Lymph node chemokines promote sustainedT lymphocyte motility without triggering stable integrinadhesiveness in the absence of shear forces

Eilon Woolf1, Irina Grigorova2, Adi Sagiv1, Valentin Grabovsky1, Sara W Feigelson1, Ziv Shulman1,Tanja Hartmann1, Michael Sixt3, Jason G Cyster2 & Ronen Alon1

Lymphocyte motility in lymph nodes is regulated by chemokines, but the contribution of integrins to this motility remains

obscure. Here we examined lymphocyte migration over CCR7-binding chemokines that ‘decorate’ lymph node stroma. In

a shear-free environment, surface-bound lymph node chemokines but not their soluble counterparts promoted robust and

sustained T lymphocyte motility. The chemokine CCL21 induced compartmentalized clustering of the integrins LFA-1 and

VLA-4 in motile lymphocytes, but both integrins remained nonadhesive to ligands on lymphocytes, dendritic cells and stroma.

The application of shear stress to lymphocytes interacting with CCL21 and integrin ligands promoted robust integrin-mediated

adhesion. Thus, lymph node chemokines that promote motility and strongly activate lymphocyte integrins under shear forces fail

to stimulate stable integrin adhesiveness in extravascular shear-free environments.

Naive and memory T cells emigrate into specialized T cell zones inlymph nodes, where they scan for cognate antigen presented by majorhistocompatibility complex molecules on dendritic cells (DCs)1. Thearrest of T cells on high endothelial venules is regulated by the integrinLFA-1 and the chemokines CCL21 and CCL19, which bind to thereceptor CCR7 on T cells2,3. In vivo imaging with confocal andmultiphoton intravital microscopy suggests that lymphocytes enteringthe T cell zones follow a ‘random walk’ over densely packed DCnetworks4 and specialized fibroblastic reticular cells (FRCs)5. Pub-lished findings suggest that CCR7-binding chemokines are involved insupporting this motility. Inhibition of chemokine signaling inT lymphocytes after they enter the lymph node considerably attenu-ates their interstitial locomotion6. In addition, large quantities of theCCR7 ligand CCL21 are expressed in the T cell zone7 and attractactivated CCR7+ B cells8. Finally, suppression of CCR7 expression onT cells decreases their entry into lymph nodes and attenuates therandom locomotion of residual T cells that enter the T cell zone6,9.

Additional cues for lymphocyte motility in the T cell zone could beprovided by integrin ligands expressed on FRCs and DCs10. The LFA-1ligand ICAM-1 is ubiquitously expressed by DCs11, and histologicalstudies suggest that 60% of T cells in lymph nodes are in contact withDCs at any given moment12. FRCs express both ICAM-1 and VCAM-1(ref. 13), the main ligands of the integrins LFA-1 and VLA-4,respectively. FRCs also display CCL21 and CXCL12, ligands ofCCR7 and CXCR4, respectively13. FRCs could therefore be ideally

suited to present both chemokine and integrin ligand cues toT lymphocytes5. In addition to their functions promoting motility,CCL19, CCL21 and CXCL12 activate LFA-1 adhesiveness on naive andmemory T lymphocytes14,15. As ICAM-1 and a second LFA-1 ligand,ICAM-3, are also expressed on T cells16, exposure of lymphocytes tolymph node chemokines, if not tightly regulated, could cause non-productive interlymphocyte clumping or premature lymphocyte arreston DCs and FRCs independently of arrest signals triggered by contactof lymphocytes with cognate antigen. To optimize their antigen-scanning ability, T cells must escape such antigen-independent,integrin-mediated arrest cues.

To delineate the individual functions of integrin and chemokinecues in mediating T cell arrest, we used a series of molecularly definedmodel surfaces ‘decorated’ with the chief T cell zone chemokineCCL21 as well as the integrin ligands ICAM-1 and VCAM-1. Wefound that surface-bound CCL21 but not its soluble counterparttriggered robust and sustained T cell locomotion in the absence ofany additional adhesive ligands. Signals from surface-bound CCL21were insufficient to trigger LFA-1 and VLA-4 adhesiveness and therebyminimized lymphocyte clumping and arrest on DCs and stroma. This‘silenced’ integrin adhesiveness was immediately reversed by theapplication of shear forces. Our data suggest that chemokine-primedlymphocyte integrins need shear forces to optimally adhere to theirligands. This mechanism may allow lymph node chemokines tomaximize lymphocyte motility and minimize integrin-mediated

Received 14 May; accepted 6 July; published online 26 August 2007; doi:10.1038/ni1499

1Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel. 2Department of Microbiology and Immunology and Howard Hughes MedicalInstitute, University of California, San Francisco, California 94143, USA. 3Max Planck Institute of Biochemistry, Munich 82152, Germany. Correspondence should beaddressed to R.A. (ronen.alon@weizmann.ac.il).

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adhesions to ligands in lymph node interstitial spaces devoid of shearflow. At the same time, identical chemokines robustly stimulate firmintegrin-mediated adhesion of lymphocytes to blood vessel ligandsunder shear flow.

RESULTS

Sustained lymphocyte motility triggered by immobilized CCL21

To determine whether prominent lymph node chemokines canpromote T cell motility in vitro, we initially tracked by video micro-scopy the motility of resting human CD3+ peripheral blood lympho-cytes (PBLs) on an adhesive matrix coated with immobilized CCL21(Fig. 1a). Although the spontaneous autonomous motility of restinglymphocytes was negligible, surface-bound CCL21 triggered robustand random T cell motility (Fig. 1a,b and Supplementary Video 1online); this motility was eliminated by pertussis toxin, an irreversibleinhibitor of Gai protein signaling (Fig. 1b). Unexpectedly, solubleCCL21, even in quantities sufficient to trigger phosphorylation of theErk1 and Erk2 mitogen-activated protein kinases (SupplementaryFig. 1 online) and transient lymphocyte polarization (SupplementaryFig. 2 online), failed to promote any T cell locomotion (Fig. 1a andSupplementary Video 2 online). T cell locomotion on immobilizedCCL21 was rapid, with velocities within the ranges reported beforein vivo5,17 (Fig. 1a). A critical density of 100 sites of immobilizedCCL21 per mm2 was needed to support motility, but above thisthreshold, locomotion velocity was not altered by increasing chemo-kine concentration (data not shown). Motile T cells were mainly naiveCD45RA+ T cells (Supplementary Fig. 3 online) and about 60–70% ofthe memory T cells present among PBLs (data not shown).

