Essential role of NKT cells producing IL-4 and IL-13 in the development of allergen-induced airway hyperreactivity

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1 Essentil role of NKT ells produing IL-4 nd IL-13 in the development of llergen-indued irwy hyperretivity OMID AKBARI 1, PHILIPPE STOCK 1, EVERETT MEYER 1, MITCHELL KRONENBERG 2, STEPHANE SIDOBRE 2, TOSHINORI NAKAYAMA 3, MASARU TANIGUCHI 3, MICHAEL J. GRUSBY 4, ROSEMARIE H. DEKRUYFF 1 & DALE T. UMETSU 1 23 Nture Pulishing Group 1 Division of Immunology nd Allergy, Deprtment of Peditris, Stnford University, Stnford, Cliforni, USA 2 Division of Developmentl Immunology, L Joll Institute for Allergy & Immunology, Sn Diego, Cliforni, USA 3 Grdute Shool of Mediine, Chi University, Chi, Jpn 4 Shool of Puli Helth, Hrvrd University, Boston, Msshusetts, USA Correspondene should e ddressed to D.T.U.; e-mil: umetsu@stnford.edu Asthm is mjor puli helth prolem tht hs inresed mrkedly in prevlene in the pst two dedes 1. Asthm is used y Th2-driven inflmmtory responses, whih enhne irwy nd peripherl lood eosinophili, indue AHR nd elevte serum IgE 2. Although Th2-driven immune responses re vitlly importnt in the development of sthm 3,4, in itself Th2 response is not suffiient to indue sthm. Th2-ised llergen sensitiztion n our independently of sthm, whih explins why only out third of individuls with llergi rhinitis (used y Th2 responses in the upper respirtory trt) develop sthm (used y Th2 responses in the lower irwys). Additionl elements tht re intrinsi to the lower respirtory trt my e required for lolizing the Th2 response in the lungs, in proess tht is distint from llergen sensitiztion per se, nd for the full development of sthm. These required speifi lol elements in the respirtory trt hve not een identified ut my involve either struturl responses of the lower respirtory trt to repeted injury (for exmple, irwy remodeling) 6,7 or immune system omponents tht re lolized to the lower respirtory trt. NKT ells onstitute lymphoyte supopultion tht re undnt in the thymus, spleen, liver nd one mrrow nd re lso present in the lung 8,9. NKT ells express surfe mrkers tht re hrteristi of oth nturl killer ells (suh s NK1.1 nd CD161) nd onventionl T ells (suh s TCRs). Severl NKT ells (referred to s V α 14i NKT ells) reognize glyolipid ntigens presented y the non-polymorphi mjor histoomptiility omplex (MHC) lss I like protein CD1d nd express n Pulished online 31 Mrh 23; doi:1.138/nm81 Using nturl killer T (NKT) ell defiient mie, we show here tht llergen-indued irwy hyperretivity (AHR), rdinl feture of sthm, does not develop in the sene of V α 14i NKT ells. The filure of NKT ell defiient mie to develop AHR is not due to n inility of these mie to produe type 2 T-helper (Th2) responses euse NKT ell defiient mie tht re immunized suutneously t non-muosl sites produe norml Th2-ised responses. The filure to develop AHR n e reversed y the doptive trnsfer of tetrmer-purified NKT ells produing interleukin (IL)-4 nd IL-13 to J281 / mie, whih lk the invrint T-ell reeptor (TCR) of NKT ells, or y the dministrtion to Cd1d / mie of reominnt IL-13, whih diretly ffets irwy smooth musle ells. Thus, pulmonry V α 14i NKT ells ruilly regulte the development of sthm nd Th2-ised respirtory immunity ginst nominl exogenous ntigens. Therpies tht trget V α 14i NKT ells my e linilly effetive in limiting the development of AHR nd sthm. invrint V α 14-J α 281 (lso lled J α 18 or J α 1) TCR in mie, or n invrint V α 24-J α 1 TCR in humns 1. A speifi mrine sponge glyolipid, α-gltosyl ermide (α- GlCer), inds to CD1d nd is reognized y nd tivtes mouse V α 14i + nd humn V α 24i + T ells 11,12, ut the speifi glyolipid ntigens tht re normlly reognized y NKT ells in vivo re not known. When