Kindlin-3 regulates integrin activation and adhesion reinforcement of effector T cells

Abstract

Activated T cells use very late antigen-4/α4β1 integrin for capture, rolling on, and firm adhesion to endothelial cells, and use leukocyte function-associated antigen-1/αLβ2 integrin for subsequent crawling and extravasation. Inhibition of α4β1 is sufficient to prevent extravasation of activated T cells and is successfully used to combat autoimmune diseases, such as multiple sclerosis. Here we show that effector T cells lacking the integrin activator Kindlin-3 extravasate and induce experimental autoimmune encephalomyelitis in mice immunized with autoantigen. In sharp contrast, adoptively transferred autoreactive T cells from Kindlin-3-deficient mice fail to extravasate into the naïve CNS. Mechanistically, autoreactive Kindlin-3-null T cells extravasate when the CNS is inflamed and the brain microvasculature expresses high levels of integrin ligands. Flow chamber assays under physiological shear conditions confirmed that Kindlin-3-null effector T cells adhere to high concentrations of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, albeit less efficiently than WT T cells. Although these arrested T cells polarize and start crawling, only few remain firmly adherent over time. Our data demonstrate that the requirement of Kindlin-3 for effector T cells to induce α4β1 and αLβ2 integrin ligand binding and stabilization of integrin-ligand bonds is critical when integrin ligand levels are low, but of less importance when integrin ligand levels are high.

Keywords: EAE; integrin affinity.

Fig. 1.

aEAE in mice lacking Kindlin-3 expression in T cells. (A) Median day of disease onset in Kindlin-3^fl/fl/CD4Cre⁺ (K3/Cre) and control (Co) mice with aEAE. (B and C) Relative weight normalized to day 0 (B) and clinical disease score (C) of mice with aEAE. Data points indicate means of 16 Co mice and 9 K3/Cre mice from two independent experiments. (D) Healthy control (PBS-treated) and diseased (MOG peptide-treated) mice with ongoing aEAE (clinical disease score 4) were killed, and spinal cord white matter was analyzed by immunostaining. Infiltrating leukocytes were stained with anti-CD45, anti-CD4, anti-Mac-1, or anti-Gr-1 Abs (green); blood vessels were stained with a pan-laminin Ab (red); and nuclei were stained with DAPI (blue). (Scale bar: 200 μm.) (E) Western blot analysis of protein lysates of FACS-sorted CD4⁺ cells from the CNS of K3/Cre and Co mice with ongoing aEAE. GAPDH served as a loading control.

Fig. 2.

Adoptively transferred autoreactive Kindlin-3–deficient T cells fail to induce EAE. (A) FACS analysis of in vitro-generated autoreactive T cells. Anti-CD4 and anti-CD8 Abs were used to identify CD4⁺ T helper cells (Left), anti-Vα3.2 and anti-Vβ11 were used to identify 2D2⁺ cells (Center), and anti-CD62L and anti-CD44 were used to identify memory (CD62L^hi/CD44^hi) and effector (CD62L^low/CD44^hi) T cells (Right). (B) Western blot analysis of in vitro-primed and MACS-sorted Kindlin-3^fl/fl/2D2⁺/CD4Cre⁺ (K3/Cre^2D2) and control Kindlin-3^fl/fl/2D2 (Co^2D2) T cells. GAPDH served as a loading control. (C and D) Relative weight normalized to day 0 (C) and clinical disease score (D) of naïve WT C57BL/6 mice injected with encephalitogenic K3/Cre^2D2 and Co^2D2 T cells. Data points represent means from three independent Co^2D2 (n = 13) and K3/Cre^2D2 (n = 20) T-cell transfer experiments. (E) Immunostaining of the lumbar spinal cord white matter from C57BL/6 mice injected with either PBS or in vitro-generated encephalitogenic Co^2D2 or K3/Cre^2D2 T cells. Infiltrating lymphocytes were stained with anti-CD4 Ab (green), blood vessels were stained with a pan-laminin Ab (red), and nuclei were stained with DAPI (blue). (Scale bar: 200 μm.)

Fig. 3.