Notably, lymphocyte locomotion triggered by CCL21, CCL19 andCXCL12 did not require the presence of any additional adhesiveligands (Supplementary Video 3a,b online and data not shown).Furthermore, blockade of b1 integrin did not alter the motility ofT cells exposed to CCL21 immobilized together with the b1 ligandscollagen and fibronectin (Supplementary Fig. 4 online and data notshown). Mouse T cells also moved on immobilized CCL21 regardless

of the presence of an integrin ligand (Sup-plementary Fig. 5 online). The failure ofsoluble CCL21 to promote motility was not

due to insufficient ligand density, as a single T cell encountered asimilar number of chemokine molecules when presented with either30 nM soluble CCL21 or 100 sites of immobilized CCL21 per mm2

(data not shown). Notably, although soluble CCL21 failed to promotemotility, it also did not antagonize locomotion on immobilizedCCL21 (Fig. 1a). Consistent with the potent activity of immobilizedCCL21 and CXCL12, T cells often simultaneously extended twopseudopodia throughout their migration period (SupplementaryVideo 3a,b).

T cell motility persisted over immobilized CCL21 for at least 3 hwithout noticeable changes in velocity (Fig. 1a and data not shown),suggesting minimal global desensitization of CCR7 by immobilizedCCL21. As CCL21 is a poor desensitizing chemokine18, we comparedtwo additional lymph node chemokines, the CCR7 ligand CCL19 andthe CXCR4 ligand CXCL12, both known to induce strong desensitiza-tion of their cognate G protein–coupled receptors19. T cell locomotionon these chemokines was sustained to a similar degree (Fig. 1c). Incontrast, the small fraction of effector memory PBLs capable oflocomotion on immobilized inflammatory chemokines such asCXCL9 (Fig. 1c) or CXCL10 (data not shown) rapidly slowed afterinitial chemokine exposure, presumably because of desensitization ofCXCR3. Thus, three important homeostatic chemokines but notinflammatory chemokines act in their surface-bound state astriggers of robust and sustained integrin-independent locomotionof resting T lymphocytes.

CCL21 polarizes integrins without activating ligand binding

LFA-1 on lymphocytes is rapidly activated by CCL21 displayed onendothelial cells14,15. We therefore next analyzed the distributionand adhesiveness of LFA-1 as well as VLA-4, a second mainintegrin expressed on T lymphocytes, on lymphocytes moving oversurface-bound CCL21. The a4 integrins VLA-4 and a4b7 clustered atthe uropods of most lymphocytes exposed to immobilized CCL21,whereas LFA-1 clustered mainly in the highly dynamic pseudopodia ofthese migrating cells (Fig. 2a and Supplementary Videos 4 and 5

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Figure 1 Immobilized but not soluble CCL21

promotes sustained locomotion of resting

peripheral blood T lymphocytes without apparent

desensitization. (a) Left, fraction of human PBLs

moving over a matrix coated with collagen I and

fibronectin alone (None) or in the presence of

soluble (Sol) CCL21 (30 nM; saturating CCR7)

or immobilized (Immob) CCL21 (600 sites/mm2).Middle, T cells interacting with immobilized or

soluble CCL21 (from Supplementary Videos 1

and 2). Original magnification, �40. Right, tracks

of individual T cells (different color for each)

normalized to their starting coordinates; Vmean,

average instantaneous velocity of total T cells

analyzed. (b) Fraction of moving T cells among

PBLs pretreated for 12 h with pertussis toxin

(+ PTX) or carrier and plated without washing on

a matrix coated with CCL21, collagen I and

fibronectin. (c) Fraction of primary PBLs moving

over CCL19, CXCL12 or CXCL9 (1 mg/ml each)

immobilized together with collagen I and

fibronectin. **, P o 0.01 (Student’s t-test).

Data are representative of ten (a; left, means

of three fields of view), three (b) or four (c)

independent experiments.

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online). The key integrin linker talin20 was distributed amongboth edges of motile T cells (Fig. 2b), but phophatidylinositol-4,5-bisphosphate, which enhances the binding of talin to integrins, wasenriched only in the leading edge (Fig. 2b), along with the actin-remodeling GTPase Rac1 (Supplementary Fig. 6 online).

To directly assess the adhesive state of LFA-1 at the leading andtrailing edges of motile T lymphocytes, we monitored both thefrequency and strength of productive encounters between motileT cells and ICAM-1-coated beads (Fig. 3a and SupplementaryVideo 6 online). Neither the number of ICAM-1 bead encountersnor the duration of each encounter was lower after LFA-1 blockade(Fig. 3a). Thus, LFA-1 is not adhesive at either the leading or trailingedges of lymphocytes migrating over immobilized CCL21. A similar

analysis with VCAM-1-coated beads indicated a lack of VLA-4adhesiveness in motile lymphocytes (data not shown). WhereasT cells express two LFA-1 ligands, ICAM-1 and ICAM-3, and bothligands clustered at the uropod of migrating lymphocytes (Fig. 3b),LFA-1 on motile T cells failed to bind to neighboring T cells (Fig. 3b,cand Supplementary Video 7 online) unless their LFA-1 was artificiallyactivated (Fig. 3c and Supplementary Video 8 online). Lymphocytesmoving over immobilized CCL19 and CXCL12 showed a similar lackof LFA-1 adhesiveness to ligand-coated beads and to other passingT cells (data not shown).

Shear forces are critical for integrin activation by chemokines

Rapid activation of LFA-1 triggered by CCL21 displayed on thevascular endothelium involves bidirectional signaling betweenICAM-1 and juxtaposed chemokines15. Thus, the failure of immobi-lized CCL21 to induce adhesiveness of LFA-1 on motile T cells couldhave been due to physical separation of the chemokine and ICAM-1,each of which were presented on a different surface in these assays.Staining of lymph node sections showed moderate but ubiquitousICAM-1 expression on all extravascular components of the T cell zone,including FRCs associated with laminin-positive conduits (Fig. 4a and

Figure 3 LFA-1 on T cells migrating on CCL21does not bind ICAM-1. (a) Top row, T cells

moving over immobilized CCL21 and

encountering beads coated with high-density

ICAM-1 (from Supplementary Video 6). The first

image (time 0) was obtained 10 min after the

introduction of T cells to the chemokine-coated

surface. Yellow stars mark the leading edge of a

T cell colliding with an ICAM-1-coated bead.

