Wiskott-Aldrich syndrome protein controls antigen-presenting cell-driven CD4+ T-cell motility by regulating adhesion to intercellular adhesion molecule-1

Abstract

T-cell scanning for antigen-presenting cells (APC) is a finely tuned process. Whereas non-cognate APC trigger T-cell motility via chemokines and intercellular adhesion molecule-1 (ICAM-1), cognate APC deliver a stop signal resulting from antigen recognition. We tested in vitro the contribution of the actin cytoskeleton regulator Wiskott-Aldrich syndrome protein (WASP) to the scanning activity of primary human CD4(+) T cells. WASP knock-down resulted in increased T-cell motility upon encounter with non-cognate dendritic cells or B cells and reduced capacity to stop following antigen recognition. The high motility of WASP-deficient T cells was accompanied by a diminished ability to round up and to stabilize pauses. WASP-deficient T cells migrated in a normal proportion towards CXCL12, CCL19 and CCL21, but displayed an increased adhesion and elongation on ICAM-1. The elongated morphology of WASP-deficient T cells was related to a reduced confinement of high-affinity lymphocyte function-associated antigen 1 to the mid-cell zone. Our data therefore indicate that WASP controls CD4(+) T-cell motility upon APC encounter by regulating lymphocyte function-associated antigen 1 spatial distribution.

Figure 1

Short hairpin (sh) RNA-mediated Wiskott–Aldrich syndrome protein (WASP) knock-down in primary human CD4⁺ T cells. Representative example of CD4⁺ T cells transduced with lentiviral vectors encoding green fluorescent protein (GFP) and either shControl or shWASP. (a) Untransduced and GFP-sorted shControl and shWASP CD4⁺ T cells were analysed for GFP and WASP expression by flow cytometry. (b) Untransduced and GFP-sorted shControl and shWASP CD4⁺ T cells were analysed for WASP expression by Western blot. (c) Anti-CD3-induced proliferation of untransduced and GFP-sorted shControl and shWASP CD4⁺ T cells was measured by [³H]thymidine incorporation. Data correspond to the mean ± SEM of three replicates. *P < 0·05; **P < 0·01; ***P < 0·001 unpaired t test.

Figure 2

Wiskott–Aldrich syndrome protein (WASP) -deficient CD4⁺ T cells are hyper-motile at the contact with JY cells. (a) short hairpin (sh) Control or shWASP CD4⁺ T cells were seeded in the upper chamber of a Transwell with or without fixed JY cells in the absence of antigen at a 1 : 2 JY cell to T-cell ratio. Percentage of transmigrating cells was calculated as the fraction of CD4⁺ T cells that migrated to the lower chambers, filled with serum-free medium, within 3 hr. The figure represents the mean ± SEM of 13 independent experiments performed with a total of six donors. (b) The shControl or shWASP CD4⁺ T cells were incubated with fixed JY cells pulsed with increasing concentrations of TSST-1 superantigen. Percentage of transmigrating cells was calculated as in (a). Data correspond to the mean ± SEM of three independent experiments performed with two donors. (c) Either shControl or shWASP CD4⁺ T cells were cultured in serum-free medium over 6 days with or without immature dendritic cells. T-cell mortality was assessed by measuring 7-AAD-positive cells. Data correspond to the mean ± SEM of five independent experiments performed with four donors. *P < 0·05; **P < 0·01 paired t test.

Figure 3

Upon contact with non-cognate immature dendritic cells (iDC), Wiskott–Aldrich syndrome protein (WASP) -deficient CD4⁺ T cells adopt a hyper-motile behaviour with reduced pause. Short hairpin (sh) Control and shWASP CD4⁺ T cells alone or with autologous iDC were recorded during 120 min with a time lapse of 46·5 seconds. (a) Motility tracks of 20 individual T cells and corresponding zooms centred around the starting location. Tracks of fast (mean velocity > 1·8 μm/min) and slow (mean velocity < 1·8 μm/min) migrating T cells are shown in red and black, respectively. (b) Mean velocity, accumulated distance and maximum distance from origin of 20 individual shControl and shWASP T cells are shown. (c) Fluctuation of instantaneous velocity over 120 min of one representative shControl T cell and one representative shWASP T cell. Corresponding analysis of total time spent in pause over 120 min of 20 individual shControl and shWASP T cells. Data are from one representative experiment out of four experiments performed with two donors. ***P < 0·001 unpaired t test.