Kindlin-3 controls firm adhesion of T cells to VCAM-1 and ICAM-1. (A and B) Spinal cords from untreated and pertussis toxin (PT)-treated animals were stained with anti–VCAM-1 (A) and anti–ICAM-1 (B) Abs (green). Blood vessels were stained with PECAM-1 Ab (red), and nuclei were stained with DAPI (blue). (Scale bar: 200 μm.) (C and D) Autoreactive CFSE⁺-labeled (green) Co^2D2 T cells (C) and K3/Cre^2D2 T cells (D) injected into recipient C57BL/6 animals with ongoing aEAE (clinical disease score 2). Sections from lumbar spinal cords were stained with an anti-CD4 Ab (red) to identify infiltrating T lymphocytes. Blood vessels were stained with a pan-laminin Ab (blue). Arrowheads indicate CSFE⁺ CD4⁺ T cells within or in close proximity to blood vessels, and arrows indicate peripheral T cells in the parenchyma or meninges. (Scale bar: 100 μm.) (E) Numbers of CFSE⁺-labeled Co^2D2 and K3/Cre^2D2 T cells in the lumbar region of the spinal cord at 18 h after transfer into mice with aEAE (clinical disease score 2). (F) Number of T cells arrested on surfaces coated with 100 nM rmVCAM-1 (n = 4 movies for Co; 4 movies for K3/Cre) per FOV. (G) Number of arrested T cells determined on surfaces coated with 100 nM (n = 4; 4), 40 nM (n = 4; 4), or 10 nM (n = 4; 4) rmVCAM-1, expressed as percent of arrested T cells on 100 nM VCAM-1. (H) Numbers of attached T cells determined at 5, 10 and 15 min of laminar flow at 1.5 dyn/cm², expressed as percent of arrested T cells determined at 15 s after shear increase. (I) Number of T cells arrested on surfaces coated with 100 nM rmICAM-1 (n = 4; 4). (J) Percentage of arrested T cells with crawling activity on 100 nM rmICAM-1 (n = 3; 3). (K) Number of arrested T cells determined on surfaces coated with 100 nM (n = 4; 4), 40 nM (n = 4; 4), and 10 nM (n = 4; 4) rmICAM-1 and expressed as percent of arrested T cells on 100 nM ICAM-1. (L) Percentage of firmly attached T cells on 100 nM (n = 3; 3) rmICAM-1. Numbers of attached T cells were determined at 5, 10, and 15 min of laminar flow at 1.5 dyn/cm² and expressed as percent of arrested T cells determined at 15 s after shear increase. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant. Data are mean ± SD.

Fig. 4.

Kindlin-3 promotes firm adhesion of T cells to primary brain microvascular endothelial cells. T-cell crawling and firm arrest on WT pMBMECs was analyzed by live cell imaging in a flow chamber experimental setup. (A) Numbers of arrested and firmly adherent T cells per FOV on WT pMBMECs determined 15 s, 5 min, and 10 min after onset of a 0.7-dyn/cm² shear (n = 7; 8). (B) Arrested K3/Cre^2D2 T cells are more roundish and less polarized compared with Co^2D2 T cells on WT pMBMECs. Arrows indicate stationary and more roundish cells, and arrowheads indicate polarized and migratory cells. (C) Mean cell lengths of Co^2D2 and K3/Cre^2D2 T cells (n = 105; 110). (D) Percentage of arrested (determined at 15 s after shear enhancement) Co^2D2 and K3/Cre^2D2 T cells with crawling activity on WT pMBMECs (n = 3 movies for Co; 3 movies for K3/Cre). (E) Percentage of arrested T cells able to resist 10 min of shear of 0.7 dyn/cm² on pMBMECs (n = 3; 3). (F) Number of arrested T cells per FOV on ICAM-1/-2 double KO pMBMECs at 15 s after onset of a 0.7-dyn/cm² shear (n = 4; 4). (G) Percentage of arrested T cells from ICAM-1/-2 double KO pMBMECs after 10 min of laminar flow at 0.7 dyn/cm² (n = 4; 4). *P < 0.05; **P < 0.01; ***P < 0.001. NS, not significant. Data are mean ± SD.