In Vivo Analysis of Uropod Function during Physiological Trafficking Silvia F. Soriano, Miroslav Hons, Kathrin Schumann, Varsha Kumar, Timo J. Dennier, Ruth Lyck, Michael Sixt This information is current as and Jens V. Stein of September 26, 2021. J Immunol published online 27 July 2011 http://www.jimmunol.org/content/early/2011/07/27/jimmun ol.1100935 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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 27, 2011, doi:10.4049/jimmunol.1100935 The Journal of Immunology

In Vivo Analysis of Uropod Function during Physiological T Cell Trafficking

Silvia F. Soriano,*,1 Miroslav Hons,*,1 Kathrin Schumann,† Varsha Kumar,* Timo J. Dennier,* Ruth Lyck,* Michael Sixt,†,‡ and Jens V. Stein*

Migrating lymphocytes acquire a polarized phenotype with a leading and a trailing edge, or uropod. Although in vitro experiments in cell lines or activated primary cell cultures have established that Rho-p160 coiled-coil kinase (ROCK)- II-mediated uropod contractility is required for de-adhesion on two-dimensional surfaces and nuclear propulsion through narrow pores in three-dimensional matrices, less is known about the role of these two events during the recirculation of primary, nonacti- vated lymphocytes. Using pharmacological antagonists of ROCK and myosin II, we report that inhibition of uropod contractility blocked integrin-independent mouse T through narrow, but not large, pores in vitro. T cell crawling on chemokine-

coated endothelial cells under shear was severely impaired by ROCK inhibition, whereas transendothelial migration was only Downloaded from reduced through endothelial cells with high, but not low, barrier properties. Using three-dimensional thick-tissue imaging and dynamic two-photon microscopy of T cell motility in lymphoid tissue, we demonstrated a significant role for uropod contractility in intraluminal crawling and transendothelial migration through lymph node, but not bone marrow, endothelial cells. Finally, we demonstrated that ICAM-1, but not anatomical constraints or integrin-independent interactions, reduced parenchymal motility of inhibitor-treated T cells within the dense lymphoid microenvironment, thus assigning context-dependent roles for uropod con-

traction during lymphocyte recirculation. The Journal of Immunology, 2011, 187: 000–000. http://www.jimmunol.org/

he high diversity of the Ag-receptor repertoire and the along the luminal surface of HEV, which is covered with high resulting low frequency of Ag-specific T cells in an or- levels of the CCL21 chemokine (4). Binding of CCL21 to its re- T ganism pose an important logistic challenge, which is to ceptor CCR7 induces rapid (.1 s) conformational activation of quickly allow for efficient encounters between APCs and rare the LFA-1 integrin required for firm arrest of rolling lymphocytes, specific T cells close to potential microbial entry sites. To meet and it induces over the next tens of seconds to few minutes the this challenge, strategically positioned secondary lymphoid organs, acquisition of a polarized phenotype (5–7). Polarized lymphocytes including spleen, peripheral lymph nodes (PLNs), and bone then crawl in an ICAM-1–dependent manner along the HEV by guest on September 26, 2021 marrow (BM), have evolved, and these secondary lymphoid organs surface prior to transendothelial migration (TEM) through narrow serve as meeting points for tissue-borne APCs and continuously pores of the endothelial barrier and negotiate their passage passing lymphocytes (1–3). Recruitment of blood-borne lympho- through the cuff of basement membrane meshworks surrounding cytes in PLNs occurs in high endothelial venules (HEVs), spe- fibroblast reticular cells (FRCs) and occasional pericytes (8–12). cialized postcapillary venules characterized by their expression FRCs constitute the stromal backbone of the T cell area in the of peripheral node addressin (PNAd). PNAd serves as a ligand for PLN parenchyma and form a three-dimensional (3D) network, L-selectin expressed on naive lymphocytes, allowing cells to roll which T cells use as contact guidance cues during their scanning of APCs inside the tightly packed lymphoid microenvironment (13). This migration is mediated, in part, by ICAM-1 and CCL21 † *Theodor Kocher Institute, University of Bern, 3012 Bern, Switzerland; Hof- presumably presented on the FRC surface, before T cells exit PLNs schneider Group Leukocyte Migration, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany; and ‡Institute of Science and Technology Austria, 3400 Klos- through efferent lymphatic vessels after an average dwelling time of terneuburg, Austria a few hours (14–16). CXCL12 and a4 are likely candi- 1S.F.S. and M.H. contributed equally to this study. dates for inducing firm adhesion and guiding naive lymphocytes Received for publication April 6, 2011. Accepted for publication June 24, 2011. through BM sinusoids into BM parenchyme (17). This work was supported by a Marie Curie Excellence grant (MEXT-CT-2005- The chemokine-driven rapid transition of spherical blood-borne 025405) from the European Union and Swiss National Foundation Grants 31-120640 lymphocytes to fully polarized cells with a leading edge and a and CRSII3-125447 (to J.V.S.). uropod is orchestrated by the concerted activation of small Address correspondence and reprint requests to Dr. Jens V. Stein, Theodor Kocher GTPases of the Ras and Rho family (7, 18). Although Rac1 and Institute, University of Bern, Freiestrasse 1, 3012 Bern, Switzerland. E-mail address: [email protected] Rac2 direct T cell protrusion at the leading edge (19), the for- The online version of this article contains supplemental material. mation of the uropod depends on the activity of Rho, and, at least in part, of its downstream effector Rho-p160 coiled-coil kinase Abbreviations used in this article: BM, bone marrow; CMAC, 7-amino-4-chlorome- thylcoumarin; CMFDA, 5-chloromethyfluorescein diacetate; CMTMR, (5-(and-6)- (ROCK) (5, 20). Nonmuscle myosin IIA also localizes to the (((4-chloromethyl)benzoyl)amino) tetramethylrhodamine); 2D, two-dimensional; 3D, uropod, where it promotes actin–myosin contractions downstream three-dimensional; 3-DIF, 3D immunofluorescence; FRC, fibroblast reticular cell; HEV, high endothelial venule; PFA, paraformaldehyde; PLN, peripheral lymph node; of ROCK-dependent phosphorylation of the myosin L chain, ei- 2PM, two-photon microscopy; pMBMEC, primary mouse brain microvascular endo- ther directly or indirectly by inactivating MLC phosphatase (21, thelial cell; PNAd, peripheral node addressin; ROCK, Rho-p160 coiled-coil kinase; 22). In vitro experiments support an important role for the Rho– TEM, transendothelial migration; WT, wild type. ROCK–myosin signaling axis and uropod contraction during Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 leukocyte migration in at least three processes. First, in two-