Figure 4

Wiskott–Aldrich syndrome protein (WASP) -deficient CD4⁺ T cells migrate in a normal proportion toward CXCL12, CCL19 and CCL21. (a) Short hairpin (sh) Control and shWASP CD4⁺ T cells treated or not with pertussis toxin were video recorded, during 120 min with a time lapse of 46·5 seconds, upon contact with JY cells and the mean velocity of 20 individual T cells was measured. Results show the mean of two experiments performed with two donors. (b) CXCL12 and CCL19 expression on immature dendritic cells (iDC) and JY cells. Staining with corresponding isotype controls is shown in grey. (c) CXCR4 and CCR7 expression on untransduced, shControl and shWASP CD4⁺ T cells. Staining with corresponding isotype controls is shown in grey. (d) Untransduced, shControl or shWASP CD4⁺ T cells were seeded in the upper chamber of a Transwell, and the percentage of migrating cells was calculated as the fraction of CD4⁺ T cells that migrated to the lower chamber, filled with the indicated chemokine, within 2 hr. Data correspond to the mean ± SEM of three independent experiments performed with three donors. No statistical difference was revealed by paired t-test.

Figure 5

Wiskott–Aldrich syndrome protein (WASP) -deficient CD4⁺ T cells display an increased adhesion on intercellular adhesion molecule 1 (ICAM-1). (a) Short hairpin (sh) Control and shWASP CD4⁺ T cells treated or not with blocking anti-lymphocyte function-associated antigen 1 (LFA-1) antibody were video recorded, during 120 min with a time lapse of 46·5 seconds, upon contact with JY cells and the mean velocity of 20 individual T cells was measured. Results show the mean of three experiments performed with three donors. (b) ICAM-1 expression on immature dendritic cells (iDC) and JY cells. Staining with corresponding isotype control is shown in grey. (c) LFA-1 expression under its total (HI111), intermediate-affinity (KIM127) and high-affinity (24) conformations on shControl and shWASP CD4⁺ T cells either unstimulated or stimulated with CXCL12. Staining with corresponding isotype controls is shown in grey. (d) KIM127 and 24 basal geometric mean fluorescence intensity (MFI) values and MFI fold increase upon CXCL12 stimulation of three independent experiments performed with five donors are shown. No statistical difference was revealed by paired t-test. (e) Percentage of adhesive shControl and shWASP CD4⁺ T cells on ICAM-1-coated slides. Data correspond to the mean ± SEM of four independent experiments performed with three donors. A total number of 265 shControl and 265 shWASP CD4⁺ T cells were counted. *P < 0·05 Mann–Whitney U-test.

Figure 6

Wiskott–Aldrich syndrome protein (WASP) -deficient CD4⁺ T cells elongate on intercellular adhesion molecule 1 (ICAM-1) and immature dendritic cells (iDC). (a) Representative images of green fluorescent protein-positive (GFP⁺) short hairpin (sh) Control and shWASP CD4⁺ T cells adhering to ICAM-1. Roundness coefficient of 83 shControl and 83 shWASP CD4⁺ T cells upon adhesion to ICAM-1. Data are from two independent experiments performed with two donors. ***P < 0·001 unpaired t-test. (b) Representative images of GFP⁺ shControl and shWASP CD4⁺ T cells in conjugation with ICAM-1-coated beads and immunostained for WASP and F-actin. Percentage of T cells displaying three distinct morphological types defined as follows are represented: type 1: spherical cells with polymerized actin surrounding the bead; type 2: elongated cells; type 3: protruded actin-rich structures away from the bead contact site. Data represent mean ± SEM of two experiments performed with three donors. (c) Movie sequences of shControl and shWASP T cells at the contact of iDC; white arrows indicate cells that round up and red arrows indicate adherent uropods. Results are representative of four experiments performed.

Figure 7

Adhesive Wiskott–Aldrich syndrome protein (WASP) -deficient CD4⁺ T cells display an abnormal localization of high-affinity lymphocyte function-associated antigen 1 (LFA-1). (a) One representative image of green fluorescent protein-positive (GFP⁺) short hairpin (sh) Control and shWASP CD4⁺ T cells adherent to intercellular adhesion molecule 1 (ICAM-1), immunostained for WASP, F-actin, and KIM127 (upper panel) or 24 (lower panel). (b) KIM127 and 24 monoclonal antibody fluorescence intensity profiles, from the leading edge to the tip of the uropod. Mean fluorescence intensity of 9–13 individual T cells is represented in red. The black bars define the cell regions that were considered for area under the curve calculations. No statistical difference was found between the KIM127 staining of shControl and shWASP CD4⁺ T cells. The 24 staining at the mid-body was reduced in shWASP CD4⁺ T cells compared with shControl CD4⁺ T cells (P = 0·025 unpaired t-test). Data are from one representative experiment out of three experiments performed with three donors.