Below, number and duration of collisions between

T lymphocytes migrating on immobilized

CCL21 and ICAM-1-coated beads. U, uropod;

L, lamelipodium. a-b2, antibody TS1.18

(anti-b2; blocking LFA-1 on T cells); a-ICAM-1,

pretreatment of ICAM-1-coated beads with

polyclonal anti-ICAM-1; Control, isotype-matched

monoclonal antibody. (b) Top row, concentration

of ICAM-1 and ICAM-3 at the uropods of T cells

moving over immobilized CCL21, among T cellsfixed and stained with anti-ICAM-1 or anti-ICAM-

3. Bottom row, collision of a T cell with the

uropod of another T cell (from Supplementary

Video 7), among T cells prelabeled with

nonblocking anti-LFA-1. (c) Frequency and

average duration of contacts between T cells

colliding with each other during locomotion over CCL21 (30 s or less) in the presence of a control antibody, antibody TS1.18 (b2-blocking) or antibody

240Q (b2-activating). Inset (below), two T cells preactivated with 240Q migrating on immobilized CCL21 and colliding into each other; 240Q also increased

by 70% the binding of ICAM-1-coated beads to T cells migrating on CCL21 (data not shown). Original magnification, �40. Data are one representative of

four (a) or three (b,c) experiments.

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Figure 2 Immobilized CCL21 triggers polarized redistribution of LFA-1 and

a4 integrins. (a) Live images of LFA-1 (top) or of a4 integrins (bottom) in

T cells migrating over CCL21 at 5 min after initial T cell exposure to the

immobilized chemokine (from Supplementary Videos 4 and 5); cells are

labeled with Alexa Fluor 568–tagged TS2/4 (nonblocking anti-aL) or

Alexa Fluor 488–tagged B5G10 (nonblocking anti-a4). Top rows, merged

differential interference contrast and fluorescence images obtained

consecutively; bottom rows, spectrum analysis of relative fluorescenceintensity corresponding to the images above. Neither antibody alters

T cell speed. (b) Intracellular staining of talin and phophatidylinositol-4,

5-bisphosphate (PIP2) in resting T cells (–) or in T cells moving for 10 min

over CCL21 (+). Original magnification, �40. Data are representative of five

(a) or three (b) independent experiments.

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data not shown). Notably, throughout the T cell zone, CCL21 stainingwas consistently localized together with FRCs and was thereforeenhanced around high endothelial venules (Fig. 4a). This stainingpattern suggested that stromal and endothelial CCL21 are physio-logically encountered by T cells in juxtaposition to ICAM-1.

To determine whether immobilized CCL21 juxtaposed withICAM-1 can activate LFA-1 on T cells, we reconstituted variousICAM-1-bearing surfaces with a fixed amount of surface-boundCCL21. ICAM-1 alone, although shown before to promote motilityof T cell blasts21, failed to support the motility of resting lymphocytes(Fig. 4b); in contrast, CCL21 triggered robust motility (Supplemen-tary Video 3a). Notably, increased ICAM-1 densities did not arrest ordecelerate T cells moving on immobilized CCL21 (Fig. 4b). Theseresults collectively indicate that T cell LFA-1 is not functionallyactivated even when T cells encounter CCL21 and ICAM-1 immobi-lized together. Indeed, blocking LFA-1 on T cells did not affect lym-phocyte locomotion (Fig. 4b), although on high-density ICAM-1,blockade of LFA-1 marginally reduced the velocity of motile T cells,consistent with a minor motility-promoting activity of chemokine-activated LFA-1 (Fig. 4b).

In contrast, when T lymphocytes were settled on identical sub-strates under continuous application of shear stress, CCL21 triggered

LFA-1-mediated adhesive interactions with ICAM-1 (Fig. 4b). Like-wise, T lymphocytes that had already spread on high-density ICAM-1and CCL21 for 3–4 min in shear-free conditions and were thensubjected to shear forces rapidly developed strong LFA-1 adhesions(Fig. 4c). Thus, LFA-1, although mainly ‘silent’ in shear-free condi-tions in the presence of ICAM-1 and CCL21 immobilized together,was readily activated by the application of shear force.

FRCs in the T cell zone express also the VLA-4 ligand VCAM-1(ref. 13). However, the motility of T cells encountering immobilizedCCL21 presented in juxtaposition to high-density VCAM-1 was notaffected by the presence of VCAM-1, and VLA-4 blockade had noeffect on the motility of cells over surfaces coated with VCAM-1 andCCL21 (Fig. 5a and data not shown). Nevertheless, similar to LFA-1,VLA-4 was readily activated in T lymphocytes in the same conditionsin the presence of shear stress (Fig. 5b). As ligand-occupied VLA-4 canaugment LFA-1-mediated T cell motility and adhesiveness22, we nextassessed whether CCL21-stimulated T lymphocytes stopped on asurface bearing equal densities of VCAM-1 and ICAM-1. In theabsence of shear stress, the presence of both integrin ligands failedto arrest or even decelerate T lymphocytes moving over surface-boundCCL21 (Fig. 5c). However, as seen on surfaces coated with individualintegrin ligands, in the presence of shear stress, CCL21 triggeredadhesion of both VLA-4 and LFA-1 to the surface coated withVCAM-1 and ICAM-1 (Fig. 5d). In shear-free conditions, blockadeof VLA-4 marginally reduced the velocity of T cells migrating oversurface-bound CCL21 and high-density VCAM-1 but did not alter themotility of T cells exposed to immobilized CCL21, VCAM-1 andICAM-1 (Fig. 5a,c). Thus, in the presence of motility-promotingCCL21 signals, both VLA-4 and LFA-1 are similarly ‘silent’ onlymphocytes migrating in shear-free conditions.

This mechanical shear stress regulation of integrins was not uniqueto T lymphocytes. We confirmed a similar effect in neutrophilsarrested on the integrin Mac-1 ligand fibrinogen immobilized togetherwith the potent chemokine interleukin 8 (IL-8). In the absence ofshear stress, neutrophil spreading and locomotion were entirelydependent on IL-8 but not on b2 integrins, whereas under shearstress, neutrophils developed robust b2-mediated adhesion to fibrino-gen in response to the same IL-8 (Supplementary Fig. 7 online anddata not shown).

