LFA-1 activates focal adhesion kinases FAK1/PYK2 to generate LAT-GRB2-SKAP1 complexes that terminate T-cell conjugate formation

Abstract

Lymphocyte function-associated antigen 1 (LFA-1) affinity and avidity changes have been assumed to mediate adhesion to intercellular adhesion molecule-1 for T-cell conjugation to dendritic cells (DC). Although the T-cell receptor (TCR) and LFA-1 can generate intracellular signals, the immune cell adaptor protein linker for the activation of T cells (LAT) couples the TCR to downstream events. Here, we show that LFA-1 can mediate both adhesion and de-adhesion, dependent on receptor clustering. Although increased affinity mediates adhesion, LFA-1 cross-linking induced the association and activation of the protein-tyrosine kinases FAK1/PYK1 that phosphorylated LAT selectively on a single Y-171 site for the binding to adaptor complex GRB-2-SKAP1. LAT-GRB2-SKAP1 complexes were distinct from canonical LAT-GADs-SLP-76 complexes. LFA-1 cross-linking increased the presence of LAT-GRB2-SKAP1 complexes relative to LAT-GADs-SLP-76 complexes. LFA-1-FAK1 decreased T-cell-dendritic cell (DC) dwell times dependent on LAT-Y171, leading to reduced DO11.10 T cell binding to DCs and proliferation to OVA peptide. Overall, our findings outline a new model for LFA-1 in which the integrin can mediate both adhesion and de-adhesion events dependent on receptor cross-linking.

Figure 1. LFA-1 induced SKAP1-LAT and reduced LAT-SLP-76 complexes.

(a) In situ proximity analysis shows anti-LFA-1 induced SKAP1-LAT proximity. Murine DC27.10T-cells were ligated with anti-CD3 and/or LFA-1 followed by in situ proximity analysis for SLP-76 and LAT (upper panels) or SKAP1 and LAT (lower panels) (n=4). (b) LFA-1 cross-linking increases SKAP1-LAT micro-cluster colocalization. Jurkat T cells transfected with mCherry-LAT and GFP-SKAP1 were stimulated on anti-CD3 or ICAM1/CD3 coated cover slips. Micro-cluster formation was monitored by confocal microscopy. Upper panel: resting; middle panel: anti-CD3; lower panel anti-CD3/ICAM1. Scale bar corresponds to 10 μM. (n=3) (c) SKAP1 co-precipitates LAT and vice versa in response to anti-LFA-1. DC27.10 T cells were ligated with anti-CD3 and/or anti-LFA-1 for 5 min followed by membrane preparation and precipitation with anti-SKAP1 and blotting with anti-SKAP1, anti-LAT and anti-SLP-76 (upper panels). Conversely, cells were separated into cytosol (lanes 3,4) and membranes (lanes 5,6) and precipitated with anti-LAT followed by blotting with anti-LAT or anti-SLP-76 (n=3). (d) ICAM1 binding to LFA-1 increases the binding of SKAP1 to LAT. DC27.10T-cells were ligated with ICAM1-Fc alone on plates (2 μg ml⁻¹) for various times followed by anti-SKAP1 precipitation and blotting with either anti-LAT, anti-SLP-76 or anti-GADs (n=3) (e) Anti-LFA-1 induced LAT-SKAP1 in human peripheral primary T-cells. Human resting T-cells ligated with plate bound ICAM1-Fc (2 μg ml⁻¹) or ICAM1-Fc plus soluble anti-CD3 (1 μg ml⁻¹) for various times followed by anti-SKAP1 precipitation and blotting with anti-SKAP1, anti-LAT, anti-GADS or anti-SLP-76 (n=3).

Figure 2. Anti-LFA-1 induced SKAP1-LAT forms independently of SLP-76.