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100935 2 IN VIVO FUNCTION OF ROCK IN T CELLS dimensional (2D) systems, Rho–ROCK activity limits the adhe- Reagents sion interface, probably by promoting cortical tension. Therefore, Y27632 and blebbistatin were purchased from Calbiochem (San Diego, upon blocking ROCK activity using the pharmacological inhibi- CA). CCL21 and CXCL12 were provided by PeproTech (London, U.K.), tor Y27632 or myosin II ATPase activity with blebbistatin, cells and CXCL13 was from R&D Systems (Minneapolis, MN). CFSE, 5- spread more, even on nonadhesive substrates, thereby limiting chloromethyfluorescein diacetate (CMFDA; CellTracker Green), 7-amino- migration speed (23–27). Second, myosin contractility is required 4-chloromethylcoumarin (CMAC; CellTracker Blue), and (5-(and-6)-(((4- chloromethyl)benzoyl)amino) tetramethylrhodamine) (CMTMR; Cell- for LFA-1 de-adhesion during migration on ICAM-1–coated 2D Tracker Orange) were purchased from Molecular Probes (Eugene, OR). surfaces. Upon cell treatment with Y27632 or blebbistatin, leu- kocytes remain attached with their uropod, restraining net cell Lymphocyte-polarization assay displacement promoted by the probing leading edge (28–30). Fi- T cells were treated with DMSO (wild type [WT]), Y27632 (20 mM), or nally, uropod contractility is required for integrin-independent blebbistatin (30 mM) for 1 h; stimulated in suspension with 100 nM dendritic cell squeezing through 3D meshworks. This is mainly CCL21 final concentration; and immediately plated on fibronectin-coated due to the trapping of the relatively large nucleus in narrow pores; chamber slides for incubation at 37˚C. After 1 h, T cells were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature, per- thus, it can be modulated by increasing the pore size of the 3D meabilized in PBS/0.2% Tween-20 for 20 min, and blocked in PBS/5% network (31). Accordingly, T cell blast migration to CCL19 and BSA for 20 min. Immunostaining was performed with biotinylated anti- CCL21 in assays using 3-mm pores and dense collagen CD44 mAb (BioLegend, San Diego, CA) or PKCz mAb (Santa Cruz matrices is significantly diminished by ROCK inhibition (25, 32). Technology, Santa Cruz, CA), followed by labeling with Alexa Fluor 488- conjugated streptavidin or Alexa Fluor 555-conjugated anti-rabbit pAb, Thus far, most studies addressing the role of uropod contraction respectively (Invitrogen). Fluorescence and phase-contrast images were and de-adhesion in lymphocytes were performed in vitro with taken with a Zeiss AxioObserver microscope equipped with an Apotome, Downloaded from preactivated cells and on 2D surfaces, in the absence of shear using a 403 objective. The T cell length and width were measured using forces. Because T cell activation dramatically alters nuclear size, Zeiss imaging software and expressed as the polarity index (i.e., the ratio actocytoskeletal activity, and overall integrin avidity, the role of of length/width of the cell). ROCK–myosin II activity in the dynamic behavior of recirculat- Chemotaxis assay ing, nonactivated lymphocytes on different vascular beds under T or B cells were purified by negative immunomagnetic cell sorting (Dynal, shear has not been thoroughly investigated. Therefore, it is not Oslo, Norway) with purity yields .95%. Chemotaxis assays were per- http://www.jimmunol.org/ known whether ROCK activity is required for TEM in vivo or formed using Transwell chambers (3- or 5-mm pore size; CoStar, whether cells are trapped in the perivascular or parenchymal space Amsterdam, The Netherlands), with 100 ml cell suspension (5 3 106 cells/ when uropod contractility and integrin de-adhesion are impaired. ml) in the top chamber and indicated concentrations of CCL21, CXCL12, or CXCL13 in the bottom chamber. After 2 h at 37˚C, 7% CO2, the per- Similarly, although a recent study revealed that myosin II is re- centage of migrated cells was calculated by flow cytometry after com- quired for efficient lymphocyte migration within lymphoid paren- paring with a precalibrated bead standard (Sigma-Aldrich) and correcting chyma (27), it remains unclear whether this is a consequence of for variations in input concentrations. ICAM-1–induced retention, impaired nuclear propelling owing to Collagen matrix assay the dense microenvironment, or nonspecific overadhesiveness to adjacent cells present within lymphoid tissue. Taken together, the Three-dimensional collagen gel chemotaxis assays were done as described by guest on September 26, 2021 3 importance of ROCK–myosin function in naive lymphocytes dur- previously (31). Briefly, PureCol (INAMED, Fremont, CA) in 1 Eagle’s MEM (Sigma-Aldrich) and 0.4% sodium bicarbonate (Sigma-Aldrich) was ing their trafficking routes through various endothelial barriers and mixed with OTII T cell blasts in RPMI 1640 (Invitrogen) and 10% FCS lymphoid parenchyme displaying site-specific microarchitectural (Invitrogen) at a ratio resulting in gels with the indicated concentration. constraints is not well described. Final cell concentrations in the assay were 106 cells/ml gel. Collagen–cell In this study, we analyzed the impact of impaired ROCK and mixtures were cast in custom-made migration chambers with a thickness of 0.5–1 mm. After 30-min assembly of the gels, cells were recorded with myosin II activity on transmigration of primary mouse lympho- custom-built brightfield video microscopy setups using a 10-s time lapse. cytes in vitro and in vivo, using pharmacological inhibitors com- Cells were manually tracked using the ImageJ chemotaxis plug-in. bined with live imaging. Our data suggested that, similar to other Flow chamber assays cell types, the in vitro efficiency of integrin-independent directed T cell migration depends on intact uropod contractility when the These assays were essentially carried out as described, using primary mouse pore diameter is small. Under flow conditions, CCL21-induced brain microvascular endothelial cells (pMBMECs) or the endothelial cell line crawling and transmigration are impaired in Y27632-treated bEnd5 (19). In brief, a custom-built laminar flow chamber was placed on the CCL21-coated pMBMEC or bEnd5 monolayer or on ICAM-1–coated slides T cells in an endothelial cell type-dependent manner in vitro, and mounted on the stage of a computer-controlled inverted microscope correlating with the endothelial barrier properties. To investigate equipped for automated fluorescence and phase-contrast imaging (Axi- the function of ROCK in vivo, we applied a novel thick-tissue– oObserver Z1; Zeiss, Feldbach, Switzerland). All flow experiments were imaging approach to examine T cell entry into PLN and BM conducted inside a heat-controlled chamber at 37˚C. DMSO- and Y27632- treated T cells (5 3 106 cells/ml) labeled with CellTracker dyes (CFSE, parenchyme and found an organ-specific defect in T cell homing 2.5 mM and CMTMR, 5 mM; 20 min at 37˚C) were perfused with an auto- in the presence of Y27632. Finally, two-photon microscopy (2PM) mated syringe pump (Harvard Apparatus) at a wall shear stress of 0.24 dyne/ experiments of T cell behavior within PLNs of live, anesthetized cm2 for 1–2 min to allow cells to accumulate on the endothelium or on 2 mice revealed that TEM and parenchymal motility were impaired ICAM-1. Subsequently, shear flow was increased to 1 dyne/cm and kept in the absence of ROCK activity, the latter in an ICAM-1– constant during the remaining recording period, while time-lapse images (one frame every 20 or 30 s) were taken. All images were recorded through dependent manner. a203 or 403 objective (LD Plan-Neofluar; Zeiss, Feldbach, Switzerland) with phase or differential interference contrast and fluorescence illumina- tion. Image analysis was performed using ImageJ software using the manual Materials and Methods tracking, chemotaxis and migration tools, and color-merging plug-ins. Mice Short-term homing and 3D immunofluorescence Five- to ten-week-old male and female C57BL/6 mice were purchased from Harlan and used for all experiments. ICAM-1–deficient mice on the These assays were essentially carried out as described (12). Purified T cells C57BL/6 background were described previously (33). All experiments (1 3 107 cells/ml) in RPMI 1640/10% FCS were labeled with CellTracker were performed with the approval of and in accordance with kantonal and Blue (CMAC; 20 mM), CellTracker Green (CMFDA; 3 mM), or Cell- federal animal-experimentation regulations. Tracker Orange (CMTMR; 5 mM) for 20 min at 37˚C. CMAC-labeled The Journal of Immunology 3