Microtubules are dispensable for CCL21-promoted motility

Microtubules are key cytoskeleton components that regulate bothpolarity and directional movement. In leukocytes, microtubules

80

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Figure 4 LFA-1 mediates stable adhesions in lymphocytes moving on

immobilized CCL21 and ICAM-1 under shear stress but not in shear-

free conditions. (a) Immunohistochemistry of lymph node sections

(antibodies, above images). Scale bars, 30 mm. Data are representative of

three experiments. (b) Left, fraction of T cells moving (Motile) or polarized

and arrested (Arrested) on CCL21 alone (600 sites/mm2) or with ICAM-1

(top, 320 sites/mm2; middle, 160 sites/mm2; bottom, 0 sites/mm2), among

T cells pretreated with control antibody (–) or the b2-blocking antibodyTS1.18 (+). Numbers above bars indicate the velocity of all motile T cells

(mean + s.e.m.). **, P o 0.01. Right, fraction of T cells establishing either

transient or firm adhesions when interacting under shear stress (0.5 dyn/

cm2) with substrates and blocking conditions identical to those at left. Error

bars: left, mean + s.d. of four fields; right, mean + range of two fields. Data

are one representative of three experiments. (c) T lymphocytes allowed

to spread for 5 min on CCL21 (600 sites/mm2) with or without ICAM-1

(320 sites/mm2) and then exposed for 60 s to a shear stress of 0.2 dyn/cm2.

Anti-b2, pretreatment of T cells with the b2-blocking antibody TS1.18. Data

represent the mean of three fields of view and are one representative of

three experiments.

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regulate directional motility23,24 and integrin adhesiveness25. Micro-tubule disassembly releases b2 integrins from cytoskeletal constraints,thereby increasing their avidity for ICAM-1. Given the key involve-ment of the actin cytoskeleton in CCR7-triggered LFA-1 adhesivenessand in lymphocyte motility over CCL21 (refs. 15,26; data not shown),we sought to delineate the function of microtubules in CCL21-stimulated T cell motility. Microtubule depolymerization induced bynocodazole (Fig. 6a) did not alter CCL21-triggered lymphocytespreading or locomotion in shear-free conditions (Fig. 6b). However,

in conditions of shear stress, disruption of microtubule polymerizationsignificantly decreased the CCL21-triggered LFA-1-induced arrest ofT cells on ICAM-1 (Fig. 6c). Notably, T cell motility involved myosinII–mediated contractility (Supplementary Fig. 8 online). These find-ings collectively indicate that CCL21-triggered T cell motility does notrequire an intact microtubule cytoskeleton; however, although integrinindependent, it still involves contractile motion.

CCL21-induced T cell locomotion over lymph node cells

Next we sought to confirm that CCL21 presented by DCs or stromalcells expressing ICAM-1 effectively promotes in vitro T cell motilityand prevents LFA-1-mediated T cell arrest. Immature DCs, abundantin the lymph node T cell zone, spread rapidly and extended manyprotrusions when settled on immobilized CCL21 or CCL19 (datanot shown). These DCs expressed ICAM-1 (Fig. 7a) but did notattract T cells unless they were overlaid with CCL21 (Fig. 7a,b andSupplementary Video 9 online). Most T cells that moved toward andover autologous DCs in response to immobilized CCL21 did soindependently of LFA-1 blocking (Fig. 7b). In addition, the smallfraction of T cells that arrested during locomotion over the DC surfacedid not require functional LFA-1 (Fig. 7c). Similar to DCs, ICAM-1-expressing human bone marrow stromal cells (Fig. 7d) supportedrobust lymphocyte motility only when overlaid with CCL21 (Fig. 7d,eand Supplementary Video 10 online). This motility and the residuallymphocyte arrest occurred independently of LFA-1 (Fig. 7e,f). Thus,lymphocyte motility over and arrest on ICAM-1-expressing autolo-gous DCs and stroma triggered by CCL21 did not require LFA-1.

Lymphocyte motility in resting lymph node T cell zones

To assess the contribution of LFA-1 and VLA-4 to in vivo T cellmotility in the resting lymph node, we next compared the interstitialmotility of wild-type T cells and CD18-deficient T cells (lackingLFA-1) adoptively transferred into wild-type or ICAM-1-deficientrecipient mice. As LFA-1 is the only CD18-containing integrin onresting T cells, this system allowed us to assess the contribution ofLFA-1 to T cell motility patterns in the T cell zone. We assessed themotility of transferred cells in the T cell zones of explanted recipientlymph nodes by two-photon microscopy at 24 h after transfer(Supplementary Video 11 online). Only a minor fraction of trans-ferred lymphocytes (1–3%) transiently arrested during migration, andthey did so independently of CD18 (data not shown). The motile

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Figure 5 VLA-4 is not adhesive in T cells migrating on CCL21 and juxtaposed integrin ligands in shear-free conditions. (a) Fraction of PBLs either moving

or polarized and arrested on CCL21 (600 sites/mm2) plus high-density VCAM-1 (320 sites/mm2) in the presence or absence of the VLA-4 inhibitor Bio1211

(1 mg/ml; VLA-4 blockade). *, P o 0.04. (b) Fraction of T cells establishing transient, rolling or firm adhesions when interacting under shear stress with

substrates and treatments identical to those in a. (c) Fraction of PBLs either moving or polarized and arrested on CCL21 (600 sites/mm2) immobilized

together with both VCAM-1 and ICAM-1 (160 sites/mm2 each), among PBLs pretreated with control antibody (–) or a blocking ‘cocktail’ (Bio1211 (VLA-4

blockade) mixed with TS1.18 (b2 blockade); +) and settled without washing. (d) Fraction of T cells establishing transient or firm adhesions when interacting

under shear stress with substrates and treatments identical to those in c. Numbers above bars (a,c) indicate the velocity of moving T cells (mean ± s.e.m.).

Data are the mean + s.d. of four fields (a,c) or the mean + range of two fields (b,d) and are from one representative of three experiments.

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Figure 6 Microtubules contribute to firm CCL21-induced LFA-1

adhesiveness under shear stress but are not required for lymphocyte

motility on CCL21 and ICAM-1. (a) Visualization of microtubules in

human PBLs pretreated for 40 min with the microtubule-depolymerizing

agent nocodazole or control reagent, then fixed and stained with anti-

tubulin. Original magnification, �40. Images are of two representative of

60 cells of each group analyzed in two experiments. (b) Effect of nocodazole

pretreatment on the fraction and velocity of PBLs moving on ICAM-1(320 sites/mm2) and CCL21 (600 sites/mm2). b2 blockade, antibody TS1.18

(blocking LFA-1). Numbers above bars indicate the velocity of moving

T cells (mean ± s.e.m.). P ¼ 0.075, nocodazole versus vehicle control.

(c) Fraction of T cells establishing transient or firm adhesions under

shear stress upon interaction with substrates identical to those in b.

**, P o 0.003 (total tethers); *, P o 0.004 (firm tethers). Data are the

mean + s.d. of four fields (b) or the mean + range of two fields (c) and are

from one representative of three experiments (b,c).

