Micropatterning of TCR and LFA-1 ligands reveals complementary effects on cytoskeleton mechanics in T cells

Abstract

The formation of the immunological synapse between a T cell and the antigen-presenting cell (APC) is critically dependent on actin dynamics, downstream of T cell receptor (TCR) and integrin (LFA-1) signalling. There is also accumulating evidence that mechanical forces, generated by actin polymerization and/or myosin contractility regulate T cell signalling. Because both receptor pathways are intertwined, their contributions towards the cytoskeletal organization remain elusive. Here, we identify the specific roles of TCR and LFA-1 by using a combination of micropatterning to spatially separate signalling systems and nanopillar arrays for high-precision analysis of cellular forces. We identify that Arp2/3 acts downstream of TCRs to nucleate dense actin foci but propagation of the network requires LFA-1 and the formin FHOD1. LFA-1 adhesion enhances actomyosin forces, which in turn modulate actin assembly downstream of the TCR. Together our data shows a mechanically cooperative system through which ligands presented by an APC modulate T cell activation.

Figure 1. LFA-1 is necessary T-cell spreading and enhances T-cell activation

A) Continuous presentation of TCR and LFA-1 ligands. 3D reconstruction of confocal micrographs of anti-myosin IIa and phalloidin stained human CD4+ cells on OKT3+ICAM-1 (left panel) and OKT3 (right panel); note the dendritic vs. circular morphology of the T-cells on the OKT3 vs. OKT3+ICAM-1 substrates. B) Phospho-tyrosine and phalloidin staining of human CD4+ cells on spin coated PDMS (70μm thickness), coated with either OKT3 or OKT3 and ICAM-1. The mean phospho-tyrosine intensity is higher on rigid (Sylgard 184, 2 MPa) than on soft (Sylgard 527, 5 kPa) substrates. The presence of ICAM-1 further enhances phospho-tyrosine levels. p values from one-way ANOVA with Tukey’s multiple comparison test: p<0.005 (**); p<0.0001 (***); not significant (ns).

Figure 2. LFA-1 is necessary for the development of cell edge tension

A) 3D reconstruction of phallodin (blue) and myosin (green) in CD4+ T-cells, spreading on OKT3 vs. ICAM-1 and OKT3 vs. OKT3 grids (1 μm wide lines, 10 μm pitch). B) Population analysis of bi-directional spreading lengths along orthogonal OKT3 lines and along OKT3 vs. ICAM-1 grids in the control cells and after blebbistatin-treatment. Blebbistatin treatment decreases the spreading length along ICAM-1 lines and increases the spreading along OKT3 lines. C) Free-cell edge curvature radii are significantly larger on OKT3 vs. ICAM-1 than on OKT3 vs. OKT3 grids. Blebbistatin treatment decreases the curvature radius on the OKT3 vs. ICAM-1 grids, indicating that LFA-1 regulates cytoskeletal tension via actomyosin contractility. Error bars indicate SD. n=121 (OKT3 vs. ICAM-1 Control), 92 (OKT3 vs. ICAM-1 blebbistatin), 130 (OKT3 vs. OKT3 Control) and 152 (OKT3 vs. OKT3 blebbistatin); p values from student’s t-test: p<0.05 (*); p<0.001 (**); p<0.0001 (***). D–F) PDMS pillar arrays. Jurkat cells were spread on PDMS pillar substrates coated with OKT3+ICAM-1 or OKT3 and imaged with 1 frame per second from the initiation of spreading. Addition of ICAM-1 enables cell spreading (D) and increases (contractile) inwards-directed forces. E) Quantification of displacement angles for all pillars over 60s after first contact. F) Contractile forces (vector component perpendicular to the cell edge of inwards displacements) are significantly higher in the presence of ICAM-1. n > 400 pillars for 5 cells per condition. Student’s t test: p<0.0001 (***).

