3 and anti-CD28 of EVL/VASP dKO T cells. (B) Representative CD69 expression on WT and EVL/VASP dKO T cells 48 h after activation. (C) Representative CFSE dilutions of WT and EVL/VASP dKO T cells 48 h after activation. Data in A are the mean of seven independent experiments (error bars are SEM); data in B and C are representative of two independent experiments.

Fig. S4.

Impairment in activated EVL/VASP dKO T-cell trafficking to peripheral lymphoid organs is maintained 24 h posttransfer, and does not result from trapping in the lung. (A) Trafficking of activated WT and EVL/VASP dKO T cells in peripheral lymphoid tissues harvested 24 h after T-cell transfer, expressed as a ratio and normalized to the ratio in the injected sample. A ratio below 1.0 (red line) indicates impaired homing of dKO T cells. (B) Number of WT and dKO T cells recovered from the indicated tissues from data in A. (C) Activated WT and EVL/VASP dKO T cells trapped in the blood vessels of the lung 2 h after transfer (measured by intravascular staining). Data expressed as a ratio and normalized to the ratio in the injected sample. Data are the mean of four independent experiments; error bars are SEM. Statistics in A and C are one-sample t test compared with a hypothetical value of 1.0; statistics in B are paired t tests. ns, not significant.

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 LPS-induced 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 one-sample t test compared with a hypothetical value of 1.0 (B, E) or paired t test (C, F). IP, intraperitoneal; ns, not significant; SC, subcutaneous.

Fig. 3.

Deletion of both EVL and VASP in activated CD4 T cells reduces chemokine-triggered actin polymerization but does not impair chemotaxis. (A) Quantification of chemokine receptor expression in WT and EVL/VASP dKO activated T cells; data shown as gMFI. (B) Time-course analysis of actin polymerization in WT and dKO activated T cells in response to 100 ng/mL CCL21 stimulation (WT vs. dKO curve comparison P < 0.0001) or 100 ng/mL CXCL10 stimulation (WT vs. dKO curve comparison P = 0.032), measured by flow cytometry quantification of fluorescent phalloidin staining. (C) Chemotactic 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 are the average of at least three independent experiments; error bars are SEM; statistics are paired t tests in A and C, or two-way ANOVA in B. ns, not significant.

Fig. S5.

Normal chemokine-induced actin polymerization in naïve EVL/VASP dKO T cells, and normal chemokinesis and chemotaxis of EVL/VASP dKO activated T cells. (A) Time-course analysis of actin polymerization in WT and dKO naïve T cells in response to 100 ng/mL CCL21 stimulation (WT vs. dKO curve comparison not significant), measured by flow cytometry quantification of fluorescent phalloidin staining. (B) Activated T-cell migration across 5-μm pore Transwell chambers, either in the absence of chemokine or with CCL21 (1 μg/mL) both in the upper and lower wells (chemokinesis), or with CCL21 (1 μg/mL) only in the lower well (chemotaxis). Data are the mean of three independent experiments; error bars are SEM. Statistics in A are two-way ANOVA, statistics in B are paired t tests. ns, not significant.

Fig. 4.

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

Fig. S6.

Integrin expression profiles on activated and naïve EVL and VASP KO T cells. (A) Examples of CD11a and CD49d expression by flow cytometry in activated VASP sKO T cells. (B) Quantification of CD11a and CD49d expression in activated VASP sKO T cells. (C) Examples of CD11a and CD49d expression by flow cytometry in activated EVL sKO T cells. (D) Quantification of CD11a and CD49d expression in activated EVL sKO T cells. (E) Quantification of expression of CD49d in naïve WT and EVL/VASP dKO T cells by flow cytometry. (F) Quantification of CD29 (β1 integrin) and β7 integrin expression in activated WT and EVL/VASP dKO T cells, shown as the percentage of expression in dKO T cells relative to WT T cells by gMFI. (G) Quantification of CD11a expression in naïve WT and EVL/VASP dKO T cells by flow cytometry. (H) Surface expression and total expression (measured by staining after permeabilization) of CD11a in activated T cells, shown as the percentage of expression in dKO relative to WT T cells by gMFI. (I) Surface expression and total expression (measured by staining after permeabilization) of CD49d in activated T cells, shown as the percentage of expression in dKO T cells relative to WT T cells by gMFI. Data in A and C are representative of three independent experiments; data in B and D–I are the mean of at least three independent experiments. Error bars are SEM; statistics are paired t tests except in F, which are one-sample t test compared with a hypothetical value of 100. ns, not significant.

Fig. 5.

EVL/VASP dKO activated CD4 T cells are competent in shear-resistant adhesion and migration. (A) Schematic of the experimental set-up for measuring T-cell adhesion and motility under physiologic shear forces. (B and C) WT and EVL/VASP dKO T-cell shear-resistant adhesion to ICAM-1 (B), or to VCAM-1 (C). (D and E) Mean WT and dKO T-cell crawling speed under flow on ICAM-1 in the presence of CCL21 (D) or on VCAM-1 in the presence of CCL21 (E). (F) Quantification of WT and dKO T-cell shear-resistant adhesion to TNF-α–activated primary microvascular brain endothelial cells in the presence of CCL21. (G) Detachment of initially adhered T cells from activated endothelial monolayers after shear flow increase from 0.2 to 2 dyne/cm². (H) Hyperadhesiveness of WT and dKO T cells to primary brain endothelial 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 endothelial monolayers. (J) Percentage of in vivo adoptively transferred WT and dKO T cells recovered from lymph nodes that remained in the vasculature 2 h posttransfer (identified by intravascular staining). (K) Percentage of transferred T cells recovered from the CNS that remained intravascular, 24 h posttransfer. Data represent the average of a minimum of three independent experiments; error bars are SEM; P values are paired t tests. ns, not significant.

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 imaged by 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.