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fractions of wild-type and CD18-deficient T cells were similar in wild-type and ICAM-1-knockout recipients (over 97%), although T cellslacking CD18 migrated slightly more slowly than their wild-typecounterparts in wild-type recipients, with a median velocity about15% lower (Fig. 8a,b). Consistent with that finding, there wassimilarly lower T cell velocity in the absence of ICAM-1 on recipientcells (stroma, DCs and neighboring lymphocytes; Fig. 8a). Asexpected, in ICAM-1-deficient lymph nodes, CD18-deficient T cellsdid not demonstrate slower movement than that of wild-type T cells(Fig. 8a). CD18-deficient T cells also moved in a less linear way thanwild-type T cells, as indicated by their slightly higher median turning

angle and lower confinement index (Fig. 8c and data not shown).Although both types of T cells migrated randomly in the T cell zone,the slightly lower velocities and more frequent turning of CD18-deficient T cells affected their scanning, as shown by mean-squaredisplacement analysis (Supplementary Fig. 9 online). Similar to CD18deficiency, blockade of a4 integrins decreased locomotion speed onlyslightly but had no additive effect with CD18 deficiency (Fig. 8a).Thus, even T cells lacking functional LFA-1 did not seem to use theira4 integrins (VLA-4 or a4b7) to promote interstitial motility in theT cell zone. These results collectively indicate that neither LFA-1 nora4 integrins support stable adhesions of naive T cells to neighboring

200

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Figure 7 LFA-1 is not required for lymphocyte motility toward and over DCs and stroma expressing ICAM-1. (a) Top,

ICAM-1 on monocyte-derived DCs spread for 15 min on collagen and fibronectin with immobilized CCL21. Bottom,

T cell motility toward and over autologous monocyte-derived DCs. BCECF-AM-labeled T lymphocytes were added to DCsand allowed to spread for 10 min over immobilized CCL21; cells were tracked for an additional 30 min. Images are

from Supplementary Video 9. (b) Fraction of T lymphocytes that persistently moved in the experiment in a. LFA-1 on

both T cells and DCs was blocked with A308296 (LFA-1 blocker), or T cell and DC integrins were blocked with

TS1.18 (Anti-b2). (c) Fate of motile lymphocytes in b that encountered DCs: some continue to move over the DC

surface and eventually detach (Motile on DC); others arrest during migration over the DC surface for at least 1 min

(Arrested on DC). (d) Top, ICAM-1 on human bone marrow stromal cells spread on collagen and fibronectin. Bottom,

tracking of T lymphocytes on stromal cells that were overlayed for 5 min with CCL21 and washed; BCECF-labeled

T lymphocytes were then allowed to settle and were tracked as described in a. Images are from Supplementary Video 10. (e) Fraction of T lymphocytes that

persistently moved over and toward the human stromal cells in d after pretreatment (key). (f) The fate of migrating lymphocytes in e that encountered human

stromal cells, assessed as described in c. Data are representative of four (a), three (c,d) or two (f) experiments or are the mean (+s.e.m.) of eight fields (b,e)

from one representative of three (b) or two (e) experiments.

*1816141210

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Figure 8 Slight increase in T cell velocity in lymph nodes by CD18. Velocities and turning angles of green fluorescent protein–positive wild-type (WT) T cells

and CMTMR-labeled CD18-deficient (CD18-KO) T cells transferred together into wild-type or ICAM-1-deficient (ICAM-1-KO) recipient mice or into wild-type

recipient mice treated with a4-blocking antibody (WT + anti-a4); 1 d later, labeled cells were simultaneously imaged in explanted lymph nodes. Cells were

tracked for a minimum of 10 min at intervals of 20 s. For wild-type recipients, at least 70 cells were tracked; in other systems, at least 40 cells were

tracked. The motility of wild-type T cells was not affected by colabeling with CMTMR. (a) Median velocities of T cells in a single imaging volume calculated

over 30-minute periods. *, P o 0.5. Data are averaged from three independent experiments (different symbol for each; bars indicate mean). (b) Medianvelocities of wild-type and CD18-deficient T cells migrating in wild-type lymph nodes (bin, 2 mm/min). (c) Overall distribution of turning angles of wild-type

and CD18-deficient T cells in recipient lymph nodes (bin, 15 degrees). Turning angles are from the same data sets as those in b. Inset, median turning angle

values in each of three experiments (different symbol for each; bars indicate mean). *, P o 0.5 (median values). Data are averages and s.e.m. calculated for

three experiments (b,c).

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T cells, DCs or stroma in lymph node T cell zones. LFA-1–ICAM-1interactions in the context of the abundance of ICAM-1 in these zones(Fig. 4a) did marginally increase the motility of a subset of T cells, inaccordance with the in vitro results (Fig. 4b). Nevertheless, we did notfind stable adhesive LFA-1–ICAM-1 interactions, which could havearrested motile T cells or slowed their movement, even in this complexT cell zone milieu.

DISCUSSION

CCR7 and integrin cues must be tightly controlled to simultaneouslymaximize the extravasation of lymphocytes from the blood andinterstitial locomotion in the search for rare antigens and minimizeantigen-independent sticking to DCs, stroma and surrounding lym-phocytes1. Our results here have demonstrated that chemokinespresented on the surfaces of lymph node stroma are sufficient topromote robust and sustained lymphocyte locomotion. Furthermore,when juxtaposed to integrin ligands ubiquitously expressed on stroma,DCs and lymphocytes in the lymph node T cell zone, these chemo-kines did not activate firm integrin adhesiveness and therefore stickingand clumping of motile T cells were prevented in this highly packedenvironment. Notably, this ‘silencing’ of firm integrin adhesivenesswas tightly regulated by mechanical signals. Thus, in the presence ofshear forces prevailing at lymphocyte-endothelium contacts, integrinadhesiveness is readily activated by the same chemokines that fail totrigger integrin adhesiveness in the extravascular space where shearforces are absent. Integrin ‘silencing’ in the presence of chemokines istherefore a unique ‘mechanoregulated’ program that optimizes inter-stitial motility while minimizing integrin activation that could resultin the sticking of lymphocytes navigating in highly dense networks ofimmune cells.

