WNK1 kinase balances T cell adhesion versus migration in vivo

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1 WNK1 kinse lnes T ell dhesion versus migrtion in vivo Roert Köhl 1, Flvin Thelen, Lesley Vnes 1, Tigo F Brzão 1, Kthryn Fountin 1, Jin Xie 3, Chou-Long Hung 3, Ruth Lyk, Jens V Stein & Vitor L J Tyulewiz 1, Adhesion nd migrtion of T ells re ontrolled y hemokines nd y dhesion moleules, espeilly integrins, nd hve ritil roles in the norml physiologil funtion of T lymphoytes. Using n RNA-medited interferene sreen, we identified the WNK1 kinse s regultor of oth integrin-medited dhesion nd T ell migrtion. We found tht WNK1 is negtive regultor of integrin-medited dhesion, wheres it ts s positive regultor of migrtion vi the kinses OXSR1 nd STK39 nd the ion o-trnsporter SLC1A. WNK1-defiient T ells home less effiiently to lymphoid orgns nd migrte more slowly through them. Our results revel tht pthwy previously known only to regulte slt homeostsis in the kidney funtions to lne T ell dhesion nd migrtion. T ells ontinuously reirulte etween lymphoid orgns vi the lood nd lymphti systems, entering lymph nodes through speilized high endothelil venules (HEVs) 1. Initil seletin-medited intertions result in rolling of T ells on the endothelium. Susequently, signling y hemokine CCL1 through its reeptor CCR7 on T ells leds to the tivtion of ell-surfe integrin dhesion moleules suh s LFA-1 (α L β ), whih inds to its lignds ICAM1 nd ICAM, leding to firm dhesion etween the T ell nd HEV. T ells then trnsmigrte ross the endothelium nd enter the prenhym of the lymph node, where they migrte rpidly under the influene of the CCR7 lignds CCL19 nd CCL1, proess tht is muh less dependent on integrins. Finlly, T ells exit lymph nodes through lymphti vessels nd return to irultion vi the venous system. This reirultion is ritil for llowing T ells to sn lymphoid tissue for ntigen-presenting ells (APCs) ering ognte ntigen in the form of omplex of peptide nd mjor histoomptiility omplex tht n ind to the T ell ntigen reeptor (TCR). Binding of ntigen to the TCR results in signling tht stops T ell migrtion nd tivtes LFA-1, using firm dhesion etween the T ell nd the APC. The formtion of suh T ell APC onjugtes is neessry for T ell tivtion nd initition of T ell immune responses. The tivtion of LFA-1 y hemokine reeptors is triggered y inside-out signls from the reeptor tht led to onformtionl hnges in the integrin 7. These onvert the integrin from onformtion with low ffinity for lignd to n extended, losed onformtion, nd then extended, open, high-ffinity onformtion. The inside-out signl lso leds to inding of tlin nd kindlin-3 to the ytoplsmi domin of the β-suunit of LFA-1, ssoition of F-tin vi tlin, nd LFA-1 lustering, whih inreses its vidity for lignd. Notly, for stle high-ffinity inding, oth LFA-1 nd its lignds need to e immoilized, suh tht inding of lignd to LFA-1 results in the exertion of fore. Inside-out signls from the TCR result in LFA-1 tivtion through similr mehnisms; however, in the sene of immoilized lignd, they do not hnge the integrin onformtion 8. One gin, for stle dhesion, the lignd needs to e immoilized on the APC, nd LFA-1 needs to e nhored to the tin ytoskeleton suh tht inding of LFA-1 to ICAM1 results in fore tht promotes high-ffinity inding. A ritil signling moleule tht trnsdues inside-out signls from oth hemokine reeptors nd the TCR is the GTPse RAP1 (ref. ). Ative inds to the effetor proteins RIAM nd RAPL, whih in turn promote inding of tlin to the β-suunit of LFA-1 nd lustering of LFA-1, respetively To identify dditionl proteins tht might ontriute to the tivtion of LFA-1, we used n RNA-medited interferene (RNAi) sreen to identify kinses tht regulte integrin-dependent inding of T ells to APCs. We found tht the kinse WNK1 is negtive regultor of oth hemokine-reeptor- nd TCR-indued tivtion of LFA-1 nd susequent dhesion nd tht it does so vi RAP1. Conversely, we found tht WNK1 is positive regultor of T ell migrtion through the kinses OXSR1 nd STK39 nd the ion o-trnsporter SLC1A. In the sene of WNK1, T ells home less effiiently to lymphoid orgns nd migrte more slowly through them. Our results revel tht pthwy previously shown to regulte slt homeostsis in the kidney 1,13 lso funtions to lne T ell dhesion nd migrtion. RESULTS WNK1 is negtive regultor of integrin-medited dhesion To identify previously unknown signling pthwys tht regulte the dhesion of T ells, we rried out n RNAi sreen in whih 1 The Frnis Crik Institute, London, UK. Theodor Koher Institute, University of Bern, Bern, Switzerlnd. 3 University of Texs Southwestern Medil Center, Dlls, Texs, USA. Deprtment of Mediine, Imperil College, London, UK. Correspondene should e ddressed to V.L.J.T. (vitor.t@rik..uk). Reeived 1 April; epted My; pulished online 11 July 1; orreted fter print Otoer 1; doi:1.138/ni.39 nture immunology VOLUME 17 NUMBER 9 SEPTEMBER 1 17

2 we knoked down expression of 719 genes enoding kinses nd kinse-relted moleules individully in the Jurkt humn T ell leukemi ell line nd nlyzed the ility of the ells to form ntigen-speifi onjugtes with the Nlm B ell line in response to the superntigen stphylool enterotoxin E (SEE) (dt not shown). One of the vlidted hits from the sreen ws WNK1, whih enodes the serine-threonine kinse WNK1; the knokdown of WNK1 resulted in inresed T ell B ell onjugtion. Jurkt T ells with redued mounts of WNK1 exhiited inresed onjugtion ross rod rnge of SEE onentrtions (Fig. 1 nd Supplementry Fig. 1,). In ontrst, knokdown of the tyrosine kinse LCK, ritil positive regultor of TCR signling, resulted in deresed onjugtion (Fig. 1). Antigen-indued dhesion etween T ells nd APCs is medited minly y inding of integrins suh s LFA-1 on T ells to their lignds suh s ICAM1 on APCs 1. To test whether the inresed T ell APC onjugtion oserved in WNK1-defiient Jurkt T ells ws used y inresed TCR-indued integrin-medited dhesion, we evluted the ility of the ells to ind ICAM1 omplexes nd to dhere to plte-ound ICAM1. In ddition, we rodened the Conjugtes (%) ICAM1 inding (MFI 1 ) f m (GMFI 1 ) NT sirna Wnk1 sirna pool Lk sirna pool NT sirna SEE (µg/ml) CCL1 Anti-CD3 MnCl WNK1 sirna pool m (GMFI 1 ) 8 ICAM1 inding (MFI 1 ) US CXCL1 US Anti- MnCl US CXCL1 CD3 Kim17 (GMFI 1) ICAM1 inding (MFI 1 ) NT sirna Wnk1 sirna pool CXCL1 Anti-CD3 MnCl Kim17 (GMFI 1 ) 3 1 US Anti- MnCl CD3 Figure 1 WNK1 is negtive regultor of T ell dhesion. () Conjugte formtion etween Jurkt T ells trnsfeted with non-trgeting (NT) sirna or sirna pools direted ginst WNK1 or LCK nd Nlm B ells pulsed with vrious onentrtions (horizontl xis) of SEE. () Binding of solule ICAM1 omplexes to Jurkt ells in response to tretment for vrious times (horizontl xis) with CXCL1, ntiody to CD3 (nti-cd3) or MnCl. () Binding of ICAM1 to mouse CD + T ells from ontrol or WNK1-defiient mie in response to tretment for vrious times (horizontl xis) with CCL1, nti-cd3 or MnCl. (d) Adhesion of mouse CD + T ells to plte-ound ICAM1 left unstimulted (US) or in response to nti-cd3 or CXCL1 (horizontl xis), normlized to the dhesion of ontrol ells in response to nti-cd3. (e) Conjugtion of mouse CD + T ells to CH7 ells pulsed with SEB. (f) Binding ICAM1 inding (MFI 1 ) ICAM1 inding (MFI 1 ) g NT sirna WNK1 sirna.. h RAP1... Time (s) 1 RAP CXCL1 Wnk1 fl/+ Wnk1 fl/fl RCE RCE 1 1 d Adhesion (%) CCL1 1 1 Wnk1 +/+ dlkcre Wnk1 fl/fl dlkcre US Anti- CCL1 CD3 NT sirna WNK1 sirna RAP Anti-CD3 7 7 ICAM1 inding (MFI 1 3 ) RAP Conjugtion (%) e Wnk1 +/+ dlkcre Wnk1 fl/fl dlkcre Wnk1 fl/+ Wnk1 fl/fl RCE RCE Anti-CD3 1 SEB (µg/ml) of ntiodies speifi for LFA-1 onformtions with high or intermedite ffinity for lignd (m or Kim17) on Jurkt T ells left unstimulted or treted with vrious stimuli (horizontl xis), presented s geometri men fluoresene intensity (GMFI). (g) Immunolot nlysis (top) of RAP1 nd preipitted from Jurkt T ells trnsfeted with non-trgeting or WNK1-speifi sirna (ove lots) nd stimulted for vrious times (ove lnes) with CXCL1 or nti-cd3; elow, undne of reltive to tht of RAP1. (h) Immunolot nlysis of RAP1 nd preipitted from mouse CD + T ells stimulted for vrious times (ove lnes) with CCL1 or nti-cd3 (elow, quntifition s in g). P <., P <.1, P <.1 nd P <.1 (two-wy nlysis of vrine (ANOVA) ( ), Mnn-Whitney test (d,e) or unpired t test (f h)). Smple sizes: (three), (four, ontrol; six, mutnt), (six), d (five), e (six, ontrol; five, mutnt), f (six; exept three in rightmost pnel), g (four), h (four). Dt re from one experiment representtive of three (,e, nd f, fr right) or four (g,h) experiments (men ± s.e.m.) or re pooled from two independent experiments (d, nd f, leftmost three pnels; men + s.e.m.). 17 VOLUME 17 NUMBER 9 SEPTEMBER 1 nture immunology

3 study y investigting the effet of WNK1 defiieny on hemokineindued integrin-medited dhesion. We found tht knokdown of WNK1 resulted in inresed dhesion of Jurkt T ells to oth solule ICAM1 nd plte-ound ICAM1 following stimultion through either the hemokine reeptor CXCR or the TCR, despite there eing no ltertion in surfe expression of CXCR, TCR or LFA-1 (Fig. 1 nd Supplementry Fig. 1 f). In ontrst, knokdown of LCK resulted in deresed dhesion. Notly, time-ourse nlysis reveled tht the inding of ICAM1 ws lredy signifintly inresed in WNK1-defiient Jurkt T ells 1 s or 1 min fter stimultion through CXCR or TCR, respetively, whih suggested tht WNK1 is involved in the erly stges of dhesion. To investigte whether WNK1 lso regultes dhesion in primry T ells, we generted mie with WNK1-defiient T ells, using either the dlk-cre trnsgene 1, for onditionl deletion of loxp-flnked llele of Wnk1 (Wnk1 fl ) 1 t lte stges of thymoyte development, or ROSA-CreERT (RCE), tmoxifen-induile Cre reominse 17. We generted one mrrow himers y reonstituting irrdited mie defiient in the RAG1 reominse (whih lk mture B ells nd T ells) with mrrow ells from either strin. This served to inrese the numer of mie from whih WNK1-defiient T ells ould e isolted nd, for the strin, it limited the omplete loss of Wnk1 to lymphoytes. In oth strins, there ws effiient loss of Wnk1 mrna in CD + T ells nd CD8 + T ells (Supplementry Fig. ). We found tht in response to stimultion through either CCR7 or the TCR, WNK1-defiient CD + T ells showed inresed inding of ICAM1 omplexes nd inresed dhesion to ICAM1-oted pltes (Fig. 1,d). Furthermore, onsistent with inresed TCR-indued dhesion through LFA-1, the mutnt T ells were more effiient t forming ntigen-speifi onjugtes with APCs (Fig. 1e). Mutnt T ells hd slightly redued surfe expression of TCR nd CCR7, ut slightly elevted expression of LFA-1 (Supplementry Fig. d,e). One gin, time-ourse nlysis reveled inresed ICAM1 inding t ll time points following stimultion through CCR7 or TCR. Thus, s in Jurkt T ells, WNK1 is negtive regultor of oth hemokinereeptor- nd TCR-indued integrin-dependent dhesion in primry mouse T ells nd ts t the erliest stges of dhesion. WNK1 is negtive regultor of RAP1 Next we investigted the mehnism y whih WNK1 regultes integrin-medited dhesion. Using ntiodies tht reognize either n Migrtion (%) 1 1 NT sirna WNK1 sirna pool CXCL1 (ng/ml) Migrtion (%) CCL1 (ng/ml) top CCL1 (ng/ml) ottom No ICAM1 ICAM1 Speed (µm/min) 1 1 CCL1 (ng/ml) intermedite-ffinity onformtion of humn LFA-1 or high-ffinity onformtion of humn LFA-1, we were le to detet n inrese in oth of these onformtions on Jurkt T ells in response to CXCL1 (Fig. 1f). In ontrst, s expeted 8, stimultion of the ells through the TCR resulted in no detetle hnge in LFA-1 ffinity (Fig. 1f). Notly, the CXCL1-indued inrese in intermedite- or high-ffinity LFA-1 onformtions ws not ffeted y loss of WNK1 (Fig. 1f); thus, WNK1 does not regulte the hemokine-indued ffinity hnge in LFA-1. In ontrst, we noted inresed dhesion to ICAM1 in oth WNK1-defiient Jurkt ells nd primry mouse T ells in response to Mn +, tion tht diretly indues the high-ffinity onformtion of LFA-1 (Fig. 1,,f). This suggests tht WNK1 my regulte other proesses tht ontriute to LFA-1-medited dhesion, suh s nhoring of the integrin to the tin ytoskeleton or its lustering. We extended the nlysis to intrellulr signling pthwys nd found no hnge in hemokine- or TCR-indued tivtion of ERK kinses, no hnge in TCR-indued lium fluxes in WNK1-defiient Jurkt T ells, nd no hnge in hemokine-indued tin polymeriztion in WNK1-defiient mouse CD + T ells (Supplementry Fig. 3 ). However, oth Jurkt T ells nd primry mouse CD + T ells defiient in WNK1 showed lrge inrese in oth hemokinend TCR-indued tivtion of RAP1, ritil regultor of integrinmedited dhesion,1 (Fig. 1g,h nd Supplementry Fig. 1g). Given pulished studies showing tht expression of onstitutively tive RAP1 in T ells results in inresed integrin-medited dhesion 18, we onlude tht WNK1 my t s negtive regultor of RAP1 tivtion nd therey influene integrin-medited dhesion. WNK1 is positive regultor of T ell migrtion Given tht the proesses of T ell dhesion nd migrtion re losely relted, with inresed dhesion potentilly leding to deresed migrtion 19, we lso exmined the effet of WNK1 knokdown on ell migrtion using Trnswell ssy. We found tht WNK1-defiient Jurkt T ells migrted less effiiently in response to the hemokine CXCL1, despite