Filamin a Is Required for Optimal T Cell Integrin-Mediated Force Transmission, Flow Adhesion, and T Cell Trafficking

Filamin A Is Required for Optimal T Cell Integrin-Mediated Force Transmission, Flow Adhesion, and T Cell Trafficking

This information is current as Terhi Savinko, Carla Guenther, Liisa M. Uotila, Marc Llort of September 28, 2021. Asens, Sean Yao, Sari Tojkander and Susanna C. Fagerholm J Immunol published online 26 March 2018 http://www.jimmunol.org/content/early/2018/03/24/jimmun ol.1700913 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 26, 2018, doi:10.4049/jimmunol.1700913 The Journal of Immunology

Filamin A Is Required for Optimal T Cell Integrin-Mediated Force Transmission, Flow Adhesion, and T Cell Trafﬁcking

Terhi Savinko,*,† Carla Guenther,*,† Liisa M. Uotila,*,† Marc Llort Asens,*,† Sean Yao,‡ Sari Tojkander,‡ and Susanna C. Fagerholm*,†

T cells traffic from the bloodstream into tissues to perform their functions in the immune system and are therefore subjected to a range of different mechanical forces. Integrins are essential for T cell trafficking into the tissues, as they mediate firm adhesion between the T cell and the endothelium under shear flow conditions. In addition, integrins are important for the formation of the contact between the T cell and the APC required for T cell activation. The actin-binding protein filamin A (FlnA) provides an important link between the integrin and the actin cytoskeleton. FlnA has been reported to function as an integrin inhibitor by competing with talin. However, its role in regulating integrin-dependent immune functions in vivo is currently poorly understood.

In this study, we have investigated the role of FlnA in T cells, using T cell-specific FlnA knockout mice. We report that FlnA is Downloaded from required for the formation of strong integrin–ligand bonds under shear flow and for the generation of integrin-mediated T cell traction forces on ligand-coated hydrogels. Consequently, absence of FlnA leads to a reduction in T cell adhesion to integrin ligands under conditions of shear flow, as well as reduced T cell trafficking into lymph nodes and sites of skin inflammation. In addition, FlnA is not needed for T cell activation in vivo, which occurs in shear-free conditions in lymphoid organs. Our results therefore reveal a role of FlnA in integrin force transmission and T cell trafficking in vivo. The Journal of Immunology, 2018,

200: 000–000. http://www.jimmunol.org/

cells are subjected to a range of different mechanical because they mediate the firm adhesion to ICAMs expressed on forces, as they spend part of their lifetime in the blood- endothelial cells under shear force conditions (blood flow). The T stream subjected to shear stress and part of it inside b2 integrin LFA-1 (aLb2) is also a component of the immuno- lymphatic or peripheral tissues where they interact mechanically logical synapse between a T cell and an APC, aiding in the sta- with other cells, such as APCs. Mechanotransduction encom- bilization of the contact, and it is required for optimal T cell passes the translation of a mechanical signal into a biochemical activation in vitro and in vivo (3, 4). signal to change cell behavior, and it is becoming increasingly To perform their functions in vivo, integrins need to be switched by guest on September 28, 2021 apparent that mechanotransduction is of fundamental importance on and off at appropriate times (e.g., following TCR activation or in many biological systems, including immune cells (1). chemokine receptor activation). In their active state, integrins are Extrinsic mechanical signals can be sensed by cells through able to bind their ligands, such as ICAMs, on the surfaces of other surface receptors, such as integrin adhesion receptors, and because cells. After binding ligands, b2 integrins can also participate in they act as bidirectional signaling molecules, integrins also signaling to change cell behavior (e.g., to mediate cell spreading, transduce mechanical signals from the actin cytoskeleton to the actin reorganization, or cell migration). Integrin activation and extracellular matrix (2). b2 integrins are leukocyte-specific adhe- signaling is regulated by the binding of cytoplasmic proteins, such sion molecules expressed on the surface of T cells and other im- as talin, filamin, kindlin-3, and 14-3-3 proteins, to the b integrin mune cells. They are essential for T cell trafficking out from the cytoplasmic domain (5–11). bloodstream and into tissues in homeostasis and inflammation Filamin A (FlnA) is an important mechanosensitive linker that binds to integrins and couples them to the actin cytoskeleton. In addition to actin and integrins, FlnA also interacts with many other *Faculty of Biological and Environmental Sciences, University of Helsinki, 00790 cytoplasmic partners and can therefore translate mechanical signals Helsinki, Finland; †Institute of Biotechnology, University of Helsinki, 00790 Helsinki, Finland; and ‡Department of Veterinary Biosciences, Faculty of Veterinary into biochemical signals, thereby acting as a mechanosensor in cells Medicine, University of Helsinki, 00790 Helsinki, Finland (12). Both extrinsic and cell-intrinsic forces (e.g., actomyosin ORCIDs: 0000-0003-4483-7235 (T.S.); 0000-0001-7915-1028 (C.G.); 0000-0003- contraction) acting on filamin can lead to changes in the interaction 4743-9013 (L.M.U.); 0000-0002-5592-9365 (M.L.A.); 0000-0003-1194-0244 (S.T.). of FlnA with its various cytoplasmic partners, providing Received for publication June 22, 2017. Accepted for publication March 4, 2018. a mechanism by which FlnA can act as a cellular mechanosensor This work was supported by the Academy of Finland (to S.C.F. and S.T.), the Sigrid (12, 13). In contrast, FlnA has also been reported to function as an Juselius Foundation, Liv och Ha¨lsa Foundation, Magnus Ehrnrooth Foundation, Bio- centrum Helsinki, and the University of Helsinki (all to S.C.F.). integrin inhibitor by competing with talin and/or by linking integrin a-andb-chains together (5, 8, 14). FlnA has also been reported to Address correspondence and reprint requests to Assoc. Prof. Susanna C. Fagerholm, Biocenter 1, University of Helsinki, Viikinkaari 9, 00790 Helsinki, Finland. E-mail play a role in T cell activation through interactions with CD28 and address: susanna.fagerholm@helsinki.fi PKC-u (15). However, the in vivo roles of this integrin interactor in The online version of this article contains supplemental material. the immune system remain poorly understood. Abbreviations used in this article: CTCF, corrected total cell fluorescence; Ctrl, CD4- In this study, we have investigated the role of FlnA in T cell + Cre ; DC, dendritic cell; FlnA, filamin A; KO, knockout; LN, lymph node; OXA, functions in vivo. Using a T cell–specific FlnA knockout (KO) oxazolone; T , effector T; Treg, regulatory T cell; WT, wild type. eff mouse model, we show that FlnA does not function as an integrin Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 inhibitor in T cells. Instead, it is required for b2 integrin–dependent