tivted, however, NKT ells rpidly produe lrge quntities of IL-4 nd interferon (IFN)-γ, whih seem to influene susequent dptive immune responses nd the polriztion of onventionl αβ-tcr T ells NKT ells therefore seem to hve importnt regultory funtions, lthough ler role for NKT ells in the respirtory trt hs not een shown. In oth humn nd mouse models of type 1 dietes mellitus, NKT ells regulte the development of disese. In individuls with dietes mellitus, oth the numer of NKT ells nd their pity to produe IL-4 hve een shown to e redued 16, lthough this finding is ontroversil 17. In mie, overexpression of NKT ells, doptive trnsfer of NKT ell enrihed popultions nd tivtion of V α 14i NKT ells with α-glcer protets ginst the development of dietes In models of experimentl utoimmune enephlomyelitis, nother Th1-medited utoimmune disese, tivtion of V α 14i NKT ells with n nlog of α-glcer protets suseptile mie ginst disese 22. Although IL-4 prodution is required for these protetive effets to our, the preise mehnisms y whih NKT ells re le to protet ginst utoimmune inflmmtory diseses re not known, prtiulrly euse the tivtion of NKT ells with α- NATURE MEDICINE ADVANCE ONLINE PUBLICATION 1

2 23 Nture Pulishing Group Anti-OVA IgE (ng/ml) , BALB/ CD1d / GlCer is required to oserve the protetive effets of V α 14i NKT ells in these models. The prodution of lrge quntities of IL-4 y V α 14i NKT ells, lthough ssoited with protetion ginst Th1- medited disese, my lso exerte the development of Th2-ised immune responses y providing soure of IL-4 for Th2 differentition. However, severl studies hve not found role for V α 14i NKT ells in enhning Th2-ised immune responses in vivo 23 2, nd the tivtion of V α 14i NKT ells hs een shown to inhiit Th2 ell differentition nd IgE prodution 1. To exmine more lerly whether V α 14i NKT ells in the lung ffet the development of Th2-ised responses in the respirtory trt, we hve studied the indution of llergen-indued AHR in the presene or sene of NKT ells in CD1d-defiient BAL ell ount ( 1 ) d TCC Mo Lym Eos Neu Fig. 1 Anlysis of ntigen-indued irwy responses in Cd1d1 / mie., Cd1d1 / mie do not develop ntigen-indued AHR. Inresing onentrtions of methholine were used to mesure AHR in sensitized mie 1 d fter the lst hllenge with OVA. Dt re the men ± s.e.m. enhned puse (Penh) vlues from 4 sensitized mie in eh group., OVA-hllenged BALB/;, PBS-hllenged BALB/;, OVA-hllenged Cd1d1 / ;, PBS-hllenged Cd1d1 /., Airwy eosinophili is redued in Cd1d1 / mie. BAL fluid from the mie in () ws nlyzed 24 h fter AHR mesurement., BALB/;, Cd1d1 /. Results re shown s the numer of ells per ml in BAL fluid. Eos, eosinophils; Lym, lymphoytes; Mo, monoyte/mrophge; Neu, neutrophils; TCC, totl ell ount., OVA-speifi IgE in Cd1d1 / mie. OVA-speifi IgE ws mesured y ELISA in ser from mie sensitized nd hllenged with OVA ( ) or from nive mie ( ), 24 h fter AHR mesurement. Dt re the men ± s.d. of 3 mie. d, Reominnt IL-13 indues AHR in Cd1d1 / mie. Mie reeived 2 µg ril-13 or PBS intrnslly dily for 3 dys nd AHR ws mesured on dy 4., ril-13 treted BALB/;, PBS-treted BALB/;, ril- 13 treted Cd1d1 / ;, PBS-treted Cd1d1 /. nd J281 / mouse strins, whih oth lk V α 14i NKT ells. Our studies show tht in the sene of V α 14i NKT ells, llergen-indued AHR does not develop in either CD1d-defiient or J281 / mie (BALB/ kground). We reonstituted AHR in J281 / mie y doptively trnsferring V α 14i NKT ells isolted with α- GlCer loded CD1d tetrmers nd found tht V α 14i NKT ells isolted from wild-type mie, ut not mie defiient in IL-4 nd IL-13, restore AHR. Our studies indite tht the development of AHR fter respirtory exposure requires the tivtion of pulmonry NKT ells tht produe IL-4 nd IL-13. They lso indite tht pulmonry NKT ells my provide n intrinsi