Two key cell types in the lymph node, high endothelial venules andFRCs5, coexpress CCR7 ligands and the chief LFA-1 ligand ICAM-1.Proximity of chemokines to integrin ligands facilitates rapid signalingand activation of LFA-1 (ref. 15) and VLA-4 on many types ofleukocytes27. Indeed, when displayed together by high endothelialvenules or on model surfaces, CCL21 and ICAM-1 drove robust LFA-1-mediated T cell sticking and firm adhesion. Nevertheless, the sameconfiguration failed to trigger LFA-1-dependent adhesiveness ofT lymphocytes in shear-free environments, a mechanism that restrictspremature antigen-independent T cell arrest on lymph node stoma orDCs coexpressing both ICAM-1 and CCR7 ligands. Our resultsindicate that an externally applied force is required for productivechemokine-induced integrin activation and that forces applied onintegrin-ligand bonds act as a critical switch, allowing full activation offirm integrin adhesiveness by ‘inside-out’ chemokine signals. Pub-lished results have suggested that force accelerates ligand-inducedrearrangements in the LFA-1 ‘headpiece’, thus stabilizing the high-affinity state of the main integrin ligand-binding site on the aIdomain28,29. Modeling studies have also suggested that ligand-bound b3 integrins can transition into a high-affinity state withinnanoseconds after the application of force on the ligand, whereas thesame ligand-driven head rearrangements may require seconds in theabsence of force30. The mechanical regulation of integrins may notonly regulate resting T cells and neutrophils, as evident in our studyhere, but also could be important for controlling integrin adhesivenessin effector lymphocytes that use CCR7 signals to enter and movethrough afferent lymphatic vessels to lymph nodes in diverse condi-tions of shear flow31,32.

Studies have suggested that immobilized chemokines promote theadhesion of T cells to ICAM-1 even in shear-free conditions33.However, those studies involved multiple washing steps, and therefore

considerable external forces were applied to cells previously spread onICAM-1- and chemokine-coated surfaces. A study of mainly mouseT lymphoblasts has suggested that these cells readily establish LFA-1-dependent tethers to CCL21 present together with ICAM-1 on thesurfaces of antigen-presenting cells34. Our results challenge the ideaof such LFA-1 activation in resting human T cells, but we cannot ruleout the possibility that activated LFA-1 on lymphoblasts or on the Bcell antigen-presenting cells used in the former study34 remainedhighly adhesive even in shear-free conditions. Our results also allowreinterpretation of an in vitro analysis of mouse T splenocytes thatsuggested that soluble CCL21 fails to promote motility unless pro-vided in the context of integrin ligands such as ICAM-1, VCAM-1or fibronectin35. We found that soluble mouse CCL21 readilyadsorbed onto collagen, fibronectin and ICAM-1; thus, these integrinligands may have contributed to lymphocyte motility by adsorbingand presenting CCL21.

Our study also suggests that surface-bound CCR7 chemokines, inaddition to restricting integrin adhesiveness, minimize global CCR7desensitization on rapidly moving lymphocytes. Minimizing homo-logous desensitization may allow persistent T cell locomotion and mayenable T cells to respond rapidly to a second homologous CCR7ligand, CCL19, which is ‘preferentially’ expressed by activated DCsentering the lymph node36. In general, immobilized chemokines,unlike their soluble counterparts, may be physically protected fromuptake and recycling but may still be able to trigger small amounts ofphosphorylation and desensitization of G protein–coupled receptors.Such observations, however, may be restricted to the homeostaticCCR7 and CXCR4 chemokines, as T cell motility over the two CXCR3chemokines, CXCL9 and CXCL10, rapidly decays; these data indicate ahigher sensitivity of CXCR3 than of CCR7 and CXCR4 to desensitiza-tion during motility.

Our results suggest a dual function for CCR7 and CXCR4 assignaling and weak adhesive receptors for their immobilized ligandsCCL21, CCL19 and CXCL12 in shear-free environments. The cell-adhesive chemokine fractalkine (CX3CL1) and its G protein–coupledreceptor CX3CR can support strong leukocyte adhesion even inconditions of shear flow37, but the ability of this chemokine-receptorpair to trigger random and sustained leukocyte motility, as shown herefor lymph node chemokines, has not been demonstrated so far. Thelocomotion of lymphocytes on immobilized lymph node chemokinesmay involve both signaling G protein–coupled receptors and non–Gprotein–coupled receptor surface molecules. P-selectin glycoproteinligand 1 (PSGL-1), for example, can bind to CCL21 and CCL19(ref. 38). Although it has been suggested to serve as a trap for solubleCCR7 chemokines, PSGL-1 may act together with CCR7 in bindingimmobilized CCL21 presented on FRCs. Notably, T cells migrated asefficiently on CCL21 as on immobilized CXCL12, which does not bindPSGL-1 (ref. 38); thus, T cell PSGL-1 is not obligatory for lymphocytemigration on immobilized lymph node chemokines, challenging theidea of a general function for CCL21–PSGL-1 bonds in the turnover ofthe T cell leading edge.

A key outstanding issue of our study is whether in the absenceof external shear forces, integrin adhesiveness in lymphocytes andneutrophils can be regulated by forces derived autonomously. In theinterstitial space, cell-derived internal forces could be loadedby integrins by means of actin polymerization and contractile machi-neries29,39. Focal adhesions are chief sites for force loading by ligand-occupied integrins. Fibroblasts can sense the physical properties ofthe substrate40 and ‘translate’ surface rigidity into the force-triggeredformation of focal adhesions through periodic extension and con-traction activity41,42. In contrast, T cell motility on immobilized

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chemokines, although a contractile, myosin II–mediated ameboid-likeprocess, did not seem to involve the formation of focal adhesion-likestructures. Although chemokine-triggered a4 and b2 integrins aresegregated into distinct compartments in motile lymphocytes, theseintegrins seem to be uncoupled from the actin cytoskeleton43 andtherefore would not be able to ‘sense’ and load any internally drivenactomyosin-based or actin polymerization–driven forces. However,this integrin ‘silencing’ should be bypassed by a strong T cell receptorsignal that activates LFA-1, possibly by coupling this integrin to theactomyosin cytoskeleton29. A remaining unresolved issue is howand when T cell receptor ‘stop signals’ can activate firm integrinadhesiveness in T cells moving on immobilized CCL21 and otherlymph node chemokines. One possibility is that distinct chemokinesmaintain integrins on migrating T cells at different degrees of ‘silen-cing’, and thus the T cell receptor threshold signals needed to activatefirm integrin adhesiveness can vary considerably in different chemo-kine environments1,44. Notably, when secreted locally in the immunesynapses of effector T cells, some chemokines can costimulate T cellreceptor signaling45, whereas other chemokines destabilize integrinadhesiveness after synapse formation44. Future studies should deline-ate how different subsets of lymphocytes ‘translate’ these opposingsignals into motility, deceleration and stoppage in diverse extravas-cular lymphoid compartments.