expressing norml levels of CXCR (Fig. nd Supplementry Fig. 1f,h). Furthermore, WNK1-defiient primry mouse CD + T ells lso showed less-effiient hemotxis in response to grdient of CCL1 in oth the sene of ICAM1 nd presene of ICAM1 nd showed less hemokinesis in response to equl onentrtions of CCL1 elow nd ove the memrne (Fig. ). Timelpse imging reveled tht in response to CCL1, WNK1-defiient Displement (µm) 1 8 CCL1 (ng/ml) + CCL1 Distne (µm) CCL1 Distne (µm) Figure WNK1 is positive regultor of T ell migrtion. () Migrtion of Jurkt T ells trnsfeted with nontrgeting sirna or WNK1-speifi sirna pool, in response to CXCL1, normlized to CXCL1-indued migrtion of ontrol ells. () Migrtion of mouse CD + T ells from the top hmer to the ottom hmer of Trnswell plte in response to vrious onentrtions of CCL1 in the presene or sene of ICAM1 (horizontl xis). () Speed nd displement of mouse CD + T ells in response to CCL1, mesured y time-lpse mirosopy (left) nd presented s n overly of migrtion trks of individul T ells (right). P <.1 nd P <.1 (Mnn-Whitney test). Smple sizes: (five), (six), (7, ontrol unstimulted; 7, ontrol with CCL1;, mutnt, unstimulted; 93, mutnt with CCL1). Dt re pooled from five independent experiments (; men + s.e.m.) or re from one experiment representtive of two experiments (,; men + s.e.m.). nture immunology VOLUME 17 NUMBER 9 SEPTEMBER 1 177

4 Rtio CD + T ells migrted muh more slowly (Fig. nd Supplementry Video 1). Thus, WNK1 ts s positive regultor of CCL1-indued migrtion in primry mouse CD + T ells, nd this migrtion my e independent of LFA-1-medited dhesion. WNK1 is required for effiient homing into lymph nodes Next we evluted whether WNK1 lso regultes physiologil T ell trffiking. We trnsferred mixture of WNK1-expressing T ells Rtio (%) d Displement (µm) CD + CD8 + LN S B No integrin loking 1 3 Wnk1 fl/+ RCE Displement (µm) PNAd Time. (min. ) Time. (min. ) 3 1 Wnk1 fl/ RCE 1: min With integrin loking 1 3 min min 1 8 L PV PC Figure 3 WNK1 is required for effiient homing into lymph nodes. () Rtio of WNK1-defiient mouse T ells to ontrol mouse T ells in the lymph node (LN), spleen (S) nd lood (B) 1 h fter intrvenous injetion into C7BL/J reipient mie, normlized to input rtio. () Three-dimensionl histology of lymph nodes showing T ells (lue nd red) nd HEVs, reveled y expression of peripherl node ddressin (PNAd) (white), t min fter trnsfer s in (left), nd reltive frequeny of T ells in the lumen (L), perivsulr re (PV) or prenhym (PC) of lymph nodes t nd min fter suh trnsfer (right). Sle r, µm. () Mirosopy of T ells (red nd green) in the spleen, s well s the oundry etween red nd white pulp, indited y MADCAM1, the lignd for integrin α β 7 T ells (%) 8 Single-ell speed (µm/min) e Frequeny T ells (%) nd WNK1-defiient T ells intrvenously into wild-type mie nd nlyzed their homing into lymphoid orgns. We found tht WNK1- defiient CD + nd CD8 + T ells homed less effiiently into oth lymph nodes nd the spleen 1 h fter trnsfer, with more ells remining in the lood (Fig. 3). Three-dimensionl histologil nlysis reveled tht lrger frtion of the mutnt T ells were loted in the lumen of the lood vessels nd in perivsulr regions of the lymph nodes or min fter trnsfer, wheres fewer hd entered + + Integrin loking L PV PC e Cirulrity Wnk1 fl/+ RCE MADCAM1 f Wnk1 fl/ RCE Dipedisis (s) 3 1 Figure WNK1 is required for migrtion in lymph nodes in vivo. () Rtio of firmly dherent T ells to rolling or dherent T ells, determined y intrvitl mirosopy of lymph node HEVs fter trnsfer of ontrol nd WNK1-defiient T ells into C7BL/J reipient mie. ( e) Migrtion of T ells in lymph node prenhym nlyzed y two-photon intrvitl mirosopy fter trnsfer of ontrol nd WNK1-defiient T ells into C7BL/J mie in the presene or sene of integrin loking. () Trks of ontrol T ell nd WNK1-defiient T ell, showing ell shpes over 1-min period. () Migrtion speed; eh symol represents n individul ell. (d) Men displement of T ells s funtion of the squre root of time (time. ). (e) Reltive frequeny of T ells s funtion of irulrity, where irulrity of 1 is perfet irle; exmple ell shpes re shown ove the grph. Smll horizontl lines indite the medin. P <., P <.1, P <.1 (unpired t test (), Mnn-Whitney (,e)). Smple sizes: (three, ontrol; four, mutnt), (13, ontrol with no loking; 13, mutnt with no loking; 7, ontrol with integrin loking; 19, mutnt with integrin loking), e (13, ontrol; 13, mutnt). Dt re pooled from two independent experiments (men ± s.e.m. in d). d Arrest oeffiient (%) g Speed (µm/min) (lue), t 1 h fter T ell trnsfer (left), nd rtio of CD + T ells in the white pulp to tht in the red pulp of the spleen (right). Sle r, 1 µm. (d) Frtion of time ontrol nd WNK1-defiient T ells rrested (< µm/min) during migrtion over n endothelil ell monolyer under flow onditions. (e) Reltive frequeny of ells tht remined dherent on endothelil ells 1 min fter rrest. (f) Reltive frequeny of ells tht underwent dipedesis through endothelil ell monolyer (left) nd durtion of dipedesis (right). (g) Speed of ell migrtion on the endothelil ell monolyer. Eh symol (d,f (right),g) represents n individul ell; smll horizontl lines indite the medin in d nd the men in, f (right) nd g. P <., P <.1, P <.1 nd P <.1 (one-smple t-test (), Mnn-Whitney test ( e, f (right), g), Wiloxon mthed-pirs test (f, left)). Smple sizes: (1, lymph node nd spleen; 11, lood), (9, min;, min), (), d (33, ontrol; 3, mutnt), e (9), f (8, perentge of dipedesis; 73 ontrol nd mutnt, time of dipedesis), g (,8, ontrol; 3,8, mutnt). Dt re pooled from two independent experiments (,e g; men + s.e.m. in,,e,f (left)) or re from one experiment representtive of two experiments (d). Adherent ells (%) 8 Dipedesis (%) 1 8 White pulp/red pulp VOLUME 17 NUMBER 9 SEPTEMBER 1 nture immunology