element in the lungs tht permits Th2 responses to develop nd to drive the development of AHR nd sthm. NKT ell defiient mie do not develop AHR To determine the speifi role of NKT ells in the development 12. BALB/ J281 / CD1d / Vβ CD1d tetrmer RL (m H2O per ml per s) Fig. 2 J281 / mie do not develop AHR., J281 / nd Cd1d1 / mie lk CD1d tetrmer stining ells. Spleen ells from nive BALB/, J281 / nd Cd1d1 / mie were stined with α-glcer loded PE-onjugted CD1d tetrmers nd monolonl ntiody ginst Vβ nd nlyzed for doule-positive ells. Neither J281 / nor Cd1d1 / mie ontined tetrmer-positive ells, wheres 4% of the BALB/ ells were doule positive., AHR does not develop in J281 / mie. AHR ws ssessed s desried in Fig. 1. Dt re the men ± s.e.m. of pek enhned puse (Penh) vlues from sensitized mie in eh group nd re representtive of 4 similr experiments., NKT ell defiient mie do not develop AHR, s ssessed y invsive mesurement of irwy resistne (see Methods). Results re shown s R L (top, in m H 2 O per ml per s) nd C dyn (ottom, in ml per m H 2 O). Dt re the men ± s.e.m. of results from 4 mie in eh group. nd,, OVA-treted Bl/;, PBS-treted Bl/;, OVA-treted J281 / () or OVA-treted Cd1d1 / ();, PBS-treted J281 / () or OVA-treted J281 / (). Cdyn (ml per m H 2O) NATURE MEDICINE ADVANCE ONLINE PUBLICATION

3 23 Nture Pulishing Group of AHR, we exmined the development of AHR in Cd1d1 / mie, whih lk the lss I restriting element of NKT ells nd therefore lk NKT ells. When sensitized nd hllenged with ntigen, ontrol BALB/ mie developed severe AHR, s expeted; in ontrst, Cd1d1 / mie tht were sensitized nd hllenged with ntigen did not develop AHR ut showed norml irwy responsiveness (Fig. 1). The defet in the Cd1d1 / mie ws ssoited with sustntil redution in irwy eosinophili, s indited y redution of eosinophils in ronholveolr lvge (BAL) fluid s ompred with BAL fluid from BALB/ mie sensitized nd hllenged with ovlumin (OVA; Fig. 1). Neither PBS-hllenged (negtive ontrol) BALB/ mie nor PBS-hllenged Cd1d1 / mie developed irwy eosinophili or norml ellulr infiltrtes in BAL fluid (dt not shown). The filure of Cd1d1 / mie to develop AHR ws not due to defiieny in Th2 responsiveness or to suoptiml stimultion or immuniztion with ntigen, euse previous studies hve shown tht Cd1d1 / mie hve norml Th2 responses 26, nd euse more intensive immuniztion with multiple intrperitonel immuniztions with OVA in lum nd intrnsl hllenges with ntigen did not indue AHR in the Cd1d1 / mie (see Supplementry Fig. 1 online). Indeed, s previously reported 26 the immunized Cd1d1 / mie produed OVA-speifi IgE (Fig. 1), lthough the mounts of IgE oserved in Cd1d1 / mie were less thn those oserved in wild-type ontrol BALB/ mie. The lk of AHR in Cd1d1 / mie ws lso not due to speifi unresponsiveness to OVA, euse sensitiztion with other ntigens, suh s ovine serum lumin (BSA) or Aspergillus fumigtus ntigen, did not indue AHR in Cd1d1 / mie ut did indue severe AHR in BALB/ mie (see Supplementry Fig. 2 online). The sene of AHR in Cd1d1 / mie ws lso not due to n intrinsi inility of the mie to develop AHR, euse dministrtion of reominnt IL-13, whih uses AHR though diret effets on irwy smooth musle ells nd muus glnd seretion 27,28, resulted in severe AHR in Cd1d1 / mie (Fig. 1d). Although the immune system in Cd1d1 / mie is thought to e norml outside the NKT ell omprtment, it is possile tht in the sene of CD1d moleules, defets in other non-nkt ell omprtments might our. To onfirm tht the filure of Cd1d1 / mie to develop AHR ws due speifilly to the sene of NKT ells, we exmined the development of llergen-indued AHR in J281 / mie, whih lk the invrint TCR of NKT ells. The spleens of J281 / mie, like those of Cd1d1 / mie, did not ontin ells tht stined with α-glcer loded CD1d tetrmers, inditing