METHODSReagents. Human ICAM-1–Fc, VCAM-1–Fc and all chemokines were from

R&D Systems. IL-4 and granulocyte-macrophage colony-stimulating factor

were from Cytolab. The following monoclonal antibodies were used: antibody

to human ICAM-1 (anti–human ICAM-1; MCA161XZ; 15.2; Serotec), anti-

human ICAM-3 (BMS111; Bender MedSystems), anti-talin (T3287; Sigma),

anti-phophatidylinositol-4,5-bisphosphate (Z-A045; Echelon), anti–human

a-tubulin (T9026; Sigma) and anti-human Rac1 (102; Pharmingen). Antibody

TS1/18 (IgG1) blocks integrin b2; TS2/4 (anti-aL; IgG1) is a nonblocking LFA-

1-specific antibody; monoclonal antibody 13 blocks integrin b1; B5G10 (IgG1)

is a nonblocking integrin a4–specific antibody; 240Q is an integrin b2–

activating F(ab) antibody; and A308926 inhibits the LFA-1 I-domain allosteric

site (control compound, A270412)15. Phycoerythrin-conjugated goat anti-

mouse was from Jackson ImmunoResearch Laboratories. BSA (fraction V),

calcium- and magnesium-free Hank’s balanced-salt solution, pertussis toxin,

the Rho kinase inhibitor Y-27632, and fibronectin were from Sigma-Aldrich.

Collagen type I (90135) was from Chemicon. Human serum albumin (fraction

V), protein A and Blebbistatin were from Calbiochem.

Mice and cells. C57BL/6J (B6) mice, ICAM1-deficient mice, UBC-GFP and

b-actin-CFP mice were from Jackson Laboratories. All mutant strains were on a

C57BL/6 background. Splenocytes obtained from 6- to 9-week-old C57BL/6

mice were cultured for 16 h; CD3+ T cells were then isolated with a negative cell

isolation kit (MACS; Miltenyi Biotec). Whole blood was obtained from healthy

human donors, and PBLs were isolated from this blood after citrate anti-

coagulation as described15; over 90% of these were CD3+ T lymphocytes. Cells

were maintained in binding medium (Hank’s balanced-salt solution containing

2 mg/ml of BSA, 10 mM HEPES, pH 7.4, and Ca2+ and Mg2+ at 1 mM each)

and were used within 1 h. For the preparation of DC populations from primary

mononuclear cells, a modification of published techniques was used46. Mono-

nuclear cells were cultured for 2 h at 37 1C in plastic tissue culture dishes in

RPMI 1640 medium containing 10% (vol/vol) FCS. The adherent population

(over 70% CD14+ cells) was cultured for 7 d in RPMI medium containing

granulocyte-macrophage colony-stimulating factor (50 ng/ml) and recombi-

nant IL-4 (1,000 U/ml); cultures were ‘fed’ every 2 d. Human stromal cells were

derived from bone marrow as follows. Bone marrow samples were obtained

from consenting patients who were scheduled for medically indicated bone

marrow biopsy. Bone marrow cells were separated with a Ficoll-Paque Plus

gradient (GE Healthcare Bio-Sciences AB). The mononuclear cell fraction

was cultured for 2–3 weeks in human Mesencult medium together with

supplements (Stem Cell Technologies). Cells were then cultured in DMEM

supplemented with 10% (vol/vol) FCS and were passaged as soon as confluence

was achieved. All animal procedures were approved by the Animal Research

Committees of the Weizmann Institute of Science and the University of

California, San Francisco. Bone marrow isolation was approved by the Sheba

Tel Hashomer Medical Center Institutional Review Board and the Israeli

Ministry of Health.

Motility and adhesion assays. For motility and adhesion assays, polystyrene

plates (60 � 15 mm; Falcon) were coated for 2 h with a spot of protein A

(1 mg/ml) and then washed, then CCL21 was overlaid for 3 h at 4 1C followed

by ICAM-1–Fc or VCAM-1–Fc at a concentration of 1–5 mg/ml in medium

supplemented with 2% (wt/vol) BSA. Plates were mounted on the automated

stage of an inverted microscope (Delta Vision Spectris RT; Applied Precision),

PBLs were added, and movies were recorded for 30 min with Softworx

3.5 (Applied Precisions) at a speed of four frames per minute with a 20�differential interference contrast objective with a numerical aperture of 0.95. All

experiments were done at 37 1C. In some experiments, motility chamber

microslides (m-Slide VI flat or m-Slide VI; Integrated BioDiagnostics) were also

coated overnight at 4 1C with chemokine (2 mg/ml) followed by 1 h of coating

with fibronectin (5 mg/ml) and collagen I (15 mg/ml). Chambers were washed

and blocked with 2% (wt/vol) human serum albumin. For measurement of the

CCL21-coating densities on the various plates and chambers, binding of125I-labeled anti-CCL21 (54111; R&D Systems) was determined as described27.

Time-lapse images were collected at 15-second intervals for 30 min or more

and the positions of individual cells over time were identified with Imaris

tracking software (Bitplane). Imaris was also used for calculation of T cell

velocities. Cells that acquired leading and trailing edges for at least 1 min were

considered ‘polarized’ and were subcategorized as ‘polarized stationary’ or

‘polarized motile’ depending on their ability to move at least 30 mm from their

original position during 10 min of tracking. All flow experiments were done as

described15. Tethers were defined as ‘transient’ if cells attached briefly (under

2 s) to the substrate and as ‘arrests’ if cells were immediately arrested and

remaining stationary for at least 3 s of continuous flow. In some experiments,

T cells were first spread on the various substrates and then shear force was

applied and the fraction of cells resisting detachment was analyzed. The statistical

significance of all adhesion and motility data was evaluated with a paired two-

tailed Student’s t-test. For blockade of Gai protein signaling, PBLs were cultured

for 15 h at 37 1C in the presence of pertussis toxin (100 ng/ml). Nocodazole was

used for 1 h at a concentration of 10 mM without washing. In some experiments,

the LFA-1 I-domain was inhibited by pretreatment of cells for 5 min with the

inhibitor A308926 or its control compound A270412.