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700913 2 FlnA IS ESSENTIAL FOR T CELL TRAFFICKING traction forces in T cells as well as for formation of strong intensity, and background were measured with ImageJ and used to calculate integrin–ligand bonds and therefore for optimal T cell adhesion to corrected total cell fluorescence (CTCF) values as previously described. integrin ligands under shear flow conditions. Consequently, FlnA Briefly, CTCF values were calculated for individual cells based on mea- surements of area, intensity, and background with ImageJ as previously KO T cell homing into peripheral lymph nodes (LNs) and traf- described [CTCF = integrated density 2 (area of selected cell 3 mean ficking into sites of inflammation is reduced in vivo. In conclusion, fluorescence of background readings)] (19). Twenty-five to one hundred cells our results show that FlnA is not an integrin inhibitor in T cells, per condition were measured for each animal. but it instead plays fundamental roles in integrin-mediated force Flow cytometry transmission and T cell trafficking in vivo. Single-cell suspensions of LN, blood, thymus, and spleen were prepared. In some experiments, CD4 T cells were isolated, activated, and stained for flow Materials and Methods cytometry. Single-cell suspensions from the ear tissue were prepared by Mice cutting the skin sample into small pieces, passing it through a 70-mm cell strainer, washing it with PBS, and filtering it again through a 40-mm cell FlnA-floxed mice (16) (The Jackson Laboratory, stock no. 010907) have a strainer (BD Biosciences). The following fluorescently labeled Abs were conditional KO allele of the filamin gene. When bred to mice expressing Cre + used: CD4 (RM4-5), CD3 (145-2C11), CD8a (53-6.7), B220 (RA3-6B2), recombinase in CD4 T cells, the floxed exons are deleted in CD4 T cells CD44 (IM7), CD69 (H1.2F3), CD25 (PC61), CD18 (C71/16), CD62L expressing Cre in the offspring. CD4-Cre+ (Ctrl) mice were used as controls (MEL-14), PSGL-1 (2PH1), CD29 (HMb1-1), CD49d (R1-2), b7 (FIB27), for FlnA KO–CD4-Cre mice. For in vivo T cell activation studies, FlnA KO and CD11a (2D7), all from eBioscience. Fc block (clone 2.4G2; BD mice were crossed with TCR–OT-II–transgenic mice (4). FlnA and CD4-Cre Biosciences) was used in all staining. Intracellular staining for Foxp3 mice were ordered from The Jackson Laboratory. All experiments were in (FJK-16s) was performed according to the manufacturer’s instructions compliance with Social and Health Services of the State Provincial Office of (eBioscience). For F-actin staining, nonadherent cultured T cells were Downloaded from Southern Finland. C57/Bl6 mice were obtained from Charles River. fixed with 1% paraformaldehyde in PBS, washed, and stained with Cell isolation and culture phalloidin-FITC (Sigma-Aldrich) in 0.1% saponin and 1% FBS/PBS. Data were acquired on an LSRFortessa (BD Biosciences) and analyzed using CD4 T cells were isolated from the spleen and LNs by positive selection FlowJo software (Tree Star). using magnetic beads to CD4 (MACS; Miltenyi Biotec, Germany). To Static adhesion culture effector T (Teff) cells, CD4-isolated T cells were cultured for 48 h with 0.5 mg/ml anti-CD3 (clone 2C11; R&D Systems, Minneapolis, MN) Static adhesion assays were performed as previously described (7). Briefly, http://www.jimmunol.org/ and 20 ng/ml IL-2 (Novartis) in RPMI 1640 medium supplemented with the integrin ligand recombinant mouse ICAM-1/CD54 Fc chimera (0.5, 1, 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin–streptomycin, and 50 3, and 6 mg/ml) was coated onto 96-well MaxiSorp plates (Nunc, Thermo mM 2-ME. Cells were washed and maintained in IL-2 for a further 5 d. 6 Fisher Scientific) by overnight incubation at 4˚C. Teff cells at 1 3 10 cells/ CD8 T cells were purified from the CD4 fraction from the spleen and LN ml were resuspended in an adhesion medium (RPMI 1640 supplemented cells and were isolated with CD8 MACS beads (Miltenyi Biotec). Neu- with 0.1% BSA, 40 mM HEPES, and 2 mM MgCl2) and added to the plate. trophils were purified by using a MACS mouse neutrophil purification kit Cells were allowed to adhere for 30 min at 37˚C before gentle washing to (Miltenyi Biotec) and B cells by negative selection using CD43 (Ly 48) remove unbound cells. Bound cells were lysed and detected with phos- beads from Miltenyi Biotec. Dendritic cells (DCs) were cultured from phatase substrate para-nitrophenyl phosphate (Sigma-Aldrich). bone marrow–derived cells and cultured for 10 d with GM-CSF (10 ng/ml; PeproTech) as described previously (17). Adhesion under shear flow by guest on September 28, 2021 Subcellular fractionation A shear flow adhesion assay was performed as in (20). ibidi VI 0.4 m-slides were coated with 6 mg/ml ICAM-1 or with 6 mg/ml MAdCAM-1 (R&D CD4 T cells were fractionated as in (18). Cell pellets were lysed on ice eff Systems). ICAM-1 or MAdCAM-1 were coated overnight at 4˚C, followed for 15 min with a lysis buffer containing 150 mM NaCl, 10 mM EDTA, by SDF-1a (1 mg/ml) or CCL25 (4 mg/ml) coating at +37˚C for 40 min. 50 mM Tris (pH 7.4), and 1% Triton X-100 (Sigma-Aldrich) in the Cells flowed over coated VI 0.4 ibidi m-slides at a 0.3–1 dynes/cm2 (0.3 presence of protease (Thermo Fisher Scientific) and phosphatase inhibitors dynes/cm2 for T cells and 1 dyne/cm2 for chemokine-activated naive (Pierce Protein Biology). The lysate was centrifuged, and the supernatant eff T cells) continuous shear flow rate over a 5 min period. Cells were was further centrifuged at 159,900 3 g for 2 h at 2˚C. The pellet contained monitored by microscopy, and the number of adhered cells in the field of the cytoskeletal fraction and the supernatant the soluble fraction. The view was determined by manual counting. Rolling rates were analyzed cytoskeletal fraction was solubilized with a buffer containing 350 mM from video recordings (Hamamatsu ORCA-Flash digital camera) with NaCl, 10 mM EDTA, 50 mM Tris (pH 7.4), and 1% Triton X-100 (Sigma- ImageJ. The average speed of 100 points per cell was measured, and a total Aldrich) in the presence of protease and phosphatase inhibitors, then of 30 cells were analyzed per condition. treated with DNase (Promega). The cytoskeletal fraction was then centrifuged, and the supernatant contained the solubilized proteins. All Traction force microscopy fractions were balanced to have the same NaCl content. Teff cells were cultured for 1 h on elastic ICAM-1–coated silicone-based Western blotting gel substrates (Young’s modulus = 2 kPa) (Matrigen). Substrates were T cells, B cells, neutrophils, and DCs were lysed in 1% Triton X-100, surface coated with green fluorescent sulfate nanobeads (diameter 200 nm; 150 mM NaCl, 10 mM EDTA, 50 mM Tris (pH 7.4), and Pierce Phos- Invitrogen, Carlsbad, CA). Single cells together with the underlying beads were imaged with the 3i Marianas imaging system (3i Intelligent Imaging phatase and Protease Inhibitor Mini Tablets (Thermo Fisher Scientific). The 3 primary Ab rabbit polyclonal FlnA was from Bethyl Laboratories. The Innovations) by using multipoint imaging. A 63 /1.2 W C-Apochromat primary Abs used for fractionation samples were against talin (8d4; Santa Corr WD = 0.28 M27 objective was used, and the dish was placed in a heated sample chamber (37˚C) controlled for CO2. Following live cell Cruz Biotechnology), CD18 (C71/16; Abcam), and b-actin (Cell Signaling 3 Technology). After incubation in the HRP-conjugated secondary Ab, blots imaging, the cells were detached from the gel substrates with 10 trypsin were developed by a standard chemiluminescence technique. (Lonza), and a second set of nanobead images, serving as reference images, were obtained in a cell-free configuration. Spatial maps of cell-exerted Immunofluorescence nanobead displacements were achieved by comparing the reference bead images together with the experimental images. With the knowledge of the 3 6 1 10 CD4 Teff cells on ICAM-1 (Bio-Techne) 6 mg/ml–coated coverslips bead displacement fields, substrate stiffness (2 kPa), and a manual trace of were incubated for 30 min and fixed with 4% paraformaldehyde. F-actin was the cell boundary, the cell-exerted traction fields were computed by using stained with FITC-phalloidin (Sigma-Aldrich). For phalloidin staining, all Fourier transform traction cytometry (21, 22). The root mean squared slides were imaged using a Leica SP5 II and LAS AF Lite Software (Leica magnitude was computed from the traction field. Microsystems), with 20% 488-Argon laser power (10% visible laser power). Z-stacks were taken with the following parameters: spectral range 496–678 T cell activation in vivo nm, QD405/488/561/635 mirror, smart gain 650 V, smart offset 0%, pinhole 111.49 mm, line average 3, zoom 1, objective 633, z-distance 10.078 mm, Purified control OT-II+ and FlnA KO/OT-II+ CD4+ T cells were labeled z-step size 0.15 mm, and format 512 3 512. The area, Feret diameter (the with CFSE (Life Technologies) according to the manufacturer’s instruc- longest distance between any two points along the cell), fluorescence tions, and 2 3 106 CD4+ T cells were injected i.v. in a volume of 200 ml The Journal of Immunology 3 into the recipient wild type (WT) mice. After 24 h, WT mice were im- FlnA has an important role in coupling the actin cytoskeleton to munized i.v. into the tail vein with 100 mg OVA peptide (AnaSpec) in 10 cell surface receptors, such as integrins, thereby influencing mg LPS (Sigma-Aldrich) in a total volume of 200 ml. Spleens were col- membrane–cytoskeleton interactions, and FlnA has previously lected, and the splenic cells were stained for the T cell activation marker CD44 and analyzed by flow cytometry. been reported to function as an integrin inhibitor in cells (5, 8). We therefore investigated b integrin expression and cytoskeletal T cell activation in vitro 2 coupling and function in FlnA KO T cells. The surface expression CD4 T cells were isolated with CD4 beads (MACS; Miltenyi Biotec) and of b2 integrins (CD18 and CD11a) in CD4 T cells was normal in stimulated with 2.5 mg/ml soluble anti-CD3 plus IL-2 for 24 h. IL-2 and the absence of FlnA (Supplemental Fig. 2A). In addition, the IL-10 were measured from supernatants by ELISA. Abs for ELISA were subcellular fractionation experiments of T cells revealed that b purified anti-mouse IL-2 (clone JES6-1A12; eBioscience) and biotinylated eff 2 anti-mouse IL-2 (clone JES6-5H4; eBioscience). IL-10 was detected with integrin distribution between soluble and cytoskeletal fractions the DuoSet ELISA kit (R&D Systems). was not significantly affected by FlnA deficiency (Fig. 2A). In + In vivo homing assay addition, static adhesion assays with CD4 T cells revealed that FlnA KO T cell adhesion to the b2 integrin ligand ICAM-1 was Control and FlnA KO CD4 T cells were labeled with CFSE and CellTrace similar to control cells (Fig. 2B), indicating that FlnA does not Violet (Life Technologies) according to the manufacturer’s instructions. function as a b integrin inhibitor in primary T cells. Reversal of fluorescent dyes yielded the same result. Ctrl and FlnA KO 2 CD4+ T were mixed at a 1:1 ratio, and 5–6 3 106 cells were injected i.v. As FlnA cross-links actin in cells, we asked whether FlnA de- into recipient WT mice. At 1.5 or 18 h later, donor cells in the LNs, spleen, ficiency affects cell morphology or the actin cytoskeleton in T cells. + and blood were identified by flow cytometry. We cultured CD4 Teff cells from FlnA KO mice (as these have a