5 Gene expression (FPKM) 8 Wnk1 Wnk d CD + CD8 + Wnk3 Wnk Oxsr1 Stk39 Sl1 Sl TUBULIN.8. f ICAM1 inding (MFI 1 ) Wnk1 fl/+ RCE CCL TUBULIN Wnk1 fl/d38a RCE CCL1 the prenhym (Fig. 3 nd Supplementry Video ); this suggested tht WNK1-defiient T ells were less effiient t extrvsting ross the endothelium nd t migrting wy from the vsulture into the prenhym. Similrly, histology of the spleen showed tht more WNK1-defiient T ells were loted in the red pulp thn in the white pulp, where most T ells reside, t 1 h fter trnsfer (Fig. 3), whih suggests tht WNK1-defiient T ells re defetive t rossing the mrginl sinus-lining ells tht lymphoytes need to ross to gin entry to the white pulp. To delinete the integrin-dependent dhesion nd trnsmigrtion steps required for entry into lymph nodes, we imged ontrol nd mutnt T ells migrting on monolyer of endothelil ells under flow nd trnsmigrting through it. We found tht WNK1-defiient T ells rrested for longer frtion of time on the endothelil ells nd more of them resisted dethment from endothelil ells (Fig. 3d,e nd Supplementry Video 3), onsistent with their hyperdhesive phenotype. Conversely, fewer mutnt T ells were le to ross through the endothelil monolyer, nd they took longer to do so (Fig. 3f). Furthermore, the mutnt T ells migrted more slowly on the endothelil monolyer (Fig. 3g). Finlly, we trnsferred either ontrol or mutnt T ells into wildtype mie nd used intrvitl imging to follow their movement in the TUBULIN Wnk1 fl/+ Wnk1 fl/d38a RCE RCE Anti-CD3 ERK e 1 Anti-CD3 3 1 ERK.9..3 Wnk1 +/+ dlkcre 1 Wnk1 fl/fl dlkcre lumen of the HEV nd in the prenhym of lymph nodes. Imging of rpid T ell dhesion in lymph node HEVs showed similr stiking frtion of firmly dherent ells for oth WNK1-defiient T ells nd ontrol T ells (Fig. ), whih suggests tht the sene of WNK1 does not ffet the ility of T ells to dhere firmly to endothelium. In ontrst, nlysis of ells in the prenhym of the lymph nodes reveled tht WNK1-defiient T ells migrted more slowly thn did ontrol T ells, hd lower motility oeffiient (.7 µm /min nd 3. µm /min in ontrol nd mutnt ells, respetively) nd were more rounded (Fig. e nd Supplementry Video ). To evlute whether this redued migrtion ws used y inresed integrinmedited dhesion, we trnsferred T ells into wild-type mie nd then loked the integrins LFA-1 nd VLA h lter. We found tht WNK1-defiient T ells still migrted more slowly thn did ontrol T ells, hd redued motility oeffiient (. µm /min (ontrol ells) nd 3. µm /min (mutnt ells)) nd displyed lrger turning ngles, hrteristi feture of slower migrting ells 1 (Fig.,d nd Supplementry Fig. ). Indeed, the mutnt ells migrted even more slowly in the presene of integrin-loking ntiodies (Fig. ), whih indited tht their deresed migrtion speed ws not result of hyperdhesiveness vi integrins. Thus, we onlude tht WNK1 is CCL1 1 ACTIN.7.. Wnk1 +/+ dlkcre DMSO PI-13 MK DMSO PI-13 MK p-akt p-pras ERK p-akt p-pras ERK CCL1 Anti-CD3 CCL1 Anti-CD3 MnCl 3 Wnk1 fl/d38a RCE Wnk1fl/+ RCE Wnk1 1 fl/d38a RCE ICAM1 inding (MFI 1 ) g Migrtion (%) 1 1 Wnk1 fl/d38a RCE 1 CCL1 (ng/ml) Figure Chemokine reeptors nd TCR tivte WNK1. () RNA-sequening nlysis of the expression of vrious genes (horizontl xis) in mouse T ells, presented s frgments per kilose per million reds (FPKM). ( e) Immunolot nlysis (top) of phosphorylted OXSR1 (), tuulin, tin or ERK in mouse CD + T ells stimulted for vrious times (ove lnes) with CCL1 or nti-cd3 using wild-type T ells (), WNK1-defiient nd ontrol T ells () or T ells expressing kinse-intive WNK1-D38A nd ontrol ells (d), nd of, phosphorylted AKT (p-akt), phosphorylted PRAS (p-pras) nd ERK in wild-type T ells treted with PI(3)K inhiitor (PI-13), AKT inhiitor (MK) or vehile (DMSO) (e). Below, quntifition of phosphorylted OXSR1 in the lnes ove, normlized to the undne of tuulin, ERK or tin in eh lne. (f) Binding of ICAM1 to mouse CD + T ells expressing kinse-intive WNK1-D38A or ontrol ells, treted for vrious times (horizontl xis) with CCL1, nti-cd3 or MnCl. (g) CCL1-indued migrtion of T ells through Trnswell system, normlized to the CCL1-indued migrtion of ontrol ells. P <., P <.1, P <.1 nd P <.1 (two-wy ANOVA (f), Mnn-Whitney test (g) or unpired t-test ( e)). Smple sizes: (three), (four), (four, CCL1; three, nti-cd3), d (four), e (four, CCL1; seven, nti-cd3), f (six), g (nine). Dt re pooled from two (f,g), three (, nti-cd3), four (,, CCL1; d,e, CCL1) or seven (e, nti-cd3) independent experiments (men ± s.e.m.). ICAM1 inding (MFI 1 3 ) Wnk1 fl/fl dlkcre Anti-CD3 nture immunology VOLUME 17 NUMBER 9 SEPTEMBER 1 179

6 NT sirna WNK1 sirna pool OXSR1/STK39 sirna pool SLC1A sirna pool NT sirna pool WNK1 sirna pool OXSR1/STK39 sirna pool SLC1A sirna pool ICAM1 inding (MFI 1 ) 8 CXCL1 Anti-CD3 MnCl Oxsr1 +/+ Oxsr1 T18A/T18A ICAM1 inding (MFI 1 ) d ICAM1 inding (MFI 1 ) Sl1 +/+ Sl1 / 1 Conjugtes (%) SEE (µg/ml) ICAM1 inding (MFI 1 ) 1 1 CCL1 Anti-CD3 MnCl CCL1 Anti-CD3 MnCl. 1 3 ICAM1 inding (MFI 1 ) ritil regultor of the homing nd migrtion of T ells in vivo. The defetive overll homing of WNK1-defiient T ells my e used y less effiient dethment from HEVs nd lower dipedesis, despite omprle primry dhesion. In ontrst, the redued prenhyml migrtion of WNK1-defiient T ells is independent of inresed integrin-medited dhesion. Chemokine reeptors nd TCR tivte the WNK1 pthwy Although WNK1 hs not een studied in lymphoytes, its funtion hs een extensively investigted in kidney tuulr epithelil ells, in whih it ontrols the uptke of sodium, potssium nd hloride ions 1. In prtiulr, WNK1 phosphoryltes nd tivtes two relted kinses, OXSR1 nd STK39, whih in turn phosphorylte nd tivte the N + -K + -Cl o-trnsporters SLC1A (NKCC1) nd SLC1A1 (NKCC) nd therey stimulte uptke of these ions. In view of the unexpeted role for WNK1 in T ell dhesion nd migrtion, we sought to determine whether the pthwy tht leds from WNK1 nd rnhes into OXSR1 or STK39, either of whih then leds to SLC1A or SLC1A1 (WNK1-OXSR1/STK39-SLC1A/SLC1A1), might lso funtion in T ells nd, if so, whether it might ontriute to the dhesion nd migrtion of the ells. Anlysis of RNA-sequening dt reveled tht oth CD + primry mouse T ells nd CD8 + primry mouse T ells expressed Wnk1 ut not other memers of the Wnk fmily (Fig. ). Both susets of T ells lso expressed Oxsr1, Stk39 nd Sl1, ut not Sl11 (Fig. ). To investigte whether the WNK1-OXSR1/STK39-SLC1A pthwy funtions in T ells, we stimulted primry mouse CD + T ells through CCR7 or the TCR nd immunolotted ell extrts with ntiodies speifi for WNK1- phosphoryltion sites on OXSR1 nd STK39 (Ser3 nd Ser383, ICAM1 inding (MFI 1 3 ) ICAM1 inding (MFI 1 ) 1 3 ICAM1 inding (MFI 1 ) 1 3 respetively) 3. We found tht eh stimulus resulted in rpid phosphoryltion of OXSR1, whih ws rogted in WNK1-defiient T ells (Fig.,). Thus, oth CCR7 nd the TCR trnsdue signls through WNK1, whih led to the phosphoryltion nd, presumly, tivtion of OXSR1. To evlute whether WNK1 s kinse tivity is required for its regultion of T ell dhesion nd migrtion, we generted strin of mie with replement of the sprti id t position 38 with lnine (D38A) in the kinse domin of the protein, hnge tht hd een shown to eliminte kinse tivity 3 (Supplementry Fig. ). Mie homozygous for this Wnk1 D38A llele did not survive to irth (Supplementry Fig. ), similr to results previously reported for mie with totl loss of WNK1 (ref. ). To nlyze T ells expressing only kinse-intive