T cells were additionally treated with DMSO, whereas CMFDA- and CCL21 through filter inserts with 5-mm pores. In contrast, the mi- CMTMR-labeled cells were incubated with Y27632 (20 mM) or blebbis- gration of Y27632 or blebbistatin-treated T cells was reduced by m 3 7 tatin (30 M) for 1 h. After washing, 1 10 cells of each was were half compared with control T cells when the pore diameter was only mixed in a 1:1:1 ratio and injected i.v. into age- and sex-matched C57BL/6 m recipient mice. Twenty minutes after cell transfer, 100 mg Mel-14 mAb 3 m (Fig. 1B). We observed a similar reduction when the inhibitors (anti–L-selectin) and 5 mg Alexa Fluor 633-conjugated MECA-79 mAb were present throughout the chemotaxis assay (data not shown). In (anti-PNAd) were injected i.v.; 20 min later, mice were killed using iso- B cells, Y27632 and blebbistatin inhibited migration through 5-mm flurane (Minrad, Buffalo, NY), perfused with 5 ml cold PBS to wash off pore sizes but with less efficacy than when using inserts with 3-mm blood-borne cells, and fixed in 10 ml cold PBS/1% PFA. PLNs were collected and carefully cleaned from surrounding fat tissue under a ste- pore diameters (Fig. 1C). When we examined the reversibility of reomicroscope. After fixation for 1 h in PBS/1% PFA, PLNs were rinsed in both inhibitors, we observed continuous inhibition of migration PBS and embedded in 1.3% low melting point agarose (Sigma). Agarose through 3 mm pores for up to 2 h after treatment in naive, non- blocks containing fixed PLNs were excised and dehydrated with 100% activated T cells, whereas the inhibitory action of both drugs was MeOH for 6 h before tissue clearing in benzyl alcohol–benzyl benzoate rapidly reversed in B cells (data not shown). (1:2 ratio) for $12 h. Blocks (500 3 500 3 400–800 mm) of agarose- embedded PLNs were imaged using a 2PM setup (TrimScope; LaVision To mimic more complex migration conditions, we performed mi- Biotec, Bielefeld, Germany) and, in some cases, were spectrally unmixed gration experiments in 3D collagen matrices with varying collagen using Imspector software. The absolute number and localization of dif- densities (31). In low-density collagen matrices, Y27632 blocked the ferentially labeled T cells within a tissue block were determined using migratory speed of T cell blasts toward CCL19 by 32% compared Volocity (Improvision; PerkinElmer, Cambridge, U.K.). For 3D immunofluorescence (3-DIF) of BM, purified T cells (1 3 107 with control T cells. With increased collagen density and reduced cells/ml in RPMI 1640/10% FCS) were labeled with CMTMR (5 mM) for pore size, Y27632 inhibited the T cell velocity by 52% (Fig. 1D).