+ Downloaded from In vitro migration assay more pronounced actin cytoskeleton than naive CD4 lymphocytes) and investigated the F-actin cytoskeleton in these cells ibidi m-slide eight-well plates were coated with 3 mg/ml ICAM-1 in PBS plated on the b2 integrin ligand ICAM-1 by phalloidin staining. overnight, after which wells were washed with an adhesion medium before + 70,000 cells in 400 ml adhesion media were added to each well. Cells were We found that the cell spreading of CD4 Teff cells was similar in imaged in doublets, with controls and KO cells on the same plate. Cells FlnA KO and in control cells plated on ICAM-1 (Fig. 2C, 2E). were imaged in brightfield with a 203 magnification using the 3i Marianas Also, cell morphology assessed by Feret diameter analysis was imaging system (3i Intelligent Imaging Innovations) with multipoint im- similar in control and FlnA KO T cells (Fig. 2C). However, FlnA http://www.jimmunol.org/ aging. Cells were imaged for 1 h in 1-min intervals. Cells were tracked KO CD4 T cells displayed significantly increased F-actin content with the manual tracking plugin from ImageJ. when compared with control cells (Fig. 2E). This result is in line Contact hypersensitivity model with previous research showing increased F-actin levels in plate- Mice were sensitized on their shaved backs with 1% oxazolone (OXA) on day lets lacking FlnA, which have one or more abnormal F-actin foci 0 and challenged with 0.3% OXA on both ears on day 5, followed by sample inside the cell when FlnA is absent (24), and in a human mela- collection and analysis 24 h later on day 6 as in (23). Ear thickness was measured noma cell line lacking FlnA (25). The abnormal actin cytoskeleton with a micrometer (Mitutoyo, Kanagawa, Japan) at 24 h after challenge, after in some FlnA KO T cells on ICAM-1 may be related to the im- which the ear lobes were collected for RNA isolation and flow cytometry. portant role of FlnA as a cross-linker of actin filaments into high- Quantitative real-time PCR angle orthogonal networks linked to membrane receptors, such as by guest on September 28, 2021 Ear samples were homogenized with T10 Ultra-Turrax (IKA) and RNA was integrins. This process may be disturbed in FlnA KO cells, extracted with the NucleoSpin RNA kit (Macherey-Nagel). cDNA was resulting in denser, more parallel actin filaments in these cells synthesized from 0.5 mg of total RNA with a High Capacity cDNA when they are spreading on integrin ligands, as has been previ- Reverse Transcription Kit (Applied Biosystems, Life Technologies). ously reported in human melanoma cells lacking FlnA (25). In TaqMan gene expression assays were performed as in (7) with TaqMan Fast Advanced Master Mix and real-time quantitative PCR with a Bio-Rad contrast, F-actin content in T cells in suspension was normal CFX96 Real-Time PCR Detection System. (Fig. 2D). In summary, our results show that FlnA-deficient T cells display Statistics relatively normal integrin-mediated static adhesion and spreading, The Student t test or Mann–Whitney U test was used for statistical and filamin therefore does not function as an integrin inhibitor in , , analysis. In all cases, p values are defined as *p 0.05, **p 0.01, primary T cells. and ***p , 0.001. FlnA deficiency leads to a reduction of b2 integrin–mediated Results traction forces in Teff cells FlnA is not required for T cell development and does not Adhesion sites mediate actin-dependent cellular traction forces on b function as a 2 integrin inhibitor in primary T cells exterior substrates, and FlnA has been reported to stabilize adhesion To investigate the role of FlnA in T cells in vivo, we generated sites under applied force (26, 27). To investigate whether the integrin/ T cell–specific FlnA KO mice. Mating of conditional Flnafl/fl mice FlnA signaling node in T cells affects transmission of force through with CD4-Cre transgenic mice generated mice lacking FlnA in the integrin to an extracellular substrate, traction force microscopy CD4 (and CD8) T cells. Western blotting confirmed the deletion experiments with WT and FlnA KO T cells on hydrogels coated with of FlnA in cultured CD4 Teff cells and in CD8 T cells (Fig. 1A). the b2 integrin ligand ICAM-1 were performed. In this method, the Deletion of FlnA did not lead to upregulation of other filamin integrin-mediated cellular traction forces are measured by assessing isoforms in T cells (e.g., FlnB or FlnC) (Fig. 1B). Furthermore, integrin-mediated fluorescent bead displacement in the ligand-coated expression of FlnA was normal in B cells, neutrophils, and cul- hydrogels. Importantly, we found that filamin-deficient CD4 Teff cells tured DCs (Supplemental Fig. 1A), showing that the deletion is generated significantly less b2 integrin–dependent traction forces than T cell specific. Deletion of FlnA in CD4 T cells did not affect the control cells (Fig. 3A). proportion of CD4 or CD8+ T cells in the LNs, spleen, or thymus (Fig. 1C, Supplemental Fig. 1B). Also, the proportions of B220+ FlnA is required for T cell adhesion under flow conditions