WNK1, we generted ompound heterozygous mie with oth loxp-flnked llele of Wnk1 nd kinse-intive llele of Wnk1, nd tmoxifen-induile Cre (Wnk1 fl/d38a RCE); tretment of suh mie with tmoxifen results in T ells tht express only kinse-intive WNK1. As ontrol, we used mie, in whih tmoxifen injetion leds to deletion of the onditionl llele of Wnk1, leving n intt wild-type llele. T ells from oth strins express similr mounts of Wnk1 (Supplementry Fig. ). T ells expressing kinse-intive WNK1 hd muh less phosphoryltion of OXSR1 nd showed no induile phosphoryltion of OXSR1 in response to stimultion through either CCR7 or the TCR (Fig. d). These results indite tht the kinse tivity of WNK1 is required for the trnsdution of signls vi CCR7 nd the TCR, whih led to phosphoryltion of OXSR1 t Ser3. Given tht this site is known sustrte of WNK1, our findings suggest tht signling vi oth CCR7 nd the TCR leds to tivtion of WNK1 s kinse tivity. ICAM1 inding (MFI 1 3 ) Figure OXSR1, STK39 nd SLC1A do not regulte integrin-medited dhesion. () Binding of ICAM1 to Jurkt T ells trnsfeted with non-trgeting sirna or sirna pools direted ginst vrious genes (key) nd stimulted for vrious times (horizontl xis) with CXCL1, nti-cd3 or MnCl. () Conjugtion of Jurkt T ells to Nlm B ells pulsed with vrious doses of SEE (horizontl xis). () Binding of ICAM1 to mouse CD + T ells of vrious genotypes (key) fter stimultion for vrious times (horizontl xis) with CCL1, nti-cd3 or MnCl. (d) Binding of ICAM1 to mouse CD + T ells of vrious genotypes (key) following stimultion for vrious times (horizontl xis) with CCL1, nti-cd3 or MnCl. P <., P <.1, P <.1 nd P <.1 (two-wy ANOVA). Smple sizes: (six), (four), (eight), d (five, ontrol ells; six, mutnt ells). Dt re pooled from three () or four () or two () independent experiments (men ± s.e.m.). 18 VOLUME 17 NUMBER 9 SEPTEMBER 1 nture immunology

7 Migrtion (%) 1 1 NT sirna WNK1 sirna pool OXSR1/STK39 sirna pool SLC1A sirna pool CXCL1 (ng/ml) Migrtion (%) 1 1 Oxsr1 +/+ Oxsr1 T18A/T18A CCR7 nd TCR tivte WNK1 vi PI(3)K nd AKT We next investigted how CCR7 nd the TCR might tivte WNK1, despite engging lrgely different signling pthwys. Pulished studies hve suggested tht AKT might tivte WNK1 (ref. ), nd given tht oth hemokine reeptors nd the TCR re known to tivte AKT vi phosphtidylinositol-,-isphosphte 3-kinse (PI(3)K), we ssessed the potentil involvement of these kinses in the tivtion of WNK1. We found tht tretment of T ells with PI-13 or MK (inhiitors of PI(3)K or AKT, respetively) loked oth CCR7- nd TCR-indued phosphoryltion of AKT nd PRAS, known sustrte of AKT, inditing tht oth inhiitors were ting s expeted (Fig. e). Notly, oth inhiitors lso loked CCR7- nd TCR-indued phosphoryltion of OXSR1 (Fig. e). Together these results suggest tht oth reeptors trnsdue signls vi PI(3)K nd AKT, whih led to the tivtion of WNK1. WNK1 s kinse tivity regultes dhesion nd migrtion Finlly, we investigted whether WNK1 s kinse tivity is required for the regultion of integrin-medited dhesion or migrtion in T ells. We found tht CD + T ells expressing kinse-intive WNK1 showed inresed inding of ICAM1 following stimultion through CCR7 or TCR nd in response to tretment with Mn + to tivte integrins from the outside (Fig. f), nd they showed deresed CCL1-indued migrtion (Fig. g). These results re very similr to those otined for WNK1-defiient T ells nd indite tht the kinse tivity of WNK1 is required for its negtive regultion of integrin-medited dhesion nd positive regultion of migrtion. OXSR1, STK39 nd SLC1A do not regulte dhesion vi LFA-1 We next investigted whether the OXSR1/STK39-SLC1A pthwy, the est-hrterized pthwy downstrem of WNK1, is lso involved in the proesses ssessed ove. Knokdown of OXSR1 or STK39 lone, oth OXSR1 nd STK39 together, or SLC1A in Jurkt T ells hd no effet on CCR7- or TCR-indued inding of ICAM1 omplexes or dhesion to plte-ound ICAM1, or on onjugtion of Jurkt T ells to SEE-pulsed APCs (Fig.,, nd Supplementry 1 CCL1 (ng/ml) Migrtion (%) 1 1 d Sl1 +/+ Sl1 +/+ Sl1 / Sl1 / 1 CCL1 (ng/ml) Speed (µm/min) 1 1 CCL1 (ng/ml) Displement (µm) 8 CCL1 (ng/ml) Figure 7 OXSR1, STK39 nd SLC1A positively regulte hemokine-indued migrtion. () CXCL1-indued migrtion of Jurkt T ells trnsfeted with sirna (s in Fig. ), normlized to migrtion of ells trnsfeted with non-trgeting sirna (results for non-trgeting nd WNK1-speifi sirna in Fig. re repeted here for lrity). () CCL1-indued migrtion of mouse CD + T ells of vrious genotypes (key). () CCL1-indued migrtion of mouse CD + T ells of vrious genotypes (key). (d) Speed nd displement of mouse CD + T ells in response to CCL1, mesured y time-lpse mirosopy (left) nd presented s n overly of migrtion trks of individul T ells (right). (e) CCL1-indued migrtion of mouse CD + T ells of vrious genotypes (key) in the presene or sene of umetnide. P <.1, P <.1 nd P <.1 (Mnn-Whitney). Smple sizes: (five), (eight), (ten, ontrol ells; 11, mutnt ells), d (3 ontrol ells no CCL1; 1,773 ontrol ells with CCL1; 3 mutnt ells no CCL1; 1,1, mutnt ells with CCL1), e (six). Dt re pooled from five (), two (,e) or four () independent experiments (men + s.e.m.)) or re representtive of two independent experiments(d; men + s.e.m.). +CCL1 Distne (µm) CCL1 Fig. 1,e). Extending these studies to primry mouse T ells, we investigted CD + T ells from mie ering point muttion in Oxsr1 tht results in replement of the threonine t position 18 with lnine (T18A) 13. This residue is phosphorylted y WNK1, whih leds to tivtion of OXSR1 (ref. ), nd OXSR1-T18A n therefore no longer e tivted y WNK1. We lso ssessed SLC1A-defiient CD + T ells. We found tht T ells expressing OXSR1-T18A or defiient in SLC1A showed no ltertion in CCR7-, TCR- or Mn + - indued inding of ICAM1 omplexes (Fig.,d). Thus, lthough the WNK1 kinse is negtive regultor of integrin-medited dhesion, it does not do this through OXSR1, STK39 or SLC1A. OXSR1, STK39 nd SLC1A regulte migrtion In ontrst with the lk of effet on dhesion, knokdown of OXSR1 or STK39 lone, oth together, or SLC1A resulted in redued CCL1-indued hemotxis of Jurkt T ells (Fig. 7 nd Supplementry Fig. 1h). Furthermore, CCL1-indued hemotxis ws deresed in mouse CD + T ells expressing OXSR1-T18A or defiient in SLC1A (Fig. 7,), nd time-lpse imging reveled tht SLC1A-defiient T ells migrted more slowly in response to CCL1 (Fig. 7d nd Supplementry Video ), despite unltered surfe expression of CCR7 (Supplementry Fig. f,g). Finlly, we sought to determine whether the movement of ions through the o-trnsporter SLC1A is required for its funtion in T ell migrtion y treting ells with umetnide, n inhiitor of ion trnsport y SLC1A. We found tht umetnide deresed the CCL1-indued hemotxis of wild-type CD + T ells, lthough the derese ws not s lrge s tht in WNK1-defiient T ells (Fig. 7e). Together, these results suggest tht WNK1 regultes T ell migrtion vi the OXSR1 STK39-SLC1A pthwy nd tht it does so in prt y ontrolling ion movement through SLC1A. DISCUSSION Using n RNAi sreen, we identified previously unknown pthwy tht regultes T ell dhesion nd migrtion in vivo. WNK1 hs een shown to ontrol slt resorption in the kidney, in prt through e Sl1 +/+ Sl1 / Migrtion (%) Bumetnide ( µm) Distne (µm) 1 1 CCL1 (1 ng/ml) nture immunology VOLUME 17 NUMBER 9 SEPTEMBER 1 181