20 min at 37˚C and additionally treated with DMSO or Y27632 (20 mM) In summary, our data showed a pore size-dependent role for Downloaded from 7 for 1 h. A total of 1 3 10 cells was injected i.v. into age- and sex-matched ROCK and myosin II-triggered uropod contraction or maintenance m C57BL/6 recipient mice. Twenty minutes after cell transfer, 300 g Alexa of cortical tension in T and B cells, in the absence of integrin- Fluor 633-conjugated tomato lectin (Sigma) was injected i.v. to label the vasculature. Twenty minutes later, mice were killed and perfused as above. mediated retention. The lack of inhibition of T cell migration Long bones (femur and tibia) were collected, frozen, and cut in pieces 1–2 through large pores also indicated unimpaired promigratory capa- mm in length. Still-frozen bone pieces were dehydrated and cleared as bilities in the presence of Y27632 and blebbistatin. Based on the above. Blocks (500 3 500 3 300–500 mm) of agarose-embedded bone reversibility of Y27632 and blebbistatin inhibition in B cells, we http://www.jimmunol.org/ pieces were imaged and analyzed as above. focused additional analyses on ROCK and myosin II function in 2PM of popliteal PLNs naive T cells. m Purified T cells were treated with DMSO or Y27632 (20 M) for 1 h and T cell crawling and TEM under flow are inhibited by Y27632 in fluorescently labeled with 2.5 mM CMTMR or CFSE for 15 min at 37˚C. After washing, labeled T cells were injected i.v. into sex-matched 5–10- an endothelium-specific manner 2/2 wk-old anesthetized C57BL/6 or ICAM-1 recipient mice surgically We next investigated the requirement of ROCK activity for TEM prepared to expose the right popliteal lymph node, as described (12, 34). Immediately after injection, 2PM imaging (TrimScope) was performed under flow, to mimic physiological barrier conditions encountered with an Olympus BX50WI fluorescence microscope and a 203 objective in vivo. Based on the above findings, we set out to examine (NA 0.95). For four-dimensional analysis of cell migration, 11–25 z-stacks dynamic T cell crawling and transmigration through endothelial by guest on September 26, 2021 (spacing 2.5–4 mm) of 250–300 3 250–300 mm x–y sections were ac- beds showing a complete, or incomplete, tight junction organi- quired every 20 or 30 s for 30–45 min. Imaging was performed in the zation and overall permeability. First, we superfused control or T cell area identified by the presence of HEVs labeled using Alexa Fluor a 633-conjugated MECA-79. Sequences of image stacks were transformed Y27632-pretreated T cells over TNF- –activated and CCL21- into volume-rendered four-dimensional movies using Volocity software, coated pMBMECs, which possess a low permeability rate (35). which was also used for semiautomated tracking of cell motility in three Under these conditions, control T cells exhibited dynamic motility dimensions. The average track velocity and turning angles (defined as the and transmigration (Fig. 2A,2B, Supplemental Video 1), with angle between the two velocity vectors before and after a measurement 6 m time point) were calculated from the x, y, and z coordinates of cell cent- a mean luminal crawling velocity of 5.7 0.2 m/min (Fig. 2B). roids, as described (12, 34). Occasionally, we observed a sudden change in the directionality of crawling T cells in line with the direction of shear flow, whereas Statistical analysis the uropod anchored crawling T cells on the pMBMECs (data not The Student test, Mann–Whitney U test, or one-way ANOVAwas used for shown). statistical analysis (Prism; GraphPad Software). Significance was set at Contrary to T cell blasts (29, 30), we rarely observed naive T cells p , 0.05. Data are presented as mean 6 SD, unless otherwise indicated. with elongated tails in the presence of Y27632 (data not shown). However, compared with control T cells, Y27632-treated T cells Results moved more slowly on pMBMECs (3.4 6 0.2 mm/min; Fig. 2B, ROCK and myosin II are required for chemokine-driven in vitro Supplemental Video 1). The overall percentage of Y27632-treated migration of lymphocytes in a pore size-dependent manner T cells undergoing transmigration in the recording period was re- Integrin-independent migration of dendritic cells through narrow duced by 74% (from 51.1 6 18.4% to 13.5 6 3.4%; Fig. 2C). 3D matrices requires uropod contractility (31). We examined Occasionally, we observed Y27632-treated T cells protruding a whether this was also relevant for CCR7-dependent migration of lamellipodium underneath the endothelium in an apparent attempt naive T cells in vitro. To block uropod contraction, we used the to transmigrate but apparently were unable to translocate the main ROCK inhibitor Y27632 or the myosin II inhibitor blebbistatin. To cell body containing the nucleus through the endothelial pore (Fig. verify the inhibitory activity, we analyzed chemokine-induced 2A, Supplemental Video 1). These data suggested that uropod polarization in the presence or absence of Y27632 and blebbis- contraction is central for efficient passage through a tight endothe- tatin. Both inhibitors significantly reduced the formation of an lial cell layer. Furthermore, Y27632-treated T cells, which managed elongated uropod in naive T cells, without affecting the polar to transmigrate, moved more slowly underneath pMBMECs com- distribution of leading and trailing edge markers (Fig. 1A). pared with control cells, indicating a role for uropod contraction or In chemotaxis experiments, we observed a pore size-dependent cortical tension in this constrained environment (Fig. 2D). effect on T cell migration after inhibition of ROCK or myosin II. The brain-derived endothelial cell line bEnd5 is characterized by Controlandinhibitor-pretreatedTcellsmigratedcomparablytoward its .10-fold greater permeability rate and its less elaborate tight 4 IN VIVO FUNCTION OF ROCK IN T CELLS Downloaded from