(a) Titration of anti-LFA-1 increases LAT-SKAP1 binding. DC27.10 T cells were ligated with anti-CD3 (2 μg ml⁻¹) in the presence of different concentrations of anti-LFA-1 followed by precipitation with anti-SKAP1 and blotting with anti-SKAP1, LAT, GADS and SLP-76 (n=3). (b) SKAP1 binds to LAT in SLP-76 deficient J14 Jurkat cells. Jurkat and J14 Jurkat T-cells were ligated with anti-CD3 (1 μg ml⁻¹) and/or anti-LFA-1 (1 μg ml⁻¹) for 5 min followed by membrane preparation, anti-SKAP1 precipitation and blotting for anti-SLP-76, anti-LAT or anti-SKAP1 (n=3). (c) SLP-76 co-precipitates LAT from SKAP1-depleted cell lysates. Upper panel: Blot showing anti-SKAP1 depletion of SKAP1. Jurkat cells ligated with anti-CD3 and/or anti-LFA-1 for 5 min followed by detergent solubilization and serial depletion of lysates with anti-SKAP1 (5 times), anti-SLP-76 precipitation and anti-LAT blotting (n=3). (d) SKAP1 co-precipitates LAT from SLP-76-depleted cell lysates. Upper panel: Blot showing anti-SLP-76 depletion of SLP-76. Jurkat T-cells were ligated with anti-CD3 and/or anti-LFA-1 for 5 min followed by detergent solubilization and serial depletion of lysates with anti-SLP-76 (five times), anti-SKAP1 precipitation and anti-LAT blotting (n=3). (e) SKAP1 does not associate with PLCγ1. Jurkat T-cells were ligated, detergent solubilized and precipitated by anti-SKAP1. Western blotting was then conducted with anti-PLCγ1 (left panel). Upper right panel: cells were cross-linked with anti-LFA-1 and sequentially precipitated with anti-SKAP1 and each precipitate was subjected to western blotting with anti-SKAP1 (lanes 1–4) or anti-PLCγ1 (lane 5). Lower right panel: Cells were depleted sequentially with anti-PLCγ1 and each were the blotted with anti- PLCγ1 (lanes 1–4) or anti-SKAP1 (lane 5) (n=3). (f) SKAP1 associates with ADAP and RapL. Jurkat T-cells were ligated with antibodies as shown, were precipitated with anti-SKAP1 and subjected to blotting with anti-LAT, GADs, SLP-76 and ADAP (upper panels). The same cells were ligated precipitated as above but then subjected to blotting with anti-RapL (n=3).

Figure 3. GRB2 binds via its SH3 domains to SKAP1.

(a) GRB2 binds to SKAP1. 293T cells were transfected with GFP-GRB2, FLAG-SKAP1 or both, followed by anti-Flag precipitation and blotting with anti-GFP or anti-Flag. Lanes 1–3: cell lysates; lanes 4–6: anti-Flag precipitations (n=3). (b) GRB2 SH3-1 and 2 domains bind to SKAP1. 293T cells were transfected with FLAG-SKAP1, followed by detergent solubilization of cells and GST pull-down with versions of GST-tagged GRB-2 constructs and blotting with anti-Flag or anti-GST (n=3). (c) GRB2 SH3 domains bind to the SK region of SKAP1. 293T cells were transfected with GFP-tagged GRB2 followed by detergent solubilization of cells, and GST pull-down with various versions of GST-SKAP1 (GST-SKAP1-WT1; GST-SKAP1-N-PH-SK; GST-SKAP1-N-PH; GST-SKAP1-PH; GST-SKAP1-SH3; GST-SKAP1-SK) and blotting with anti-GFP or anti-GST (n=3). (d) GST-SKAP1 co-precipitates LAT, GRB-2 and ADAP from Jurkat T-cells. Jurkat cells were ligated with anti-LFA-1 for 5 min. followed by lysis of cells, pull down with GST, GST-SKAP1 WT or GST-SKAP SH3 domain followed by blotting with anti- PLCγ1, anti-LAT, anti-GRB-2, anti-ADAP and anti-SLP-76 and anti-GST. GST-SKAP WT pulled down LAT, GRB-2, ADAP, but neither PLCγ1 nor SLP-76 (n=3).

Figure 4. LFA-1, FAK1 and PYK2 phosphorylate restricted site Y-171 on LAT.