8 tivtion of OXSR1 nd STK39 nd the o-trnsporters SLC1A nd SLC1A1 (ref. ). Thus, it ws surprising to find tht WNK1 regultes T ell dhesion, tht the WNK1-OXSR1/STK39-SLC1A pthwy ontrols ell migrtion, nd tht this pthwy is tivted y stimultion of the ells through the TCR or CCR7. Our results suggest tht oth reeptors trnsdue signls vi PI(3)K nd AKT, whih led to the rpid tivtion of WNK1. AKT hs een reported to phosphorylte WNK1 t Thr (refs. 7,8); however, this phosphoryltion does not led to tivtion of WNK1 s kinse tivity, so it remins unler how AKT tivtes WNK1. We found tht WNK1 is negtive regultor of LFA-1-medited dhesion nd tht WNK1 s kinse tivity is required for this funtion. WNK1 does not regulte hemokine-indued onformtionl hnges of LFA-1. Insted, we found tht WNK1-defiient ells hd lrge inrese in tivted RAP1, whih we propose might e responsile for the inresed dhesion. Consistent with this, T ells expressing onstitutively tive RAP1 re hyperdhesive 18, inluding fter tretment with Mg + nd EGTA, whih indues high-ffinity onformtion of LFA-1 similr to Mn + tretment. Thus the inresed in WNK1-defiient T ells might ount for the inresed dhesion in response to Mn +. Ative inds to the effetor proteins RIAM nd RAPL, whih in turn indue inding of tlin to the β-suunit of LFA-1 nd lustering of LFA-1, respetively, therey promoting dhesion It is unler how WNK1 might regulte RAP1 tivtion, ut it is possile tht it diretly or otherwise regultes RAP1-speifi gunine nuleotide exhnge ftors or GTPse-tivting proteins. Our results lso indite tht WNK1 is positive regultor of T ell migrtion in response to hemokines. This migrtion funtion required WNK1 s kinse tivity ut ws distint from its role s negtive regultor of dhesion for two resons. First, geneti studies reveled tht the OXSR1/STK39-SLC1A pthwy downstrem of WNK1 is required for the regultion of migrtion, ut not dhesion. Seond, if the redued migrtion of WNK1-defiient T ells were result of inresed dhesion, loking dhesion should hve llowed mutnt T ells to migrte more rpidly. However, intrvitl imging of T ells in lymph nodes reveled tht loking integrin inding did not use WNK1-defiient T ells to speed up; on the ontrry, they migrted even more slowly, whih suggests tht their hyperdhesive phenotype prtilly resues the migrtion defet. Our results indite tht WNK1 regultes migrtion through the OXSR1/STK39-SLC1A pthwy nd tht t lest prt of this might e medited y the movement of ions ross the memrne through SLC1A. One possiility is tht ion trnsport ontriutes to ell migrtion y using osmoti uptke of wter, whih results in hnges in ell volume tht re required for ell movement 9. In this ontext, we note the proposed osmoti engine model, in whih polrized uptke of ions nd wter t the leding edge of ell nd efflux of ions nd wter t the triling edge result in ell movement 3. In support of the proposed role for SLC1A in ell migrtion, this o-trnsporter lolizes to the leding edge of migrting gliom ells, nd inhiition of its iontrnsport funtion results in redued migrtion 31. Future studies should investigte loliztion of the proteins in the WNK1-OXSR1/ STK39-SLC1A pthwy in migrting T ells. Finlly, we note tht the dhesome of firolsts shows enrihment for WNK1 nd WNK1 expression is upregulted in migrting firolsts, whih suggests tht it might e involved in these proesses in other ell types 3,33. In summry, we hve identified WNK1 s previously unknown regultor of T ell dhesion nd migrtion tht, to the est of our knowledge, is the first regultor of its kind to inversely ontrol these two proesses. In ontrst, for exmple, T ells defiient in the GTPses RAC1 nd RAC show derese in oth integrinmedited dhesion nd migrtion 3. T ells with hypertive LFA-1 show inresed dhesiveness ut redued migrtion 3,3 ; however, this redution in migrtion is onsequene of hyperdhesiveness nd seen only in the presene of integrin lignds, wheres interstitil migrtion in lymph nodes is unffeted. In ontrst, loss of WNK1 results in inresed dhesion nd deresed migrtion, ut the ltter phenotype is independent of the dhesion phenotype. Thus, WNK1 inversely nd independently regultes dhesion nd migrtion nd my t to lne these two relted proesses. Methods Methods nd ny ssoited referenes re ville in the online version of the pper. Aession odes. Sequene Red Arhive: RNA-sequening dt, SRP9. Note: Any Supplementry Informtion nd Soure Dt files re ville in the online version of the pper. Aknowledgments We thnk S. Ley for ritil reding of the mnusript; D. Alessi (University of Dundee) for Oxsr1 T18A mie, nti-pser3-oxsr1 ntiodies nd disussions; R. Zmoysk (University of Edinurgh) for Jurkt ells, D. Bell (Frnis Crik Institute) for help with imge nlysis; M. Adier (Theodor Koher Institute) for isoltion nd ultivtion of primry mouse rin mirovsulr endothelil ells; N. Hogg (Frnis Crik Institute) for Kim17 nd m ntiodies; nd L. Stlin nd C. Else (Mount Sini Medil Center) for ess to Sl1 mutnt mie (supported y the NIH NIDDK grnt P3 DK7937, Pittsurgh Center For Kidney Reserh, Core B). Supported y the Frnis Crik Institute (V.T.), whih reeives its ore funding from the Medil Reserh Counil, Cner Reserh UK nd the Wellome Trust, the Medil Reserh Counil (U1177 to V.T.), the Wellome Trust (8918 to R.K. nd V.T.), the Biotehnology nd Biologil Sienes Reserh Counil (BB/L8X/1 to R.K. nd V.T.), the US Ntionl Institute of Helth (DK93 to C.-L.H.) nd the Swiss Multiple Slerosis Soiety (R.L.). AUTHOR CONTRIBUTIONS R.K., F.T., L.V., T.F.B., K.F. nd R.L. designed nd performed experiments nd nlyzed dt. J.X. nd C.-L.H. provided mouse strin. J.V.S. nd V.L.J.T. designed experiments nd nlyzed dt. R.K., F.T., R.L., J.V.S. nd V.L.J.T. wrote the mnusript. COMPETING FINANCIAL INTERESTS The uthors delre no ompeting finnil interests. Reprints nd permissions informtion is ville online t reprints/index.html. 1. Girrd, J.P., Moussion, C. & Förster, R. HEVs, lymphtis nd homeostti immune ell trffiking in lymph nodes. Nt. Rev. Immunol. 