FIGURE 1. ROCK and myosin inhibition reduces chemokine-induced in vitro migration of naive T and B cells through narrow pores. A, Left panels, Y27632 and blebbistatin block CCL21-induced uropod induction in naive T cells. Although uropod formation is inhibited by Y27632 and blebbistatin, the CCL21-induced segregation with PKCz at the leading edge and CD44 at the trailing edge is largely intact in these cells. Scale bar, 10 mm. Right panel, Polarity index in control and Y27632- and blebbistatin-treated T cells. The data are from one experiment with three individual slides per condition. We analyzed 134 individual control T cells, 137 Y27632-treated T cells, and 123 blebbistatin-treated cells. ***p , 0.001. B, T cell migration to CCL21 through http://www.jimmunol.org/ Transwell membranes with pore diameters of 5 mm(left panel)or3mm(right panel). Purified DMSO-, blebbistatin-, or Y27632-pretreated naive T cells were allowed to migrate for 2 h toward 100 nM CCL21. C, B cell migration to CCL21 through Transwell membranes with pore diameters of 5 mm(left panel)or3mm(right panel). Purified DMSO-, blebbistatin-, or Y27632-pretreated B cells were allowed to migrate for 2 h toward 100 nM CXCL13. Data represent mean 6 SD of three independent experiments. D, 3D migration velocity of T cell blasts in collagen matrices of low (1.6 mg/ml) and high (4.27 mg/ml) density, corresponding to large and small pore sizes, respectively. Data are from four experiments. *p , 0.05, ***p , 0.001, compared with DMSO-treated cells (ANOVA). n.s., not significant. junction organization compared with pMBMECs (35). Superfused in vitro. We next wished to determine, in vivo, whether the im- control T cells moved efficiently on and through CCL21-coated portance of ROCK function for TEM differs in vascular beds with by guest on September 26, 2021 bEnd5 cells (Fig. 2E,2F, Supplemental Video 2), with a mean variable permeability characteristics. We chose to compare HEV migration velocity of 3.9 6 0.3 mm/min. Inhibition of ROCK by and BM sinusoids, because the latter are fenestrated with a high Y27632 reduced luminal T cell crawling velocity to 3.3 6 0.3 level of permeability and, therefore, may present an incomplete mm/min (Fig. 2F, Supplemental Video 2). Nonetheless, in marked barrier for transmigrating T cells (36). We performed short homing contrast to pMBMECs, both T cell populations showed a compa- assays of fluorescently labeled control or blebbistatin- or Y27632- rable TEM efficiency (Fig. 2G), indicating that uropod contrac- treated T cells, followed by thick-tissue imaging of PLNs and BM tility is not absolutely required for crossing a “permissive” en- slices using 3-DIF reconstructions (Fig. 3A) (12). The short total dothelial cell layer. transfer time (40 min) ensured continued efficacy of the inhibitors, Because activated endothelial cells express high surface levels of whereas the large tissue volume permitted the detection of a suf- ICAM-1, and based on the observed anchoring of luminal crawling ficient number of adoptively transferred cells for a meaningful T cells, we reasoned that the reduced luminal crawling velocity in analysis. Furthermore, the analysis of single z planes permitted the Y27632-treated T cells might be due, at least in part, to deficient precise intra- or perivascular localization of transferred T cells LFA-1 de-adhesion. To directly test this hypothesis, we superfused (Fig. 3B,3C). control and Y27632-treated T cells on coated rICAM-1 and CCL21. When we analyzed thick BM sections, we observed an efficient After allowing their accumulation at low shear, we increased the accumulation of control T cells in BM parenchyma, with 10.1 6 shear rates to physiological levels and analyzed the dynamic craw- 4% of transferred T cells being intravascular and 22 6 3.8% being ling behavior of control and inhibitor-treated cells. Y27632 signi- perivascular (Fig. 3D). Y27632 pretreatment did not alter the ficantly reduced T cell migration velocity on purified ICAM-1 + distribution of transferred T cells (9.7 6 2.6% intravascular and CCL21 from 9.2 6 6.2 to 4.7 6 2.4 mm/min (Fig. 2H), supporting 24.3 6 6.4% perivascular; Fig. 3D), indicating that ROCK func- the notion that the reduced motility on endothelial cells in the tion is not required for efficient TEM through the single layer of presence of Y27632 is due to reduced detachment during crawling. BM sinusoidal cells. In summary, although efficient cell crawling on endothelium Although the total number of T cells per PLN volume, as seemed to require LFA-1 de-adhesion, defective TEM of Y27632- detected by 3-DIF, was not affected by inhibition of ROCK or treated T cells through pMBMECs was reminiscent of impaired myosin II (data not shown), we observed a shift in the micro- propulsion of the main cell body through the endothelial pore. compartmental distribution of adoptively transferred Y27632- or blebbistatin-treated T cells within lymphoid tissue. A significantly T cell homing through HEVs, but not BM sinusoids, is reduced higher percentage of transferred inhibitor-treated T cells was pre- by inhibition of ROCK and myosin II sent in the intraluminal compartment and the perivascular space The data accumulated thus far argued for a role for uropod con- (Fig. 3E) compared with control T cells. Similar results were ob- tractility in the passage of naive T cell through narrow pores tained when the short-term homing assay was limited to a total of The Journal of Immunology 5