(a) Anti-LFA-1 cross-linking selectively phosphorylates LAT Y-171. Jurkat cells were ligated with anti-CD3 or anti-LFA-1/CD3 for 2–20 min, followed by membrane preparation and blotting for pY-171-LAT, pY-191-LAT, pY-132-LAT and with anti-LAT (n=3). (b) LFA-1 associates with and activates FAK1 and PYK2. Jurkat cells were ligated with anti-CD3 and/or anti-LFA-1 prior to anti-LFA-1 precipitation and blotting with anti-LFA-1, anti-pY-397-FAK, anti-pY-402-PYK2, anti-FAK1 and anti-PYK2 (n=3). (c) FAK1 in vitro kinase phosphorylation of LAT is dependent on the Y-171 residue. 293T cells were transfected with Flag-tagged LAT-mutants, precipitated with anti-Flag and subjected to a cold in vitro kinase assay with recombinant FAK kinase (Millipore), followed by blotting with ant-pY-171-LAT, anti-pTyr (4610) and anti-Flag (n=3). (d) Co-expression of LAT with FAK1 or PYK2 selectively phosphorylates Y-171. 293T cells were transfected with Myc-tagged LAT and Flag-tagged FAK1 or PYK2 and subjected to blotting with anti-phospho-specific antibodies against LAT. Left panel: Myc-LAT and Flag-FAK1; right panel: Myc-LAT and Flag-PYK2 (n=3). (e) Anti-LFA-1 induced pY-171 is inhibited by FAK1/PYK2 inhibitor PF 431396. Jurkat T-cells transfected with either vector control (mock) or Flag-PYK2 were ligated with anti-LFA-1 (1 μg ml⁻¹) in the absence or presence of FAK1/PYK2 inhibitor PF 431396, followed by blotting with anti-Flag, anti-pY-171-LAT or anti-LAT (n=3). (f) Anti-LFA-1 induced SKAP1-FAK1/PYK2 complex is inhibited by FAK1/PYK2 inhibitor PF573228. Jurkat T cells were ligated with rabbit anti-hamster (control), anti-CD3 (1 μg ml⁻¹) or anti-LFA-1 (1 μg ml⁻¹) in the absence or presence of PF573228, followed by precipitation with anti-SKAP1 and blotting with anti-FAK1, anti-pY-397-FAK, anti-PYK2 or pY-402-PYK2 (n=3). (g) Anti-LFA-1 induced LAT-GRB2-SKAP1 depends on LAT Y-171 site. 293T cells were transfected with Flag-SKAP1 and various Myc-tagged LAT mutants (LAT-WT; LAT-Y132F; Lat-Y171F; LAT-Y191F; LAT-Y226F: LAT-Y171/191F) followed by anti-GRB-2 precipitation and blotting with anti-Flag. GRB-2 is endogenously expressed in 293T cells (n=3). (h) Anti-SKAP1 co-precipitates FAK1/PYK2 and LAT dependent on LAT-Y-171. 293T cells were transfected with LAT-WT, PYK2 and SKAP1, LAT-Y-171F, PYK2 and SKAP1, LAT-WT, FAK1 and SKAP1 or LAT-Y-171F, FAK1 and SKAP1 followed by precipitation with anti-SKAP1 and blotting with anti-FLAG, anti-LAT or anti-SKAP1. Lysate (lanes 1–4) anti-SKAP1 IP (lanes 5–8) (n=3).

Figure 5. LFA-1 cross-linking needed for pY-171 and FAK auto-phosphorylation.

(a) MgCl₂/EDTA induced the adhesion of T cells to ICAM-1 on plates. Left panels: images of T cells bound to ICAM-Fc (left: untreated controls; right: MgCl₂/EDTA treated cells stained with DAP1 and TRITC-phalloidin). Right panel: histogram showing MgCl₂/EDTA induced increases in T-cell binding to ICAM1-Fc (n=3). (b) Antibody cross-linking is needed to induce pY-171 and FAK/PYK2 auto-phosphorylation. Jurkat cells were either incubated with soluble anti-LFA-1 Fab’ or Fab’ plus MgCl₂/EDTA or full sized anti-LFA-1 for various times followed by blotting with anti-pY171- LAT, anti-LAT, anti-pY-397-FAK, anti-FAK or anti-pY-402-PYK2 and anti-PYK2. Assay was performed in the absence of ICAM1 on plates (n=3). (c) Mono-valent ICAM1 failed to induce pY-171 and FAK/PYK2 auto-phosphorylation. Jurkat cells were either incubated with soluble ICAM1-Fc or ICAM1-Fc plus MgCl₂/EDTA or bivalent anti-LFA-1 for various times followed by blotting with anti-pY171- LAT, anti-LAT, anti-pY-402 PYK2 and anti-PYK2. Assay was performed in the absence of ICAM1 on plates (n=3).