B cells in the spleen and the LNs were similar in control and FlnA b2 Integrins are essential for firm T cell adhesion to ligands on KO/CD4-Cre mice (Fig. 1C). Therefore, filamin does not appear endothelial cells under conditions of shear flow. We hypothesized to be essential for T cell development in vivo. that the reduced integrin-mediated traction forces generated by 4 FlnA IS ESSENTIAL FOR T CELL TRAFFICKING

FIGURE 1. Filamin KO T cells develop normally. (A) Mating of conditional FlnAfl/fl and Ctrl mice generated mice lacking FlnA in CD4 and CD8 T cells, as assessed by Western blotting of T cell lysates with FlnA Abs and described in the Materials and Methods section. (B) Filamin B and filamin C expression levels were measured by quantitative PCR both in freshly isolated CD4 T cells and in cultured CD4 T cells. (C) Total cells were counted from thymus, spleen, peripheral, and mucosal LNs. CD4, CD8 T cells, and B cells in peripheral LNs and spleens of Ctrl and Flnafl/fl /CD4-Cre mice were investigated by flow cytometry. Lymphocytes were identified and gated by Downloaded from their forward and side scatter. The CD3+ T cells were then further identified and gated by the expression of CD4 and CD8 or for detection of B cells; cells were gated to lymphocytes and to B220+ cells (n = 3). http://www.jimmunol.org/