Figure 3. Actin assembly at TCR and LFA-1 adhesions is regulated by ARP2/3 and FHOD1, respectively

A) CD4+ T-cell blasts were stained for ARP2/3, HS1, or FHOD1. ARP2/3, as well as HS1 (quantification on the right) localize to OKT3 lines and FHOD1 is enriched at ICAM-1 lines. Quantification in (B). C) CD4+ T-cell blasts were pre-treated with 5 μM smiFH2 and spread on OKT3/ICAM-1 grids in the presence of the drug. D) Measurement of the T-cell bi-directional spreading lengths along orthogonal OKT3 and ICAM-1 lines. Formin inhibition disables T-cell adhesion and spreading along ICAM-1 lines. n=165 and 149 cells for control and smiFH2 treated cells respectively. E–H) FHOD1 is needed for contractile forces. FHOD1 knockdown or treatment with smiFH2 decreases the forces to the level observed without ICAM-1. FHOD1-shRNA transfected cells were identified by the expression of GFP under control of a CMV promoter on the shRNA plasmid (E, inset), validation of knock down efficiency in (G). F) Pillar displacement directions for all pillars over 60s after first contact; H) contractile forces (vector component perpendicular to the cell edge of inwards displacements); n > 400 pillars for 5 cells per condition. p values from student’s t-test: p<0.0001 (***).

Figure 4. TCR-nucleated actin cluster are transformed into a coherent network by LFA-1

3D reconstructions of the human CD4+ T-cells on micropatterend surfaces of OKT3 dots on ICAM-1 background, BSA-blocked glass without ligands, or poly-L-lysine (PL) (1 μm dots, 5 μm pitch). CD4+ T-cells spread out over multiple OKT3 dots on ICAM-1 background. Without ICAM-1 (uncoated glass) or when substituted with poly-L-lysine (PL) spreading was suppressed, and cells formed thin projections that connected to the OKT3 dots.

Figure 5. TCR and LFA-1 promote actin assembly by different mechanisms

A) GFP-Actin dynamics in primary human CD4+ (upper panels) and Jurkat T-lymphocytes (lower panels) on OKT3-dotted ICAM-1 substrates (1 μm OKT3 dots, 5 μm pitch). From left to right – single capture of cell at the moment of spreading bifurcation (white arrow), time average density diagram, kymographs of the white line on the left panels showing OKT3 dot-induced bifurcation, T-cell spreading area (black lines) and derivative plots (red). Sub-stable cell spreading areas – black asterisks, spreading bifurcations – black arrows, cell spreading acceleration peaks – red asterisks. B) Actin-GFP density profile average (n=17) across OKT3 dot and surrounding ICAM-1 area. C) F-actin polymerization spiral unfolding from a nucleating TCR adhesion site in a Jurkat cell. Right panel: Kymograph and corresponding actin-GFP (black curve) and OKT3 dot (red curve) density profiles along the indicated line. D–F) FRAP analysis of stable transfected GFP-actin Jurkat cells. D) GFP-Actin bleach areas and kymograph of recovery (right panels, asterisks mark the bleach event); E) Actin-GFP fluorescence recovery curves over OKT3 dot (top panel) and ICAM-1 adhesion areas (bottom panel) for control cells and CK-666 or smiFH2 treated cells. F) Actin-GFP semi-recovery times (mean and standard deviation in red). Because of the lack of fluorescence recovery, curves could not be fitted for CK-666 treated cells. p values from student’s t-test: p<0.001 (**); p<0.0001 (***); not significant (ns).

Figure 6. Myosin contractility modulates TCR activity

A) Examination of Actin-GFP expressing Jurkat cells shows a reduction of actin polymerization after blebbistatin treatment. Top panel: image taken immediately after bleaching; middle panel: actin density kymograph, starting after the bleach event (blebb: time point of blebbistatin addition); lower panel: actin density profiles at the time points indicated in the kymograph. B) Calyculin A (ClcA) hyper-contractility-driven cell collapse is accompanied by formation of TCR-anchored retraction tethers. Note the continuous actin polymerization events (arrowheads) at TCR adhesions inside the retraction tether (white line). Addition of ClcA is marked on the kymograph. C–D) Introduction of spatial anisotropy demonstrates the mechanosensitive nature of TCR. C) Immunofluorescence analysis of CD4+ primary T-cells: F-actin cluster formation at the apices of multi-apical OKT3 adhesions sites within ICAM-1 field. D) Live cell imaging of CD4+: actin-GFP forms apical clusters with periodic density bursts (arrows) that consequently ignite F-actin polymerization waves (combs). Time average actin density map shows all three clusters at the tri-dent OKT3 adhesion patch. Note the apical actin periodic density bursts indicated in the kymograph.