Y27632-treated T cells show reduced luminal crawling velocity and prolonged TEM and perivascular dwelling time on HEVs in vivo T cell homing to PLNs is a multistep process in which T cells adhere via LFA-1–ICAM-1/2–mediated interactions, followed by an optional ICAM-1–dependent crawling step and a rapid TEM event, which occurs efficiently in the absence of ICAM-1 or ICAM-2 (10–12). Successfully transmigrated T cells remain in the perivascular space for a few minutes, presumably negotiating their passage into the lymphoid parenchyme (12). Based on the ob- servation of impaired T cell accumulation within parenchymal lymphoid tissue after Y27632 and blebbistatin treatment, we set out to delineate, in more detail, the role for ROCK functionality during intraluminal crawling, transmigration, and perivascular motility of single cells. We adoptively transferred fluorescently labeled control and Y27632-treated T cells into recipient mice surgically prepared to expose the popliteal PLN and that had re- ceived fluorescently labeled MECA-79 to identify the HEVoutline 15 min prior to T cell transfer, as well as fluorescent dextran as Downloaded from plasma marker in some experiments. Using 2PM of popliteal PLNs, we carefully tracked the transferred T cells from the site of adhesion until their appearance in the lymphoid parenchyme (Fig. 4A, Supplemental Video 3). In accordance with published data, we found that most control T cells directly egressed from the site of adhesion into the underlying perivascular space. Similarly, the http://www.jimmunol.org/ majority of Y27632-treated T cells directly egressed from the site of adhesion into the underlying tissue (Supplemental Videos 3, 4). The percentage of intraluminally crawling T cells, defined as lu- minal postadhesion migration for .20 mm (12), was not reduced FIGURE 2. T cell transmigration through endothelium with low, but not by Y27632 treatment (13/46 total tracks = 28% for control versus high, permeability requires uropod contractibility. Purified T cells were 13/48 total tracks = 27% for Y27632-treated T cells). However, superfused on CCL21-coated pMBMECs, bEnd5, or purified ICAM-1 and CCL21 and tracked from the start of high shear conditions. A, Represen- we found that the intraluminal crawling velocity was reduced significantly from 10.5 6 1.9 mm/min in control T cells to 6.5 6 tative micrographs of control (green) and Y27632-treated (red) T cells by guest on September 26, 2021 during transmigration through pMBMECs cells. Arrowheads indicate 0.6 mm/min (mean 6 SEM) in Y27632-treated T cells (Fig. 4B). lamellipodia protrusion underneath the endothelium. The same endothelial In inhibitor-treated T cells, the time required for HEV trans- spot was filmed during this image sequence (Supplemental Video 1). Time migration was increased significantly (Fig. 4C), whereas the per- is shown in minutes and seconds. Scale bar, 10 mm. B, Crawling velocity ivascular velocity was decreased (Fig. 4D), and the meandering of control and Y27632-treated T cells on pMBMECs. C, Percent TEM of index as an indicator of directionality showed a nonsignificant control and Y27632-treated T cells on pMBMECs. The TEM efficiency of increasing tendency in Y27632-treated T cells (Fig. 4E). control and Y27632-treated cells is expressed as the percentage of trans- Taken together, our dynamic single-cell analysis uncovered migrating cells over total adherent cells at the beginning of the recording. multiple roles for ROCK during LFA-1–dependent intraluminal D, Migration velocity of control and Y27632-treated T cells below pMBMECs after TEM. Data in B–D are pooled from five videos in three crawling and regulating the transmigration time through HEV. independent experiments, with 222 control and 111 Y27632-treated total T Similarly, the ICAM-1–independent exit from the perivascular cell tracks. E, Representative micrographs of control (green) and Y27632- area was delayed in Y27632-treated T cells. treated (red) T cells transmigrating through bEnd5 cells. Arrowheads in- dicate lamellipodia protrusion underneath the endothelium. Time is shown Y27632 treatment reduces parenchymal T cell motility in an m in minutes and seconds. Scale bar, 10 m. F, Crawling velocity of control ICAM-1–dependent manner and Y27632-treated T cells on bEnd5 cells. G, Percent TEM of control and Y27632-treated T cells on bEnd5 cells. The TEM efficiency of control and T cells rapidly migrate inside lymphoid parenchyma, guided by Y27632-treated cells is expressed as percentage of transmigrating cells over the FRC network (13). We explored whether ROCK function total adherent cells at the beginning of the recording. Data in F and G are was required to move through the densely populated lymphoid pooled from five videos in three independent experiments, with 351 control microarchitecture. We transferred fluorescently labeled control and 259 tracked Y27632-treated T cells, respectively. H, Migration velocity and Y27632-treated T cells and observed their parenchymal mo- of control and Y27632-treated T cells on purified ICAM-1 + CCL21 under tility immediately after transfer (,70 min) using 2PM of pop- shear. Each dot in B, D, F, and H represents one track. *p , 0.05, ***p , liteal PLNs. Although both populations showed comparable di- 0.001, compared with control T cells. n.s., not significant; Y27, Y27632. rectionality, as assessed by turning angles, Y27632-treated T cells moved significantly slower than did control T cells (13.2 6 3.8 20 min, with anti–L-selectin mAb treatment 10 min after adoptive mm/min and 15.8 6 4.0 mm/min, respectively; Fig. 5A). Al- transfer (data not shown). though this seems to be a modest decrease, the difference in ve- In summary, analogous to our results from the in vitro experi- locity distributions resulted in a 45% reduction in the motility ments, our thick-tissue–image analysis uncovered a role for ROCK coefficients, from 130 mm2/min to 71 mm2/min for control and and myosin II activity during T cell homing through the more Y27632-treated T cells, respectively. We then investigated restrictive endothelium of lymphoid tissue, although numerous whether the reduced migration velocity was due to spatial con- T cells were still able to cross HEVs. straints imposed by the dense lymphoid microenvironment, thus 6 IN VIVO FUNCTION OF ROCK IN T CELLS Downloaded from http://www.jimmunol.org/

FIGURE 3. 3-DIF reveals a role for uropod contractility during T cell crossing of the HEVs, but not the BM sinusoid barrier. A, Schematic outline of 3- DIF. Fluorescently labeled T cells pretreated with DMSO, blebbistatin, or Y27632 were adoptively transferred into wild-type recipient mice. For BM 3-DIF (i), Alexa Fluor 633-conjugated tomato lectin was injected after 20 min to label the vasculature, whereas for PLN 3-DIF (ii), Alexa Fluor 633-conjugated MECA-79 and anti–L-selectin were injected to block further homing. Twenty minutes later, long bones (femur and tibia) or PLNs were collected from recipient mice and prepared for 3-DIF. B, 3-DIF xyz projection of BM sinusoids (yellow) embedded in autofluorescent BM parenchyme (blue) and sur-