1, (1).. Lämmermnn, T. et l. Rpid leukoyte migrtion y integrin-independent flowing nd squeezing. Nture 3, 1 (8). 3. Bosi, R.T. et l. Comprehensive nlysis of lymph node strom-expressed Ig superfmily memers revels redundnt nd nonredundnt roles for ICAM-1, ICAM- nd VCAM-1 in lymphoyte homing. Blood 11, 91 9 (1).. Woolf, E. et l. Lymph node hemokines promote sustined T lymphoyte motility without triggering stle integrin dhesiveness in the sene of sher fores. Nt. Immunol. 8, (7).. Alon, R. & Feigelson, S.W. Chemokine-triggered leukoyte rrest: fore-regulted i-diretionl integrin tivtion in quntl dhesive ontts. Curr. Opin. Cell Biol., 7 7 (1).. Shulmn, Z. et l. Lymphoyte rwling nd trnsendothelil migrtion require hemokine triggering of high-ffinity LFA-1 integrin. Immunity 3, (9). 7. Springer, T.A. & Dustin, M.L. Integrin inside-out signling nd the immunologil synpse. Curr. Opin. Cell Biol., (1). 8. Feigelson, S.W. et l. Oupny of lymphoyte LFA-1 y surfe-immoilized ICAM- 1 is ritil for TCR- ut not for hemokine-triggered LFA-1 onversion to n open hedpiee high-ffinity stte. J. Immunol. 18, (1). 9. Ktgiri, K., Immur, M. & Kinshi, T. Sptiotemporl regultion of the kinse Mst1 y inding protein RAPL is ritil for lymphoyte polrity nd dhesion. Nt. Immunol. 7, (). 18 VOLUME 17 NUMBER 9 SEPTEMBER 1 nture immunology

9 1. Ktgiri, K., Med, A., Shimonk, M. & Kinshi, T. RAPL, Rp1-inding moleule tht medites Rp1-indued dhesion through sptil regultion of LFA-1. Nt. Immunol., (3). 11. Lee, H.S., Lim, C.J., Puzon-MLughlin, W., Shttil, S.J. & Ginserg, M.H. RIAM tivtes integrins y linking tlin to rs GTPse memrne-trgeting sequenes. J. Biol. Chem. 8, (9). 1. MCormik, J.A. & Ellison, D.H. The WNKs: typil protein kinses with pleiotropi tions. Physiol. Rev. 91, (11). 13. Rfiqi, F.H. et l. Role of the WNK-tivted SPAK kinse in regulting lood pressure. EMBO Mol. Med., 3 7 (1). 1. Burh, B.J., Medeiros, R.B., Mueller, K.L. & Shimizu, Y. T-ell reeptor signling to integrins. Immunol. Rev. 18, 81 (7). 1. Zhng, D.J. et l. Seletive expression of the Cre reominse in lte-stge thymoytes using the distl promoter of the Lk gene. J. Immunol. 17, (). 1. Xie, J. et l. Endothelil-speifi expression of WNK1 kinse is essentil for ngiogenesis nd hert development in mie. Am. J. Pthol. 17, (9). 17. de Lu, C. et l. Complete resue of oesity, dietes, nd infertility in d/d mie y neuron-speifi LEPR-B trnsgenes. J. Clin. Invest. 11, (). 18. Sezd, E., Brke, M., Tugl, T., Hogg, N. & Cntrell, D.A. Rp1A positively regultes T ells vi integrin tivtion rther thn inhiiting lymphoyte signling. Nt. Immunol. 3, 1 8 (). 19. Friedl, P. & Weigelin, B. Interstitil leukoyte migrtion nd immune funtion. Nt. Immunol. 9, 9 99 (8).. Steiner, O., Coisne, C., Engelhrdt, B. & Lyk, R. Comprison of immortlized End nd primry mouse rin mirovsulr endothelil ells s in vitro loodrin rrier models for the study of T ell extrvstion. J. Cere. Blood Flow Met. 31, (11). 1. Miuri, P. et l. Atin flows medite universl oupling etween ell speed nd ell persistene. Cell 11, (1).. Alessi, D.R. et l. The WNK-SPAK/OSR1 pthwy: mster regultor of tionhloride otrnsporters. Si. Signl. 7, re3 (1). 3. Vitri, A.C., Dek, M., Morrie, N.A. & Alessi, D.R. The WNK1 nd WNK protein kinses tht re mutted in Gordon s hypertension syndrome phosphorylte nd tivte SPAK nd OSR1 protein kinses. Biohem. J. 391, 17 ().. Zmrowiz, B.P. et l. Wnk1 kinse defiieny lowers lood pressure in mie: gene-trp sreen to identify potentil trgets for therpeuti intervention. Pro. Ntl. Ad. Si. USA 1, (3).. Cheng, C.J. & Hung, C.L. Ativtion of PI3-kinse stimultes endoytosis of ROMK vi Akt1/SGK1-dependent phosphoryltion of WNK1. J. Am. So. Nephrol., 71 (11).. Flgell, M. et l. Mie lking the solterl N-K-Cl otrnsporter hve impired epithelil hloride seretion nd re profoundly def. J. Biol. Chem. 7, 9 9 (1999). 7. Jing, Z.Y. et l. Identifition of WNK1 s sustrte of Akt/protein kinse B nd negtive regultor of insulin-stimulted mitogenesis in 3T3-L1 ells. J. Biol. Chem. 8, 1 18 (). 8. Vitri, A.C. et l. WNK1, the kinse mutted in n inherited high-lood-pressure syndrome, is novel PKB (protein kinse B)/Akt sustrte. Biohem. J. 378, 7 8 (). 9. Cuddph, V.A. & Sontheimer, H. Ion hnnels nd trnsporters [orreted] in ner.. Ion hnnels nd the ontrol of ner ell migrtion. Am. J. Physiol. Cell Physiol. 31, C1 C9 (11). 3. Strok, K.M. et l. Wter permetion drives tumor ell migrtion in onfined miroenvironments. Cell 17, 11 3 (1). 31. Hs, B.R. & Sontheimer, H. Inhiition of the Sodium-potssium-hloride otrnsporter isoform-1 redues gliom invsion. Cner Res. 7, 97 (1). 3. Lee, J.H. et l. Highly multiplexed suellulr RNA sequening in situ. Siene 33, (1). 33. Shiller, H.B., Friedel, C.C., Boulegue, C. & Fässler, R. Quntittive proteomis of the integrin dhesome show myosin II-dependent reruitment of LIM domin proteins. EMBO Rep. 1, 9 (11). 3. Froudi, M. et l. Critil roles for R GTPses in T-ell migrtion to nd within lymph nodes. Blood 11, 3 7 (1). 3. Prk, E.J. et l. Distint roles for LFA-1 ffinity regultion during T-ell dhesion, dipedesis, nd interstitil migrtion in lymph nodes. Blood 11, (1). 3. Semmrih, M. et l. Importne of integrin LFA-1 detivtion for the genertion of immune responses. J. Exp. Med. 1, (). nture immunology VOLUME 17 NUMBER 9 SEPTEMBER 1 183

10 ONLINE METHODS Mie. Mie with onditionl llele of Wnk1 (Wnk1 tm1clhu, Wnk1 fl ), expressing the dlk-cre trnsgene (Tg(Lk-re)Nik), with tmoxifen-induile Cre in the ROSA lous (Gt(ROSA)Sor tm1(re/esr1)thl, ROSA-CreERT, RCE), mie expressing OXSR1-T18A mie (Oxsr1 T18A, Oxsr1 tm1.1arte ), or RAG1-defiient (Rg1 tm1mom ) were mintined on C7BL/JNimr kground nd hve een desried efore 13,1 17,37. Mie with deleted llele of Wnk1 (Wnk1 tm1.1clhu, Wnk1 ) were generted y rossing Wnk1 fl/+ mie with C7BL/J.19S-Tg(Prm-re)7Og/Nimr mie tht delete in the mle germline 38. SLC1A-defiient mie were mintined on n FVB/N kground (Sl1 tm1ges ). Both mle nd femle mie were used. In ll ses ontrol nd experimentl mie were ge- nd gender-mthed. No rndomiztion or linding ws done. All mie were red nd mintined t the MRC Ntionl Institute for Medil Reserh (now The Frnis Crik Institute). All experiments were rried out under the uthority of Projet Liense grnted y the UK Home Offie. Genertion of Wnk1D38A mie. ES ells (C7BL/J kground) were trgeted y stndrd methods with trgeting vetor onsisting of two regions of homology to the Wnk1 gene ontining exon 3 nd exon respetively (genomi oordintes in GRCm38 Mmu: nd Mmu: ), seprted y n Frt-flnked neomyin resistne gene (Neo). Exon 3 in the