FlnA-deficient T cells may influence the ability of the cell to either OT-II+–Ctrl or OT-II+FlnA KO mice with CFSE and resist shear forces. Therefore, adhesion of freshly isolated adoptively transferred them into WT mice. Recipient mice were FlnA-deficient and control CD4 T cells was studied under shear immunized with OVA peptide, which is recognized by the OT-II by guest on September 28, 2021 flow conditions. FlnA-deficient CD4 T cells adhered slightly less TCR transgene specific for OVA peptide (OVA 323–339). We to ICAM-1 in the presence of the chemokine SDF-1 (CXCL12) found that the percentage of labeled cells in the spleen recovered under low shear forces, although statistically significant reduction 5 d postimmunization was increased, rather than decreased, in the was only seen after 2 min of starting the flow (Fig. 3B). However, FlnA KO group compared with the control group (Fig. 4A, FlnA-deficient T cells displayed significantly faster integrin- Supplemental Fig. 3A). This reflects increased proliferation of mediated rolling on ICAM-1+SDF-1 than control cells, indicat- FlnA KO T cells, as demonstrated by reduced expression of CFSE ing that the bonds between integrin and ICAM-1 are weaker in in FlnA KO T cells. In addition, the expression of the T cell ac- FlnA KO than in control T cells. Because FlnA is known to in- tivation marker CD44 was also increased in the Flna KO group teract also with b7 integrins, which mediate homing into mucosal (Fig. 4A). In vitro–stimulated T cells showed no difference in sites, adhesion to the b7 integrin ligand MAdCAM-1 in the expression of CD69, CD25, or CD44 (Fig. 4B, Supplemental Fig. presence of the chemokine CCL25 under shear flow conditions 3B) or IL-2 production (Fig. 4C). Therefore, FlnA is not required was explored. We found that FlnA-deficient T cells also rolled for T cell activation; instead, it appears to restrict T cell activation + faster on the b7 integrin ligand MAdCAM-1 (Fig. 3B). in vivo. In addition, the amount of Foxp3 regulatory T cells We further investigated the adhesion of cultured Teff cells to (Treg) was reduced in the LNs of nontreated FlnA KO mice ICAM-1 under flow conditions, which does not require chemokine (Fig. 4C), and IL-10 production in nontreated FlnA KO T cells activation (20), and found that FlnA is indeed required for optimal was reduced (Fig. 4C). These results demonstrate that FlnA de- Teff cell firm adhesion to the b2 integrin ligand ICAM-1 under ficiency does not lead to reduced T cell activation in vivo, but shear flow conditions (Fig. 3C).Our results therefore show that rather, T cell activation is increased in FlnA KO mice. In addition, FlnA is required for formation of strong integrin–ligand bonds and Treg numbers and functionality (e.g., IL-10 production) are therefore for firm adhesion or cell arrest under the conditions of slightly reduced in these mice (Fig. 4C). shear flow. FlnA is important for T cell homing into lymphoid organs FlnA is not important for T cell activation As integrin functionality was affected in FlnA KO T cells, we next FlnA binds to the LFA-1 integrin (5, 8), and we show above that asked the question whether FlnA plays a role in T cell homing into FlnA is necessary for the formation of strong LFA-1–ICAM-1 lymphoid organs in vivo. Therefore, control and FlnA KO T cells bonds under force and for integrin force transmission. Func- were purified and labeled with fluorochromes, and the labeled cells tional LFA-1 is important for T cell activation in vivo (4). were injected i.v. at a 1:1 ratio into the tail veins of WT mice. Therefore, to investigate the role of the integrin regulator FlnA in Lymphoid organs were collected either 1.5 or 18 h later, and labeled T cell activation in vivo, FlnA KO mice were crossed with TCR- cells were detected by flow cytometry. Interestingly, FlnA KO transgenic (OT-II) mice. We purified and labeled CD4 T cells from T cell trafficking was indeed significantly reduced into axillary LNs The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/

FIGURE 2. FlnA KO T cells spread normally. (A) Subcellular frac- 2/2 tionation of Ctrl and Flna Teff cells was performed, and b2 integrin and talin distribution was analyzed by Western blotting (n = 3). (B) Static adhesion of Teff cells to ICAM-1 (0.5–6 mg/ml) (n = 2). (C) Cell area and Feret diameter were measured from Teff cells plated on ICAM-1. (D)F- actin content in nonadhered Teff cells was measured by flow cytometry with phalloidin-FITC–stained T cells (p = 0.3095). (E) Representative by guest on September 28, 2021 microscopy images (original magnification 363) of phalloidin stained Teff cells plated on ICAM-1 were used to analyze F-actin content (CTCF). Cell size from at least 25 cells per condition was determined. The actin content is shown as CTCF (n = 3). Mean 6 SEM is shown. **p , 0.01. C, cytoskeletal fraction; S, soluble. at both time points (Fig. 5). In addition, FlnA KO T cell homing was also decreased into cervical and inguinal LNs at the shorter time point, although this was not statistically significant after 18 h. FlnA KO T cell homing was decreased to mesenteric LNs only after 18 h. The different results for the short-term and long-term experiments may reflect effects other than homing in the long- term experiment (e.g., T cell egress, migration of T cells between different LNs, or T cell apoptosis). In contrast, similar numbers of control and FlnA-deficient T cells were detected in the blood and spleen, which is not surprising, as trafficking of T cells to the spleen is integrin independent. FlnA deficiency did not affect the expression of other T cell homing receptors important for T cell trafficking, such as a4 integrin, b1 integrin, b7 integrin, CD62L, or CD44 (Supplemental Fig. 2). In contrast to in vivo migration and shear flow adhesion experiments, in vitro two- dimensional migration on ICAM-1 was similar for FlnA KO FIGURE 3. FlnA is needed for b2 integrin–mediated traction forces and for optimal T cell adhesion under shear flow conditions. (A) Traction force T cells and control cells (Supplemental Fig. 1C). These data experiments with T cells on hydrogels coated with b integrin ligand confirm that FlnA is required for normal integrin function during eff 2 ICAM-1. Heatmaps representing traction force generated by Teff cells on in vivo homing to LNs, most likely by affecting the formation of ICAM-1–coated 2 kPa hydrogels. Twenty-five cells were measured each strong integrin–ligand bonds under conditions of shear stress. time (n = 3). **p , 0.01. Original magnification 363. (B) SDF-1 induced FlnA is important in T cell trafficking into the inflamed skin adhesion of freshly isolated CD4 T cells to ICAM-1, and CCL25 induced adhesion to MAdCAM-1 under shear flow conditions (n = 2, representative T cells play fundamental roles in the pathogenesis of allergic skin of n = 3). *p , 0.05, **p , 0.01. (C) T cell adhesion to ICAM-1 under inflammation, and b2 integrins are important in the trafficking of shear flow conditions (n = 6). Mean 6 SEM is shown. 6 FlnA IS ESSENTIAL FOR T CELL TRAFFICKING