rounded by bone (black). The precise localization of adoptively transferred T cells (red) is identified in each z-slice for the intravascular (arrow) and by guest on September 26, 2021 perivascular (arrowhead) compartment. C, 3-DIF xyz projection of PLN HEVs (yellow) with control (blue) and blebbistatin (green)- or Y27632 (red)- treated T cells. Each individual cell is assigned an intravascular (arrow), perivascular (arrowhead), or parenchymal location, as defined by its position relative to the MECA-79+ lining. D, Percentage of control and Y27632-treated T cells in the intraluminal (left panel) and perivascular (right panel) compartment, defined by their position relative to the tomato lectin+ lining. Each dot represents the percentage of an individual BM scan from a total of two recipient mice for control cells and two recipient mice for Y27632-treated T cells. E, Percentage of control or blebbistatin- or Y27632-treated T cells in the intraluminal (left panel) and perivascular (right panel) compartment, defined by their position relative to the MECA-79+ lining. Each dot represents the percentage of an individual PLN scan from a total of two recipient mice for control and inhibitor-treated T cells. **p , 0.01, ***p , 0.001, compared with control cells (ANOVA). Blebb, blebbistatin; n.s., not significant; Y27, Y27632. impairing nucleus propulsion in the absence of uropod contraction other hematopoietic and nonhematopoietic cells. Within tissue, (31), or to integrin-independent promiscuous cell attachment due polarized lymphocytes acquire a largely integrin-independent mi- to reduced cortical control (27) or, alternatively, whether impaired gratory phenotype characterized by a forward-pushing lamelli- uropod detachment from ICAM-1 caused T cell slowing in the podium and a contractile uropod under control of ROCK and presence of Y27632. To this end, we carried out 2PM experiments myosin II. In this study, we analyzed the importance of a contractile with control and Y27632-treated T cells in ICAM-12/2 PLNs, uropod and the maintenance of cortical control for efficient traf- which have an overall cell density, microarchitecture, and, thus, ficking of naive T cells under physiological conditions in vitro and spatial constraints comparable to wild-type PLNs (12). As repor- in vivo, thereby indirectly examining the anatomical constraints ted (12, 14), the absence of LFA-1–ICAM-1 interactions resulted imposed by varying endothelial barriers and lymphoid microen- in reduced migration speed and increased turning angle distribu- vironments. To distinguish between the various roles for ROCK and tion in control T cells. Of note, Y27632-treated T cells migrated myosin II during lymphocyte–stroma interactions, such as integrin with speeds and turning angles comparable to control T cells de-adhesion, decrease of promiscuous cell attachment, and nucleus (12.8 6 3.9 mm for control versus 12.6 6 3.5 mm for Y27632- propulsion, we carefully dissected potential pathways using a com- treated T cells; Fig. 5B). In summary, these data suggested that, bination of in vitro and in vivo experiments in the presence or ab- somewhat unexpectedly, the dense lymphoid microarchitecture does sence of integrin ligands and shear. Our findings confirmed that, in not constitute an obstacle for the rigid nucleus of migrating T cells, the absence of shear and integrin-mediated adhesion, the pore rather the specific detachment from adhesive LFA-1–ICAM-1 in- size determined efficient lymphocyte migration toward homeostatic teractions requires ROCK–myosin II action. chemokines in vitro. Furthermore, we found that ROCK–myosin II- induced uropod contractility and preservation of cortical tension are Discussion invariably required for efficient T cell crawling on various vascular As an adaptation to their motile life style, recirculating lympho- beds in vitro, probably, in part, to overcome LFA-1–ICAM-1 re- cytes are undergoing numerous transient adhesive interactions with tention of the uropod. In contrast, efficient in vitro TEM in the The Journal of Immunology 7

chyme, whereas the tightly packed, yet dynamic, microenvironment does not strongly interfere with the propulsion of T cell nuclei. In line with a central role for actin polymerization at the leading edge of migrating cells, intraluminal crawling and interstitial motility are virtually abolished in the absence of the Rac guanine exchange factor DOCK2 or its downstream effectors Rac1 and Rac2, although the shear-induced transmigration capacity was less affected (12, 19, 37, 38). Although it has long been recognized that ROCK or myosin II inhibition resulted in impaired crawling of activated T cells on 2D surfaces coated with integrin ligands, resulting in a strongly elon- gated cell shape (29, 30), the requirement for ROCK–myosin II functionality during physiological migration of nonactivated naive T cells has only recently started to be investigated (27). In this study, we followed up on in vitro observations to investigate whether ROCK–myosin II function is also central during recirculation pathways of naive T cells. To this end, we used the ROCK inhibitor FIGURE 4. 2PM analysis of control and Y27632-treated T cell crawling Y27632 and the myosin II inhibitor blebbistatin, and exploited the and HEV transmigration. A, Representative images of control (green) and fact that these compounds remained active in naive T cells for up to