tht oth J281 / nd Cd1d1 / mie lk V α 14i NKT ells (Fig. 2). In J281 / mie, s in CD1d-defiient mie, sensi- IL-13 (ng/ml) BAL ell ount ( 1 ) TCC Mo Lym Eos Neu IL-4 (pg/ml) IFN-γ (ng/ml) Fig. 3 Comprison of ytokine prodution, BAL fluid nd IgE in J281 / nd BALB/ mie., Airwy eosinophili is redued in J281 / mie. BAL fluid ws nlyzed s in Fig. 1., BALB/;, J281 /., OVA-speifi IgE prodution is redued in J281 / mie. OVA-speifi IgE ws mesured s in Fig. 1., nive mie;, OVA-sensitized mie. Dt re the PBS lung.4% OVA lung 3% men ± s.d. of 3 mie., IL-13 nd IL-4 prodution is redued in ronhil lymph node T ells. Bronhil lymph nodes were removed nd nlyzed, 24 h fter AHR mesurement. f V β.8% 1% 41% 38%, J281 / ;, BALB/. d, Cytokine prodution is norml in J281 / mie immunized suutneously with OVA., J281 / ;, BALB/. e, Numer of NKT ells in the lungs of wild-type BALB/ mie inreses on irwy hllenge with OVA. BALB/ mie were sensitized nd hllenged with OVA (right) or PBS (left). Lung IL-4 IL-13 ells were isolted nd stined with α-glcer loded CD1d tetrmers nd monolonl ntiody ginst TCR Vβ. f, Pulmonry NKT ells produe IL-4 nd IL-13. Lung ells isolted from the OVAhllenged mie in (e) were enrihed for CD1d tetrmer positive NKT ells y MACS. NKT ells were stimulted with PMA nd ionomyin, nd stined with α-glcer loded CD1d tetrmers nd monolonl ntiodies ginst IL-4 or IL-13. e d IL-13 (ng/ml) Tetrmer Tetrmer Anti-OVA IgE (ng/ml) 1, tiztion nd hllenge with OVA did not indue AHR, wheres similr immuniztion in wild-type BALB/ mie used severe AHR (Fig. 2). The lk of mesurle AHR ws not due to lk of sensitivity in our method of detetion, euse irwy retivity to methholine ws essentilly norml, mesured s irwy resistne (R L ) nd dynmi ompline (C dyn ) in nesthetized, trheostomized nd mehnilly ventilted CD1d-defiient nd J281 / mie tht hd een previously sensitized nd hllenged with OVA (Fig. 2). As shown y redution of eosinophils in BAL fluid, the sene of AHR in J281 / mie ws ssoited with redued irwy eosinophili s ompred with sensitized nd hllenged BALB/ mie (Fig. 3). Neither PBS-hllenged BALB/ nor PBS-hllenged J281 / mie developed irwy eosinophili or norml ellulr infiltrtes in BAL fluid (dt not shown). Sensitized nd hllenged J281 / mie lso produed less OVA-speifi IgE in their ser (Fig. 3) nd less IL-4 nd IL-13 in their ronhil IL-4 (pg/ml) 1, 7 2 BALB/ J α281 / IFN-γ (ng/ml) 1 NATURE MEDICINE ADVANCE ONLINE PUBLICATION 3

4 23 Nture Pulishing Group lymph nodes (Fig. 3) thn did similrly sensitized nd hllenged BALB/ mie. The redution in Th2 ytokine prodution fter pulmonry exposure to ntigen ws restrited to responses in the lung, euse suutneous immuniztion of the footpds of J281 / mie resulted in norml prodution of IL-4, IL-13 nd IFN-γ s ompred with immunized wild-type ontrol BALB/ mie (Fig. 3d). This result my reflet the ft tht NKT ells, lthough present only in smll numers in ronhil lymph nodes (..12%; see Supplementry Fig. 3 online), onstitute s muh s 3% of the pulmonry mononuler ells fter ntigen hllenge (Fig. 3e) ut my not e present in suutneous tissue. The NKT ells found in the lungs produed lrge mounts of IL-4 nd IL-13, s shown y intrellulr ytokine stining (Fig. 3f), whih most proly enhned the susequent development of AHR. from J281 / mie, lso reonstituted the J281 / reipients (Fig. 4). Beuse J281 / mie lk only V α 14i NKT ells nd re otherwise identil to wild-type mie, these studies verify tht V α 14i NKT ells re required for the development of llergen-indued AHR. Requirement for IL-4 nd IL-13 prodution y NKT ells To evlute the mehnisms y whih V α 14i NKT ells medite the development of AHR, we reonstituted J281 / mie with ells tht we purified from ytokine knokout mie nd seleted for inding to α-glcer loded CD1d tetrmers. Tetrmer-inding