FIGURE 4. FlnA is not important for T cell activation in vivo. (A) Purified Ctrl and FlnA2/2 OT-II–transgenic CD4 T cells were labeled with CFSE and injected i.v. into the tail vein of recipient WT mice. Twenty-four hours later, recipient mice were immunized i.v. with OVA-peptide Ag in LPS adjuvant. Spleens of recipient WT mice were harvested 5 d postimmunization. The percentage of donor CFSE+ cells, mean fluorescence intensity of CFSE, and T cell activation marker CD44 were measured by flow cytometry. Cells were gated to live lymphocytes and CFSE+ cells (n = 6). Mean 6 SEM is shown. *p , 0.05. (B) Purified Downloaded from CD4 T cells were activated with soluble anti-CD3 overnight, and activation markers CD69, CD25, and CD44 were measured by flow cytometry. (C) IL-2 and IL-10 pro- ductions were measured by ELISA from anti-CD3–activated T cells (n = 3). The http://www.jimmunol.org/ percentage of Foxp3 Treg in LNs of nontreated FlnA KO Cre mice was measured by flow cytometry (n = 6). *p , 0.05, **p , 0.01. by guest on September 28, 2021

T cells into the inflamed skin (23, 28). The mouse model of contact of T cells to the inflamed skin. OXA-sensitized Ctrl and FlnA hypersensitivity can be used to study T cell migration into inflam- KO/CD4-Cre mice had similar ear swelling (Fig. 6A), and there was matory sites in vivo. We therefore used a contact hypersensitivity no difference in the expression levels of inflammatory cytokines in model to investigate whether FlnA plays a role in recruitment the inflamed skin (Fig. 6C, Supplemental Fig. 4). Importantly,

FIGURE 5. FlnA deficiency results in impaired homing of CD4 T cells. Naive CD4 T cells were isolated from Ctrl and FlnAfl/fl-Cre mice, labeled with CFSE and CellTrace Violet, and mixed at 1:1 ratio. Reversal of fluorescent dyes yielded the same results. CD4 T cells were adoptively transferred into recipient WT mice, and the localization of Ctrl and FlnA KO donor cells in the organs indicated was analyzed (A) 1.5 (n = 3) and (B)18h(n =8) posttransfer by flow cytometer. Cells were gated to live lymphocytes and CFSE+ and CellTrace Violet+ cells. Plotted values indicate the recovery of donor cells as percentage of total recovered cells. Mean 6 SEM is shown. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 7

integrin and the actin cytoskeleton. FlnA is a large (280 kDa) actin cross-linking protein with important roles in cellular mechanosensing. It has a key role in regulating the structural stability, adhesion, and motility of cells by cross-linking actin. FlnA links to transmembrane proteins, including integrins, as well as many other proteins in cells (12). FlnA binding to the integrin b-chain cytoplasmic domain has been previously shown to inhibit integrin activation by competing with talin, a well-known integrin activator (5, 8), and/or by linking a and b integrin chains together (14). However, the role of FlnA in integrin regulation in primary immune cells has remained incompletely understood. Our results clearly demonstrate that FlnA does not function as an integrin inhibitor in primary T cells, and it is not required for T cell spreading on integrin ligands or for T cell adhesion or migration under static conditions. However, it has an important role in integrin-mediated mechanotransduction in T cells, and it is needed for optimal T cell trafﬁcking in vivo. FlnA has been proposed to have an important role in CD28-

mediated costimulation of human T cells in vitro (15, 29, 30). Downloaded from However, the role of ﬁlamin in regulating T cell responses in vivo has remained poorly understood. In this article, we show that in vivo, FlnA is not required for T cell activation but instead negatively regulates T cell activation in the spleen, although ex- actly how ﬁlamin regulates T cell activation is still unclear. In-