Y27632-treated (red) T cells transmigrating through an HEV (shown 2 h after washing off. This allowed us to follow inhibitor-treated Downloaded from brown in the top panel and omitted for clarity in the bottom panel). naive T cells immediately after superfusion onto endothelial cells Arrowheads show examples of successfully emigrated T cells. Time is in vitro or posttransfer into recipient hosts in vivo. After adhesion shown in minutes and seconds. Scale bar, 20 mm. B, Migration velocity of control and Y27632-treated T cells crawling on the intraluminal side of to a CCL21-loaded endothelial cell surface, blood-borne non- HEVs for .20 mm prior to TEM. C, Transmigration time of control and polarized T cells undergo a rapid polarization, which is charac- Y27632-treated T cells through HEVs. D, Migration velocity of control terized by the acquisition of a protruding lamellipodium and a and Y27632-treated T cells in the perivascular area. E, Meandering index contractile uropod. Although naive T cells crawling on ICAM-1 do http://www.jimmunol.org/ of control and Y27632-treated T cells in the perivascular area. Each dot in not show an elongated cell shape, as observed in T cell blasts, B–E represents a single cell track. Data are pooled from three independent perhaps owing to reduced overall adhesiveness and lower lamelli- experiments with 13 (B) and 33–49 tracks (C–E) of control and Y27632- podia protrusive activity, our findings suggested that uropod con- treated T cells. *p , 0.05, **p , 0.01, ***p , 0.001, compared with tractility facilitates trailing edge release during physiological mi- control T cells. n.s., not significant; Y27, Y27632. gration on 2D surfaces. We also showed that Y27632 reduced crawling velocity on HEVs, supporting a general role for ROCK absence of ROCK function is only affected through endothelial function during intraluminal crawling. cells with low permeability. Similarly, in vivo T cell TEM was more Our data uncovered an endothelial-dependent requirement for dependent on ROCK function when crossing PLN HEVs than BM intact ROCK–myosin II signaling for efficient TEM, which cor- by guest on September 26, 2021 sinusoids. Finally, our data suggested that tissue-expressed ICAM-1 related with the degree of tight endothelial junction formation. In retains Y27632-treated T cells migrating inside lymphoid paren- the bEnd5 endothelial cell line, the tight junction formation is less prominent than in pMBMECs, with the latter expressing higher levels of the tight junction protein occludin-1 (35). We took ad- vantage of these differences to correlate their permeability prop- erties with the requirement of ROCK activity for TEM. A novel finding reported in this article is that endothelial properties impose differing constraints on efficient lymphocyte TEM. Although this suggests that lymphocytes use the paracellular, rather than the transcellular, TEM pathway in vitro, we cannot exclude that fac- tors in addition to the increased tight junction functionality of primary endothelial cells compared with immortalized cell lines affect the transcellular TEM efficiency. This is particularly rele- vant because pMBMECs display different F-actin networks, which may influence transcellular TEM (35). Irrespective of the TEM pathway, we hypothesize that the pore size is the decisive factor for the requirement of ROCK activity. Because anatomical studies identified a pore size of 2.2–2.5 mm through HEVs (9), and our in vitro studies indicated a strong reduction in directed motility of Y27632-treated T cells through 3-mm pores, we anticipated a FIGURE 5. 2PM analysis of uropod contractility during parenchymal strong inhibition through Y27632 for TEM through HEVs. Al- T cell migration in control and ICAM-1–deficient hosts. A, Average mi- though we observed significant defects in T cell crawling, TEM, gration velocity (left panel) and turning angles (right panel) of control and and passage of the perivascular space by ROCK inhibition, the Y27632-treated T cells migrating inside lymphoid parenchyme of wild- blocking effect was less pronounced than predicted. This indicated type mice. Data are pooled from five mice/six videos/177 and 138 tracks that, under physiological conditions, HEVs are more flexible than for control and Y27632-treated T cells, respectively. B, Average migration velocity (left panel) and turning angles (right panel) of control and anticipated by morphological studies and/or the reduced thickness Y27632-treated T cells migrating inside lymphoid parenchyme of ICAM- of HEVs compared with Transwell filter inserts masks the re- 12/2 mice. Data are pooled from four mice/eight videos/314 and 204 quirement for uropod contractility under these conditions. tracks for control and Y27632-treated T cells, respectively. ***p , 0.001. Previous studies uncovered a central role for ROCK–Rho– n.s., not significant; Y27, Y27632. myosin II-mediated uropod contractility for squeezing the rigid 8 IN VIVO FUNCTION OF ROCK IN T CELLS leukocyte nucleus through narrow pores in reconstituted 3D 2. Cyster, J. G. 2005. Chemokines, sphingosine-1-phosphate, and cell migration in secondary lymphoid organs. Annu. Rev. Immunol. 23: 127–159. microenvironments (25, 31). Furthermore, efficient dendritic cell 3. Junt, T., E. Scandella, and B. Ludewig. 2008. Form follows function: lymphoid entry into afferent lymphatic vessels requires myosin II function tissue microarchitecture in antimicrobial immune defence. Nat. Rev. Immunol. 8: (39), and Y27632 reduces neutrophil motility in inflamed tissue 764–775. 4. Stein, J. V., A. Rot, Y. Luo, M. Narasimhaswamy, H. Nakano, M. D. Gunn, (40). In line with these observations, the increased transmigration A. Matsuzawa, E. J. Quackenbush, M. E. Dorf, and U. H. von Andrian. 2000. time through HEVs and the delayed motility of Y27632-treated The CC chemokine thymus-derived chemotactic agent 4 (TCA-4, secondary cells in the perivascular space, which is characterized by a porous lymphoid tissue chemokine, 6Ckine, exodus-2) triggers lymphocyte function- associated antigen 1-mediated arrest of rolling T lymphocytes in peripheral and thick basement membrane and stromal cuffs (12, 13), support lymph node high endothelial venules. J. Exp. Med. 191: 61–76. the concept of nuclear trapping in environments with small pores. 5. Sa´nchez-Madrid, F., and M. A. del Pozo. 1999. Leukocyte polarization in cell Furthermore, our data indicated that Y27632 reduces parenchy- migration and immune interactions. EMBO J. 18: 501–511. 6. Kinashi, T. 2005. Intracellular signalling controlling integrin activation mal T cell motility. A recent study showed that myosin II deficiency in lymphocytes. Nat. Rev. Immunol. 5: 546–559. induced the overall adhesiveness of preactivated T cells through 7. Thelen, M., and J. V. Stein. 2008. How chemokines invite leukocytes to dance. Nat. Immunol. 9: 953–959. increased contact areas with surrounding interfaces, rather than 8. Miyasaka, M., and T. Tanaka. 2004. Lymphocyte trafficking across high endo- through specific ICAM-1–mediated interactions, thereby causing thelial venules: dogmas and enigmas. Nat. Rev. Immunol. 4: 360–370. defects in T cell motility (27). However, in this study, naive T cells 9. Azzali, G., M. L. Arcari, and G. F. Caldara. 2008. The “mode” of lymphocyte extravasation through HEV of Peyer’s patches and its role in normal homing and adhered well to ICAM-1 but not to other proteins tested. Within the inflammation. Microvasc. Res. 75: 227–237. dynamic and highly motile microenvironment of the T cell area that 10. Shulman, Z., V. Shinder, E. Klein, V. Grabovsky, O. Yeger, E. Geron, is difficult to recreate in simplified in vitro systems, we were unable A. Montresor, M. Bolomini-Vittori, S. W. Feigelson, T. Kirchhausen, et al. 2009. Lymphocyte crawling and transendothelial migration require chemokine trig- to identify a role for integrin-independent promiscuous cell attach- gering of high-affinity LFA-1 integrin. Immunity 30: 384–396. Downloaded from ment in the absence of ROCK function. In ICAM-1–deficient PLNs, 11. Park, E. J., A. Peixoto, Y. Imai, A. Goodarzi, G. Cheng, C. V. Carman, U. H. von Y27632 did not reduce lymphocyte-migration parameters compared Andrian, and M. Shimaoka. 2010. 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