ells from Il4 / mie nd Il13 / mie were muh less effetive thn tetrmer-inding ells from wild-type BALB/ mie in reonstituting AHR when doptively trnsferred to J281 / mie (Fig. ). Tetrmer-inding ells from mie lking oth IL-4 nd IL-13 did not reonstitute AHR in the J281 / mie (Fig. ). By ontrst, tetrmer-inding ells from Ifng / mie fully restored Adoptive trnsfer of NKT ells restores AHR To show tht the lk of V α 14i NKT ells speifilly used the filure of J281 / mie to develop AHR, we reonstituted OVAsensitized J281 / mie with V α 14i NKT ells efore hllenge with OVA. Adoptive trnsfer of ell popultion enrihed for ells tht ound α-glcer loded CD1d tetrmer (>7% tetrmer positive; Fig. 4) fully reonstituted the pity of AHR (dt not shown). These results indite tht the indution of AHR my require V α 14i NKT ells tht produe oth IL-4 nd IL-13, ut not IFN-γ. We next exmined whether V α 14i NKT ells were required for the development of Th2 ells or more speifilly for permitting Th2 ells to drive the development of AHR. We therefore isolted OVA-primed CD4 + T ells from the spleens of wild-type J281 / mie to develop AHR (Fig. 4). Binding of α- BALB/ or J281 / mie tht hd een immunized intrperitonelly GlCer loded CD1d tetrmer to the NKT ells ws not required for reonstitution of AHR, euse the doptive trnsfer of unmnipulted spleen ells from wild-type BALB/ mie, ut not with OVA in lum to indue polrized Th2 responses. These ells were doptively trnsferred into severe omined immunodefiient (SCID) mie, whih were then hllenged with OVA intrnslly nd exmined for the development of AHR. Wheres OVA-primed CD4 + T ells from wildtype BALB/ mie (ontining NKT ells) trnsferred 1 AHR into the SCID mie, OVA-primed CD4 + ells from Dt.1 76% J281 / mie (ontining no NKT ells), or CD4 + ells V Vβ from nive BALB/ mie, did not (Fig. ). When V α 14i NKT ells from nive BALB/ mie, ut not from mie lking oth IL-4 nd IL-13, were trnsferred 2. with the OVA-primed CD4 + T ells from J281 / mie, however, AHR ws restored in the SCID mie reipients. Trnsfer of purified NKT ells y themselves into SCID mie did not indue AHR. Tken together, these studies indite tht Th2-ised ells n develop in the sene of V α 14i NKT ells, ut Th2 ells CD1d tetrmer tht develop in J281 / mie re not ompetent to indue AHR exept in the presene of V α 14i NKT ells. Fig. 4 Adoptive trnsfer of NKT ells restores AHR in J281 / mie., Trnsfer 12. of purified NKT ells to J281 / mie restores AHR. CD1d tetrmer positive 1 ells (3 1 6 ) positively seleted from the spleen ells of BALB/ mie were 7. doptively trnsferred into OVA-sensitized J281 / mie ( ) nd hllenged with OVA. Control J281 / ( ) nd BALB/ ( ) non-reipient mie were lso 2. sensitized nd hllenged with OVA, nd AHR ws mesured fter 24 h., Purity of the trnsferred NKT ell popultion. Flow ytometry showed tht 76% of the trnsferred ells in () stined with oth α-glcer loded CD1d tetrmer nd monolonl ntiody ginst Vβ., Trnsfer of spleen ells to J281 / mie restores AHR. Trnsfer of spleen ells (4 1 6 ) from nive BALB/ mie ( ), ut not from J281 / mie ( ), into OVA-sensitized J281 / reipients restored AHR. Reipients were given OVA nd ssessed for AHR s in (). Control BALB/ mie ( ) nd J281 / mie ( ) were lso sensitized nd hllenged with OVA. Disussion Using oth Cd1d1 / mie, whih lk the MHC restrition element required y NKT ells, nd J281 / mie, whih lk the invrint TCR of NKT ells, we hve shown tht llergen-indued AHR does not develop in the sene of V α 14i NKT ells. The lk of AHR in these mie ws not due to struturl normlities in the pulmonry prenhym euse AHR ws reonstituted in Cd1d1 / mie y the dministrtion of reominnt IL-13, whih indues AHR though diret effets on irwy smooth musle ells nd muus glnds, nd euse AHR ws reonstituted in J281 / mie y the doptive trnsfer of V α 14i NKT ells. Our study therefore shows tht V α 14i NKT ells re required for the development of AHR. Previous studies hve shown tht Cd1d1 / mie tht re immunized with ntiodies ginst IgD produe 4 NATURE MEDICINE ADVANCE ONLINE PUBLICATION