terestingly, FlnA deficiency resulted in decreased levels of Foxp3 http://www.jimmunol.org/ expression in inflamed skin and in decreased numbers of Treg in LNs of nontreated mice. Therefore, the absence of FlnA leads to abnormalities in Foxp3+Treg numbers or functionality. Interest- ingly, b2 integrins and the integrin activator talin-1 have also been previously shown to be important for Treg development in vivo (31, 32). Furthermore, it was previously shown that T cell–specific deletion of talin leads to spontaneous lymphocyte activation (33). We did not see spontaneous activation in FlnA–CD4-Cre mice, which may be explained by the higher percentage of Foxp3+ by guest on September 28, 2021 T cells in the absence of filamin (5% decrease as compared with control) than talin (10% decrease as compared with control). However, it is clear that the role of filamin in T cell activation and Treg function needs further investigation. To enter LNs or inflamed tissues, T cells need to be able to arrest on endothelial cells surrounding the blood vessels under conditions FIGURE 6. T cell trafficking is reduced in a contact hypersensitivity of shear flow. Integrins mediate this firm adhesion and transmit model in T cell–specific FlnA KO mice. (A) Ear thickness is similar in Ctrl mechanical forces by integrating the cell exterior and the cyto- and Flna KO/CD4-Cre mice after OXA-induced contact hypersensitivity skeleton inside the cell. In this article, we show that rather than + + + model. (B) CD3 , CD4 , and CD8 T cells from the lymphocyte gate in being an integrin inhibitor, FlnA is required for the formation of , , inflamed skin were analyzed by flow cytometry. *p 0.05, **p 0.01, strong integrin–ligand bonds and for optimal T cell adhesion to , C ***p 0.001. ( ) mRNA expression of IL-2 and IFN-g was investigated integrin ligands under conditions of shear flow. LFA-1–ICAM-1 by quantitative real-time PCR (n = 8). Mean 6 SEM is shown. bonds are so-called catch bonds, which become stabilized under force, such as shear flow in blood vessels (34, 35). External shear however, we found decreased numbers of CD4 and CD8 T cells in force stabilizes LFA-1 in the extended conformation, leading to the inflamed skin of FlnA KO/CD4-Cre mice (Fig. 6B). In sum- slow rolling or arrest on the endothelium in blood vessels. In the mary, our results show that FlnA is important for integrin-mediated absence of the link between integrin, FlnA, and actin, this stabi- adhesion under shear flow, and it is important not only for CD4+ lization of the integrin-extended conformation may not occur. As a T cell homing into LNs but also for T cell trafficking into sites of result of weakened integrin–ligand bonds, FlnA-deficient T cells inflammation. display increased rolling rates of T cells on integrin ligands ICAM-1 and MadCAM, and Teff cells display reduced adhesion to Discussion ICAM-1 under shear flow conditions. In addition, we show that in T cell trafficking between the blood, LNs, and peripheral tissues is the absence of FlnA, traction forces generated by T cells on essential for their functions in the immune system. Integrins are integrin–ligand-coated substrates are significantly weaker than in essential for T cell trafficking, as they mediate firm adhesion under the presence of FlnA, indicating that FlnA is essential for integrin shear flow conditions (blood flow) between the T cell and the force transmission in these cells. Therefore, FlnA is required for endothelium. In addition, integrins are important for the formation optimal T cell adhesion to b2 and b7 integrin ligands under shear of the immunological synapse between the T cell and the APC, and flow conditions. Consequently, we show that in the absence of therefore, integrins are required for T cell activation. The actin- FlnA, T cell trafficking into lymphoid organs is significantly re- binding protein FlnA provides an important link between the duced. Our results therefore clearly demonstrate the physiological 8 FlnA IS ESSENTIAL FOR T CELL TRAFFICKING importance of FlnA in integrin-mediated mechanotransduction in 12. Razinia, Z., T. Ma¨kela¨,J.Yla¨nne, and D. A. Calderwood. 2012. Filamins in mechanosensing and signaling. Annu. Rev. Biophys. 41: 227–246. T cells. 13. Stossel, T. P., J. Condeelis, L. Cooley, J. H. Hartwig, A. Noegel, M. Schleicher, Talin and kindlin-3 are integrin interactors that have previously and S. S. Shapiro. 2001. Filamins as integrators of cell mechanics and signalling. been shown to be of fundamental importance in integrin-mediated Nat. Rev. Mol. Cell Biol. 2: 138–145. 14. Liu, J., M. Das, J. Yang, S. S. Ithychanda, V. P. Yakubenko, E. F. Plow, and leukocyte adhesion under shear flow (7, 31, 36). In the absence of J. Qin. 2015. Structural mechanism of integrin inactivation by filamin. Nat. these proteins, integrin-mediated shear flow adhesion is com- Struct. Mol. Biol. 22: 383–389. pletely abolished (36). In addition, both the absence of talin and 15. Wang, X. D., Y. Gong, Z. L. Chen, B. N. Gong, J. J. Xie, C. Q. Zhong, Q. L. Wang, L. H. Diao, A. Xu, J. Han, et al. 2015. TCR-induced sumoylation of the mutation of the kindlin-3 binding site in b2 integrin lead to the kinase PKC-u controls T cell synapse organization and T cell activation. Nat. reduced amounts of T cells in peripheral LNs (7, 31) and reduced Immunol. 16: 1195–1203. 16. Feng, Y., M. H. Chen, I. P. Moskowitz, A. M. Mendonza, L. Vidali, F. Nakamura, homing of T cells in vivo. In this study, we also show that FlnA D. J. Kwiatkowski, and C. A. Walsh. 2006. Filamin A (FLNA) is required for plays an important role in T cell shear flow adhesion cell-cell contact in vascular development and cardiac morphogenesis. Proc. Natl. and lymphocyte trafficking. However, the absence of FlnA results Acad. Sci. USA 103: 19836–19841. 17. Morrison, V. L., M. J. James, K. Grzes, P. Cook, D. G. Glass, T. Savinko, in increased integrin-mediated rolling rather than a complete H. S. Lek, C. Gawden-Bone, C. Watts, O. R. Millington, et al. 2014. Loss of abolishment of adhesion, showing that these integrin-interacting beta2-integrin-mediated cytoskeletal linkage reprogrammes dendritic cells to a proteins regulate different phases of integrin-mediated shear flow mature migratory phenotype. Nat. Commun. 5: 5359. 18. Fagerholm, S. C., A. Prescott, P. Cohen, and C. G. Gahmberg. 2001. An essential adhesion. Also, although homing of FlnA-deficient lymphocytes is role for calmodulin in regulating human T cell aggregation. FEBS Lett. 491: clearly reduced in short-term homing assays, the numbers of 131–136. 19. Abashidze, A., V. Gold, Y. Anavi, H. Greenspan, and M. Weil. 2014. Involve- T cells in peripheral LNs are normal in these mice, indicating that, ment of IKAP in peripheral target innervation and in specific JNK and NGF over time, FlnA-deficient T cells can accumulate in LNs. In ad- signaling in developing PNS neurons. PLoS One 9: e113428. Downloaded from dition to its role in lymphocyte homing, we also show that FlnA, 20. Lek, H. S., V. L. Morrison, M. Conneely, P. A. Campbell, D. McGloin, S. Kliche, C. Watts, A. Prescott, and S. C. Fagerholm. 2013. The spontaneously adhesive like b2 integrins (28) and kindlin-3 (23, 37), is required for T cell leukocyte function-associated antigen-1 (LFA-1) integrin in effector T cells trafficking into sites of inflammation (e.g., to the inflamed skin in mediates rapid actin- and calmodulin-dependent adhesion strengthening to li- a contact hypersensitivity model). gand under shear flow. J. Biol. Chem. 288: 14698–14708. 21. Tolic´-Nørrelykke, I. M., J. P. Butler, J. Chen, and N. Wang. 2002. Spatial and Taken together, our results show that FlnA regulates force temporal traction response in human airway smooth muscle cells. Am. J. Physiol. transmission through integrins in T cells and T cell shear flow Cell Physiol. 283: C1254–C1266. http://www.jimmunol.org/ 22. Krishnan, R., C. Y. Park, Y. C. Lin, J. Mead, R. T. Jaspers, X. Trepat, adhesion. Because of this role in integrin force transmission, FlnA G. Lenormand, D. Tambe, A. V. Smolensky, A. H. Knoll, et al. 2009. Rein- is required for T cell homing and trafficking into sites of inflam- forcement versus fluidization in cytoskeletal mechanoresponsiveness. PLoS One mation. However, it is not required for T cell activation in vivo, a 4: e5486. 23. Savinko, T. S., V. L. Morrison, L. M. Uotila, C. H. J. Wolff, H. T. Alenius, and process that occurs in lymphoid organs in the absence of shear flow. S. C. Fagerholm. 2015. Functional Beta2-Integrins restrict skin inflammation Our data therefore provide novel insights into how FlnA regulates in vivo. J. Invest. Dermatol. 135: 2249–2257. the function of T cells in vitro and in vivo. 24. Falet, H., A. Y. Pollitt, A. J. Begonja, S. E. Weber, D. Duerschmied, D. D. Wagner, S. P. Watson, and J. H. Hartwig. 2010. A novel interaction between FlnA and Syk regulates platelet ITAM-mediated receptor signaling and Disclosures function. J. Exp. Med. 207: 1967–1979.

