The β2 integrin-kindlin-3 interaction is essential for T-cell homing but dispensable for T-cell activation in vivo - PubMed (original) (raw)

The β2 integrin-kindlin-3 interaction is essential for T-cell homing but dispensable for T-cell activation in vivo

Vicky Louise Morrison et al. Blood. 2013.

Abstract

Kindlin-3 is mutated in the rare genetic disorder, leukocyte adhesion deficiency type III, which is characterized by deficient integrin-mediated adhesion of leukocytes and platelets. However, the specific roles of kindlin-3-β2-integrin interactions in T-cell adhesion and homing and immune responses in vivo remain unclear. Here, we show that the TTT motif in β2 integrins controls kindlin-3 binding. Mutation of the kindlin-3 binding site in β2 integrins caused a loss of firm adhesion of T cells under both static and shear flow conditions and a reduction of T-cell homing to lymph nodes in vivo. However, atomic force microscopy studies of integrin-ligand bonds revealed that initial ligand binding could still occur, and 2-dimensional T-cell migration was reduced but not abolished by the TTT/AAA mutation in the β2 integrin. Importantly, dendritic cell-mediated T-cell activation in vivo was normal in TTT/AAA β2 integrin knock-in mice. Our results reveal a selective role of the kindlin-3-integrin association for lymphocyte functions in vivo; the integrin-kindlin-3 interaction is particularly important in adhesion strengthening under shear flow, and for T-cell homing to lymph nodes, but dispensable for T cell activation which occurs in a shear-free environment.

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Figures

Figure 1

Figure 1

The TTT motif in the β2 integrin tail mediates kindlin-3 binding. (A) The interaction between human WT β2 integrin (TTT, left) or mutant β2 integrin (AAA, right) with human kindlin-3 was measured in transfected HEK293 cells using FRET. In each case, representative graphs are shown with 3 measurements before and 3 measurements after acceptor photo-bleaching. Error bars indicate standard deviation. (B) FRET results of the β2–kindlin-3 interaction are summarized as percentage FRET efficiency (N = 23 cells, mean ± SEM). (C) The interaction of talin with WT-β2-integrin or TTT/AAA β2-integrin was assessed by GST pulldowns with GST alone, GST-WT-β2-integrin (WT), and GST-TTT/AAA-β2-integrin (TTT/AAA) from T-cell lysates followed by immunoblotting with an antitalin antibody. Lys, T cell lysate. Experiment is representative of N = 2.

Figure 2

Figure 2

Generation and phenotype of β2TTT/AAA integrin knock-in mice. (A) Arrangement of the murine Itgb2 genomic locus (top), targeting vector (second from top), targeted Itgb2 allele after homologous recombination (second from bottom), and the constitutively expressed Itgb2 knock-in allele after Flp recombination (bottom). F3–Flp recombination target site. (B) The size of secondary lymphoid tissues (spleen, inguinal lymph nodes, and mesenteric lymph nodes) in 6- to 10-week-old WT and β2TTT/AAA integrin knock-in (KI) homozygote mice, presented as cell number (N = 5 mice). (C) Proportions of B cells and CD4 and CD8 T cells in the spleen and inguinal lymph nodes of 6- to 10-week-old WT and KI mice (N = 3). (D) Circulating neutrophil numbers were identified by forward/side scatter profiles, with representative plots (left) and pooled data from 6 mice (right). (E) Gr-1 staining of blood samples to identify circulating neutrophils (N = 6 mice). (F) Splenic regulatory T cells were identified by intracellular Foxp3 staining (N = 6 mice). In all cases, Student t test was used to calculate significance values. NS, not significant. In all cases, mean ± SEM are presented.

Figure 3

Figure 3

β2TTT/AAA integrin knock-in T cells display impaired adhesion. (A) Adhesion of WT and KI-naïve CD4 T cells to ICAM-1 under static conditions. Cells were unstimulated, or stimulated with 200 nM PDBu or 10 μg/mL anti-CD3 (N = 4). (B) SDF-1–induced adhesion of naïve CD4 T cells to ICAM-1 under shear flow conditions (N = 4). (C) The expression of αL (left) and β2 (right) integrin subunits in WT (solid line) and KI (dashed line)-naïve CD4 T cells ex vivo (top) and cultured effector CD4 T cells (bottom). Plots are representative of N = 3 to 5. Shaded line represents the isotype control. (D) Kindlin-3, β2 integrin and 14-3-3 protein levels in WT and KI effector CD4 T cells, with and without stimulation with 200 nM PdBu. N = 1 is shown as representative of N = 2. (E) β2 integrin mRNA levels in WT and KI-naïve and effector CD4 T cells were determined by reverse-transciptase quantitative PCR (N = 4). (F) Effector CD4 T-cell adhesion to ICAM-1 under static conditions, using unstimulated, PDBu– or anti-CD3–stimulated cells (N = 5). (G) Effector CD4 T-cell adhesion to ICAM-1 under shear flow conditions (N = 4). (H) Effector CD4 T-cell adhesion to bEnd.3 cells under shear flow, after 10 minutes of adhesion (N = 3). (I) Rolling rates of effector CD4 T cells on ICAM-1 under shear flow (N = 3). (J) _p_-PLCγ and Src levels in effector CD4 T cells were determined by Western blot analysis. N = 2 is shown and is representative of N = 4. Student t test was used to calculate significance values in panels A,E-F,H-I. Two-way analysis of variance was performed in panels B,G. NS, not significant. In all cases, mean ± SEM is shown.