trgeting vetor ws mutted t two se pirs (Mmu: ) to hnge sprtte 38 to lnine (wild-type sequene -GGAGACCTT-3, D38A mutnt -GGAGCTCTT- 3, mutted ses shown in old). ES ells ering the orretly trgeted llele (Wnk1 D38A-neo, Wnk1 tm1ty ) with oth the Neo gene nd the D38A muttion were used to generte mie y stndrd proedures. Wnk1 D38Aneo mie were rossed to mie expressing the Flp reominse in the germline (B.19S- Gt(ROSA)Sor tm1(flp1)dym /RinJ) to delete the Neo gene nd generte mie expressing WNK1-D38A (Wnk1 D38A, Wnk1 tm1.1ty ), whih were mintined on C7BL/JNimr kground. Corret trgeting ws verified y Southern lotting, PCR nd DNA sequening. Constrution of the trgeting vetor, trgeting of ES ells nd genertion of mie ws rried out y Bioytogen. Rdition himers. To generte rdition himers, either one mrrow ells were hrvested from Wnk1 +/+ dlk-cre, Wnk1 fl/fl dlk-cre, Wnk1 fl/+ RCE,, Wnk1 fl/fl RCE, Wnk1 fl/d38a RCE, or from Sl1 +/+ or Sl1 / mie, or fetl livers were hrvested from E1. emryos generted y interrossing Oxsr1 T18A/+ mie. RAG1-defiient nimls ( 8 weeks of ge) were irrdited with Gy using 137 Cs-soure, nd then reonstituted intrvenously with t lest 1 one mrrow ells/reipient or. 1 fetl liver ells/reipient. All himeri nimls reeived Bytril in their drinking wter (.%, Byer Helthre) for t lest weeks post-trnsplnttion. If required, 8 weeks fter reonstitution himeri mie were injeted intrperitonelly for 3 d with mg/d of tmoxifen (Sigm) resuspended t mg/ml in orn oil (Sigm) nd nlyzed 7 d fter strt of tmoxifen tretment. RNA interferene. E.1 Jurkt T ells (gift from R. Zmoysk, negtive for Myoplsm) were trnsfeted using 9-well Shuttle Nuleofetor devie (Lonz) nd the SE Cell Line 9-well Nuleofetor Kit (Lonz). For eh nuleofetion 1 Jurkt ells were spun down, resuspended in µl of Nuleofetor solution ontining 3 ng of totl short interfering RNAs (sirnas, Life Tehnologies), trnsfeted with progrm 9-CL-1 nd then pled into ulture for 7 h. Quntittive PCR. Jurkt ells were proessed using the Cells to Ct Kit (Life Tehnologies). For mouse T ells, up to 1 CD low spleni T ells from spleens of ontrol or Wnk1-defiient nimls were sorted diretly into RLT lysis uffer, totl RNA ws extrted with n RNAEsy Plus Miro Kit (Qigen) nd DNA ws synthesized with Supersript III kit (Life Tehnologies). Smples were nlyzed on n ABI 79 using TqMn gene expression ssys (Life Tehnologies). Dt ws normlized to HPRT1 nd nlyzed using the omprtive threshold yle method. RNA sequening (RNAseq). CD + nd CD8 + spleni T ells from femle C7BL/J mie were sorted into Trizol (Life Tehnologies) using Bekmn Coulter MoFlo XDP ell sorter. RNA ws isolted using Trizol nd lened up using RNEsy mini kit (Qigen). Strnded polya-enrihed lirries were mde using the Strnded TruSeq RNA Smple Preprtion Kit (Illumin) nd sequened in the HiSeq (Illumin), olleting million piredend reds of 1 ses per smple. Pired-end reds were ligned ginst the mouse genome (uild mm9) y Topht v..9 (defult settings, exept strnded protool ws used) followed y ssessment of expression vlues in FPKM (frgments per k per million reds). Dt hs een deposited in the Sequene Red Arhive (SRA, ession numer SRP9). Flow ytometry, ntiodies, ytokines nd other mterils. Flow ytometry ws rried out using stndrd tehniques with pre-titered ntiodies. Antiodies for stimultions nd flow ytometry ginst the following proteins were otined from BioLegend or ebiosiene: B (RA3-B), CCR7 (B1), CD3ε (C11 or OKT3), CD (RM- or GKL-), CD8 (3-.7), CD11 (M1/7), CD11 (N18), CD19 (MB19-1), CD (3C7 or PC-1.), CD (IM7), CDL (MEL-1), LFA-1 (M17/), TCRβ (H7-97 or IP) nd Vβ8 (F3.1). Further regents: nti-cxcr (ID9, BD Phrmingen); nti-humn LFA-1 (38, Ad Serote); nti-armenin hmster ntiody (19-11-, Roklnd); murine TNF (PromoKine); murine CCL1 (PeproTeh nd R&D Systems); humn nd mouse ICAM1-F, humn CXCR (R&D systems); ell dyes CMFDA, CFSE, CMTMR, CMAC nd CTV (Life Tehnologies). Unless otherwise stted, ntiody dilutions were 1: for flow ytometry nd T ell enrihment, nd 1:1, for immunolotting experiments. Dilutions or onentrtions for ll other ntiodies n e found in the pproprite methods setion. All ntiodies listed here nd in susequent setions hve een vlidted y their suppliers nd referenes n e found on their wesite or on the online vlidtion dtses Antiodypedi nd 1DegreeBio. To mesure intermedite- nd high-ffinity onformtions of LFA-1, Jurkt T ells were trnsfeted with sirnas s desried erlier nd 7 h lter were stimulted for s t 37 C in CCl nd MgCl ontining HBSS,.% BSA with either 1 µg/ml nti-cd3 or. µm CXCL1 in the presene of 1 µg/ml Kim17 or m ntiodies (kind gifts of N. Hogg). The retion ws stopped with ie-old CCl nd Mg Cl ontining HBSS,.% BSA nd Kim17 nd m inding ws reveled y stining with nti-mouse IgG1 APC nd nlyzed y flow ytometry. To mesure F-tin, T ells were rested for h t 37 C in RPMI/1% FCS. T ells t 1 ells/ml were stimulted with CCL1 (1 µm) t 37 C for the indited times, fixed in old % prformldehyde (PFA) nd stined intrellulrly with Phlloidin-FITC (Millipore) efore flow ytometri nlysis. Purifition of T ells. Nïve CD low CD + T ells from peripherl nd mesenteri lymph nodes were purified y negtive seletion. Single-ell suspensions were inuted with iotin-onjugted ntiodies ginst CD8, CD11, CD11, CD, CD nd B, nd were then wshed nd inuted with Streptvidin-onjugted mgneti eds (Dyneds, Life Tehnologies). Purity of CD low CD + T ells (typilly 9%) ws onfirmed y flow ytometry. Alterntively, T ells were isolted using n EsySep Mouse T ell Negtive Seletion Kit (StemCell). CD + lymphoytes were depleted using nti-cd mgneti eds in 1:1 ells:eds rtio (Life Tehnologies). CD low T ells purity (typilly >9%) ws verified y flow ytometry. Conjugtion. Jurkt T ells nd Nlm B ells (DSMZ, negtive for Myoplsm) were leled with. µm CMFDA nd 1 µm CMTMR, respetively, for min in PBS t 37 C. Jurkts (. 1 ells) were mixed with 3 1 Nlm ells in the sene or presene of stphylool enterotoxin E (Toxin Tehnology In.) in totl volume of µl RPMI in 9 well v-ottom pltes. After -min onjugtion ws stopped y dding 8 µl of % PFA. Smples were nlyzed y flow ytometry. Perentge of onjugtes ws lulted y dividing the numer doule positive onjugtes y the totl numer of Jurkt ells. Mouse T ells: 1 CD low CD + T ells purified from Wnk1 +/+ dlk- Cre or Wnk1 fl/fl dlk-cre mie were mixed with 1 CH7 B ells in the sene or presene of 1 µg/ml stphylool enterotoxin B (Sigm) in totl volume of µl RPMI in 9 well V-ottom pltes. After -min onjugtion ws stopped y dding 8 µl of % PFA nd ells were stined with ntiodies ginst CD, CD, Vβ8, CD19 nd B nd nlyzed y flow ytometry. nture immunology doi:1.138/ni.39