25. Flanagan, L. A., J. Chou, H. Falet, R. Neujahr, J. H. Hartwig, and T. P. Stossel. by guest on September 28, 2021 The authors have no financial conflicts of interest. 2001. Filamin A, the Arp2/3 complex, and the morphology and function of cortical actin filaments in human melanoma cells. J. Cell Biol. 155: 511–517. 26. Glogauer, M., P. Arora, D. Chou, P. A. Janmey, G. P. Downey, and References C. A. McCulloch. 1998. The role of actin-binding protein 280 in integrin- 1. Chen, W., and C. Zhu. 2013. Mechanical regulation of T-cell functions. Immu- dependent mechanoprotection. J. Biol. Chem. 273: 1689–1698. nol. Rev. 256: 160–176. 27. Lynch, C. D., N. C. Gauthier, N. Biais, A. M. Lazar, P. Roca-Cusachs, C. H. Yu, 2. Sun, Z., S. S. Guo, and R. Fa¨ssler. 2016. Integrin-mediated mechano- and M. P. Sheetz. 2011. Filamin depletion blocks endoplasmic spreading and transduction. J. Cell Biol. 215: 445–456. destabilizes force-bearing adhesions. Mol. Biol. Cell 22: 1263–1273. 3. Miller, M. 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Blood 119: 4275–4282. integrin alpha and beta chain phosphorylations regulate LFA-1 activation 37. Cohen, S. J., I. Gurevich, S. W. Feigelson, E. Petrovich, M. Moser, G. Shakhar, through affinity-dependent and -independent mechanisms. J. Cell Biol. 171: R. Fassler, and R. Alon. 2013. The integrin coactivator Kindlin-3 is not required 705–715. for lymphocyte diapedesis. Blood 122: 2609–2617. Supplemental results

Savinko T et al. Filamin A is required for optimal T cell integrin-mediated force transmission, flow adhesion and T cell trafficking A B CD4Cre FlnA-CD4Cre

e e e r r r C C C 4 4 4 D D e D e e r r r f / f C /f C f/ C f f C C C

4 A 4 A 4 A 4 n D n D n D l

l l D C F C F C F C Filamin A B cells Neutrophils DCs

C CD8

S1. A. Filamin A is expressed in normally in B cells, neutrophils and dendritic cells in filamin A floxed/CD4Cre+ mice. B. CD4 and CD8 T cell subsets in thymus in control (CD4Cre) and Filamin floxed CD4-Cre mice. C. Migration of cultured effector CD4 T cells on ICAM-1. Savinko T et al. Filamin A is required for optimal T cell integrin-mediated force transmission, flow adhesion and T cell trafficking

A CD18 F CD11a CD29 CD49d beta7 I FITC+ ctrl PE+ 99,9 T 100 FITC+ PE+ 41,3 91,4 PerCP+ 4,06

FITC+ 99,9 PE+ FlnA-/- 100 FITC+ PE+ 37,7 91,8 PerCP+ 4,23

S2. Activation markers and homing receptors are normally expressed in the absence of filamin A in LNs of nontreated mice. A. The surface expression of beta2-integrins (CD18 and CD11a), beta1 (CD29), alpha4 (CD49d) and beta7-integrin in CD4 T cells is normal in the absence of filamin A. B. Activation markers and P-selectin are normally expressed in the absence of filamin A in T cells. Savinko T et al. Filamin A is required for optimal T cell integrin-mediated force transmission, flow adhesion and T cell trafficking A

Donor WT Donor FlnA-/-

S3. A. Representative flow cytometry data from in vivo T cell activation experiment shown in Fig 4 A. Cells were gated on live lymphocytes. B. Representative histograms showing CD69, CD25 and CD44 expressions in ctrl T cells (solid line) and in FlnA KO T cells (dashed line) in in vitro stimulated T cells. Savinko T et al. Filamin A is required for optimal T cell integrin-mediated force transmission, flow adhesion and T cell trafficking

IL-4 IL-13 A 15000.0 15000.0

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Foxp3 skin B 15000.0

10000.0 P=0.0599 U R 5000.0

0.0 Ctrl FlnaA-/- Oxa S4. Cytokine expression in the sensitized skin of Cre+ control and Flna-Cre mice in CHS model. A. IL-6, IL-4 and IL-13 mRNA expression and B. Foxp3 mRNA expression was measured by RT-PCR.