Figure 6. FAK1 reduces T cell contact with DCs.

(a) FAK1 siRNA increased contact times while reducing velocity. DO11.10 T cells were pre-stimulated for 24 h, resting for 12 h and transfected with siRNA for FAK1. Mature DCs were labelled with SNARF-1 and pre-incubated with OVA peptide (DC-OVA) prior to incubation with T cells on LN slices, as described. DO11.10 T cells were labelled with CFSE and tracked for migration on LN slices. Upper left panel: image example of T cells interacting with DCs; upper middle panel: dot plots showing that FAK1 siRNA increases T-cell dwell times with DCs; upper right panel: anti-FAK1 blot showing reduction in FAK1 expression (lower: FITC siRNA uptake as shown by flow cytometry); lower left panel: dot plot showing that FAK1 siRNA reduces the velocity of T cells; lower right panel: dot plots showing that FAK1 siRNA reduces the displacement of T cells (n=3). (b) siRNA knockdown of FAK1 decreased the amoeboid morphology of DO11.10 T cells. siRNA expression increased the percentage of cells with rounded morphology while decreasing those with an amoeboid shape (4 × length relative to width). Left panel: histogram of ratio of rounded versus amoeboid cells; right panels: images of cells (n=2). (c) FAK1 overexpression decreases dwell times with increased motility and displacement. DO11.10 T cells were prepared as above and transfected with GFP-FAK1. Mature DCs were labelled with SNARF-1 and pre-incubated with OVA peptide (DC-OVA) prior to incubation with T cells. Left panel: dot plot showing that FAK1 expression reduces contact times; middle panel: dot plot showing that FAK1 expression increases velocity; right panel: dot plot showing that FAK1 expression increases displacement (n=3). P values were calculated by one-way analysis of variance (ANOVA) between groups followed by a series of t-tests.

Figure 7. FAK1 reduces T-cell contact times with DCs, an effect dependent on the LAT Y-171.

(a) Dot plots showing that LAT-Y-171F expression reverses FAK1 inhibition of T-cell dwell times. DO11.10 T cells cells were prepared as above and co-transfected with FAK1 and LAT or LAT-Y171F. Mature DCs were labelled with SNARF-1 and pre-incubated with OVA peptide (DC-OVA) prior to incubation with T cells. (b) Dot plots showing that LAT-Y-171F expression reverses FAK1 increase in T-cell velocity. DO11.10 T cells were prepared as above. (c) Dot plots showing that LAT-Y-171F expression reverses FAK1 increase in T-cell displacement. DO11.10 T cells were prepared as above. (d) LAT-Y-171F expression alone increases T-cell dwell times. LAT-Y171F increases T-cell contact (left panel); LAT-Y171F decreases T-cell motility (middle panel); LAT-Y171F decreases T-cell displacement (middle panel). D011.10 T cells were prepared as above and co-transfected with LAT-Y171F prior to a measure of T-cell contact with DCs. (e) LAT-Y171F expression reverses FAK1 inhibition of DO11.10T-cell proliferation in response to OVA peptide. DO11.10 T cells were prepared as above except that they were pre-labelled with CFSE. Left panels: FACs histograms of CFSE labelling; right histogram: panels showing the inhibition of T-cell cycling by FAK1 LAT-WT (gold columns showing most cells in M1–M3, whereas FAK1 LAT Y-171F shows the increased presence of cells in M3-M6 (n=4). (f) LAT-Y-171F expression blocks T-cell motility/migration on ICAM1-Fc coated plates. J14 Jurkat cells were transfected with LAT or LAT-Y-171F and tracked for migration. Left panel: immunoblots of lysates transfected with LAT or LAT-Y-171F; middle panel: tracking profiles of LAT transfected J14 T cells; right panel: tracking profiles of LAT-Y-171F transfected J14 T cells (n=3). P values were calculated by one-way analysis of variance (ANOVA) between groups followed by a series of t-tests.