Figure 4

Figure 4

Loss of β2–kindlin-3 interactions in CD4 T cells results in impaired adhesion strengthening and impaired migration on ICAM-1 in vitro. (A) AFM measurements of the forces required to detach WT and KI effector CD4 T cells from an ICAM-1–coated surface, presented as maximum unbinding force. The control readings (Ctr) indicate WT cell adhesion to bare plastic, and WT cell adhesion to ICAM-1 in the presence of EDTA. At all contact times, adhesion of both WT and knock-in cells to ICAM-1 was significantly above binding to bare plastic or ICAM-1 in the presence of EDTA. N = 50 individual T cells, with mean ± SEM. (B) Still images of WT and KI effector CD4 T cells plated onto ICAM-1–coated surfaces. Scale bars represent 50 μm. Images are representative of cells from N = 8 mice. (C) WT and KI effector CD4 T-cell spreading on ICAM-1 was quantified in terms of area, longest axis, and perimeter. Cells are from N = 3 mice. (D) Effector CD4 T-cell 2-dimensional migration on ICAM-1 was viewed using time-lapse microscopy over a 15-minute period and videos analyzed to quantify the parameters indicated. Cells are from N = 8 mice, with mean ± SEM. Student t test was used to calculate significance values. NS, not significant.

Figure 5

Figure 5

Loss of β2–kindlin-3 interactions in CD4 T cells results in altered homing in vivo. (A) Expression levels of the adhesion molecules PSGL-1, CD43, β1 integrin, CD44, and CD62L in WT (solid line) and KI (dashed line)-naïve CD4 T cells. (B) Naïve CD4 T cells were isolated from WT and KI mice, labeled with CFSE and CellTrace Violet, respectively, and mixed at a 1:1 ratio. (C) After adoptive transfer into recipient WT mice, the localization of WT and KI donor cells in the organs indicated was analyzed 16 hours post transfer. Plotted values indicate the recovery of WT or knock-in donor cells as a percentage of total recovered cells. Data are pooled from 2 independent experiments: 5 mice per group per experiment. Each data point represents an individual mouse, with the mean indicated. The dashed line shows 1:1 recovery of WT and KI T cells. Deviation from the dashed line indicates differential homing of WT and KI cells. The minimal, but significant, defect in KI T-cell homing to the Peyer’s patches likely reflects the involvement of other integrins in access to gut-associated lymphoid tissue. Student t test was used to calculate P values. NS, not significant.

Figure 6

Figure 6

β2TTT/AAA integrin knock-in CD4 T-cell activation in vitro and in vivo is normal. (A) Purified naïve CD4 T cells were activated in vitro using soluble anti-CD3 (2.5 μg/mL) plus IL-2 (20 ng/mL), plate-bound anti-CD3 (2.5 μg/mL at 37°C for 4 hours), or phorbol ester (PdBu, 50 nM). WT (solid line) and KI (dashed line) T-cell activation was measured 24 hours later by expression of the typical activation markers: CD69, CD44, CD25, and CD62L. Histograms are representative of N = 4. (B) IL-2 production by WT and KI T cells after 24-hour stimulation with plate-bound anti-CD3 or PdBu. Data are pooled from N = 4, with mean ± SEM displayed. (C) WT and KI CD4 T cells were labeled with CFSE before activation with soluble anti-CD3 plus IL-2. Proliferation was measured by flow cytometry. Data are pooled from N = 2 and are representative of N = 4. (D) WT and KI mice received an intravenous adoptive transfer of LPS-matured, peptide-loaded WT DCs, and the antigen-specific CD4 T-cell response in the spleen was measured using MHC class II tetramers 7 days later. Data are pooled from 2 independent experiments, with 5 mice per group per experiment. Each data point represents an individual mouse, with mean indicated. The control group received no adoptive transfer. Isotype shows staining of the WT immunization group with an irrelevant peptide-tetramer. Student t test was used to calculate P values. NS, not significant.

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