Fluorescent imaging and staining. Antibodies TS2/4 (anti-LFA-1) and B5G10

(nonblocking anti-a4) were conjugated to Alexa Fluor 488 or Alexa Fluor 568

dyes as recommended by the manufacturer (Molecular Probes). T cells were

labeled for 10 min with each conjugated antibody at a concentration of

10 mg/ml, then were washed and visualized by live video microscopy as

described above. The antibodies used did not interfere with lymphocyte

motility in the experimental settings tested. For live imaging of T cell motility

over DCs or stromal cells, T cells were labeled with a solution of BCECF-AM

(2¢,7¢-bis(carboxyethyl)-4(or 5)-carboxyfluorescein diacetoxymethyl ester;

1 mM; Dojindo Molecular Technologies) by incubation for 5 min at 37 1C

followed by washing. For staining after fixation, cells were allowed to migrate

for 15–20 min on immobilized chemokine (or an extracellular matrix control)

and then were immediately fixed with 4% (wt/vol) paraformaldehyde in PBS

and were stained with primary antibody. Mouse anti–human ICAM-1 and

anti–human ICAM-3 were used together with phycoerythrin-conjugated goat

anti-mouse for signal detection. After cells were made permeable for 5 min with

0.1% (vol/vol) Triton X-100 (Sigma), they were stained with monoclonal anti-

talin, anti-phophatidylinositol-4,5-bisphosphate, anti-a-tubulin and anti-Rac1

followed by phycoerythrin-conjugated goat anti-mouse. Ex vivo staining of

mouse lymph nodes for ICAM-1 (hamster anti–mouse ICAM-1; 3E2; BD

Biosciences), CCL21 (rabbit anti–mouse CCL21; 500-P114; Peprotech) and

‘pan-laminin’ (rabbit anti–mouse pan-laminin; L9393; Sigma) were done as

described47. For this, sections were cut 80–150 mm in thickness on a vibratome

and were fixed paraformaldehyde, then were stained according to a standard

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whole-mount staining protocol, followed by subsequent image acquisition with

a confocal microscope (DMIRE2; Leica). Images were merged and ‘average-

projected’ from stacks of six z layers (z ¼ 1.7 mm) with Leica confocal

software version 2.5.

Integrin ligand bead-binding assay. For analysis of the encounter rate and

duration of T lymphocytes moving on immobilized CCL21 and ICAM-1-

coated beads, magnetic protein A beads (Dynabeads) were coated with

recombinant ICAM-1–Fc (100 mg/ml). T cells and beads were injected

together into chambers (Integrated BioDiagnostics) and T cell–bead

encounters were recorded with the DeltaVision real-time imaging system.

For blocking of ICAM-1 on beads, anti-ICAM-1 was used at a concentration

of 10 mg/ml.

Adoptive transfer and two-photon microscopy. Spleens and lymph nodes

of donor mice were isolated and purified and then were labeled with

CellTracker Orange CMTMR (5-(and-6)-(((4-chloromethyl)benzoyl)amino)

tetramethylrhodamine; Invitrogen–Molecular Probes) as described8. Cells

were adoptively transferred by intravenous injection into the lateral tail vein.

After 12–24 h, inguinal lymph nodes were isolated for two-photon microscopy,

and axillary and brachial lymph nodes were isolated for flow cytometry. Some

mice were treated intraperitoneally with 100 mg PS/2 (anti-a4) at least 8 h

before analysis; flow cytometry confirmed that lymph node T cells were

saturated with PS/2. Explanted lymph nodes were prepared as described8.

Inguinal lymph nodes were maintained in RPMI medium at 36–37 1C,

‘bubbled’ with 95% O2 and 5% CO2, and were imaged from the cortical side

of the lymph node through the capsule. A ‘custom’ two-photon microscope

system was used for imaging. For comparison of the migration of CD18-

deficient and wild-type T cells, C57BL/6 green fluorescent protein–positive

T cells were transferred together with CMTMR-labeled CD18-deficient cells

and the same time-lapse movies were used to track both cell types in the T cell

zone. B cells labeled with cyan fluorescent protein were transferred together to

delineate positioning of the follicles and of the T cell zone. The emissions of

cyan fluorescent protein–labeled cells (and second-harmonic emission of

collagen fibers), green fluorescent protein–labeled cells and CMTMR-labeled

cells were collected in three distinct channels spanning emission wavelengths of

455–485 nm, 500–540 nm and 567–640 nm, respectively. Images were acquired

with Video Savant software (IO Industries). Flow cytometry confirmed that

over 90% of both wild-type and CD18-deficient T cells in the lymph nodes

were CD62LhiCD44loCD69lo. For time-lapse imaging, typically 36 planes

spaced 3 mm apart were collected every 20 s. Each xy plane spanned 240 mm

� 288 mm at a resolution 0.6 mm per pixel. Data were further processed

with MetaMorph software (Molecular Devices). Imaris software (Bitplane

AG) was used for three-dimensional volume rendering and cell tracking.

Matlab software was further used to calculate apparent cell velocities and turn

angles. Statistical significance was evaluated with an unpaired one-tailed

Student’s t-test.

Note: Supplementary information is available on the Nature Immunology website.

ACKNOWLEDGMENTSWe thank D. Zipori and A. Nagler for providing human stromal cells;M. Krummel for help with the two-photon microscope; G. Shakhar fordiscussions; and S. Schwarzbaum for editorial assistance. CD18-deficient micewere provided by E. Brown (University of California, San Francisco); antibodiesTS1/18 and TS2/4 were from T. Springer (Harvard University); monoclonalantibody 13 was from K. Yamada (National Institutes of Health); antibodyB5G10 was from M. Hemler (Harvard University); and 240Q and A308926(control compound, A270412) were from D.E. Staunton (ICOS). Supported bythe Cancer Research Institute (I.G.), the German Research Foundation (M.S.),the Max Planck Society (M.S.), the National Institutes of Health (AI45073 toJ.G.C.), the Linda Jacobs Chair in Immune and Stem Cell Research (R.A.), theIsrael Science Foundation (R.A.), the Minerva Foundation (R.A.) and MAIN,the EU6 Program for Migration and Inflammation (R.A.).

AUTHOR CONTRIBUTIONSE.W., most in vitro assays; I.G., multiphoton imaging; A.S., assistancewith lymphocyte motility studies; V.G., flow chamber studies; S.W.F., motilityanalysis and integrin staining; Z.S., cytoskeleton staining; T.J., immunoblots;

M.S., immunohistochemistry; J.G.C., in vivo experimental design andcontributions to manuscript preparation; R.A., conceptual design andmanuscript preparation.

COMPETING INTERESTS STATEMENTThe authors declare no competing financial interests.

Published online at http://www.nature.com/natureimmunology/

Reprints and permissions information is available online at http://npg.nature.com/

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