5 23 Nture Pulishing Group Mx Penh norml mounts of IgE 26, nd tht Cd1d1 / mie on 129/C7BL/6 kground develop irwy eosinophili 23. We onfirmed here tht Cd1d1 / mie n produe IgE nd lso showed tht J281 / mie hve the pity oth to produe IgE on immuniztion with ntigen nd to produe norml quntities of IL-4 nd IL-13 when immunized suutneously. In ddition, our ssessment of AHR in Cd1d1 / nd J281 / mie hs shown tht AHR, rdinl feture of sthm tht does not lwys orrelte with irwy eosinophili 29,3, does not our in mie defiient in NKT ells. Although β2 mirogloulin defiient mie, whih lk ll lss I moleules inluding CD1d, hve een shown to develop irwy eosinophili 24 nd AHR 2, the sene of CD8 + T ells in these mie my use immune dysregultion in severl tissue omprtments tht ffet irwy responses, mking interprettion of the results diffiult. Tken together, however, these studies indite tht Th2-ised immune responses n our in mie defiient in NKT ells, nd tht Th2 ell differentition nd prodution of IgE do not require the presene of V α 14i NKT ells. Beuse Th2 response is not suffiient in itself to indue the full development of sthm nd AHR (tht is, Th2 response in peripherl lymph nodes or in the upper irwys is not lwys ompnied y sthm nd Th2 response in the lower irwys),6, V α 14i NKT ell must regulte dditionl elements in the lower respirtory trt tht re required for the development of AHR. In other words, pulmonry NKT ells, whih represent s muh s 3% of lung mononuler ells nd hve ruil role in resistne to pthogeni teri in the respirtory trt 31, seem to eome tivted y ntigens enountered in the lung, providing neessry prerequisite for the development of AHR. By ontrst, ntigens enountered in suutneous tissue do not seem to tivte NKT ells, presumly euse NKT ells re present in muh lower numers in these tissues, where prodution of the NKT ell growth ftor IL-1 (ref. 32) my e limited. Mx Penh Fig. IL-4 nd IL-13 prodution y NKT ells is required for development of AHR., NKT ells from wild-type BALB/ mie, ut not mie defiient in IL-4 nd IL-13, restore AHR. CD1d tetrmer positive ells were positively seleted from spleen ells of Il4 / ( ), Il13 / ( ), Il4 / Il13 / ( ) or wild-type BALB/ ( ) mie nd doptively trnsferred (3 1 6 ) into OVA-sensitized J281 / mie, whih were then hllenged nd ssessed for AHR, together with non-reipient BALB/ ( ) nd J281 / ( ) mie, s in Fig. 4., OVA-sensitized T ells from J281 / mie require NKT ells to indue AHR. MACS-purified CD4 + ells from spleens of OVA-sensitized BALB/ ( ) or J281 / ( ) mie, or nive BALB/ mie ( ), were doptively trnsferred into SCID mie (8 1 6 ells/mouse). Some mie lso reeived tetrmer-purified NKT ells (3 1 6 ) from nive wild-type BALB/ ( ) or Il4 / Il13 / ( ) mie, or reeived wild-type NKT ells lone ( ). Reipients were hllenged with OVA nd AHR ws mesured 24 h fter the lst hllenge. The preise mehnisms y whih ntigen dministrtion into the lungs tivtes NKT ells to indue AHR re not ler. We found here tht when trnsferred with wild-type NKT ells, llergen-sensitized CD4 + αβ-tcr T ells from NKT ell defiient mie ould indue AHR in SCID mie, wheres the trnsfer of either ell popultion on its own ould not, inditing tht NKT ells hve the pity to lolize the Th2 response to the lungs nd to indue AHR. Beuse the immuniztion of NKT ell defiient mie with more ggressive protools nd with different ntigens did not indue AHR, we do not think tht NKT ells simply influene the mgnitude of the pulmonry Th2 response, ut rther tht they liense Th2 ells tht enter the pulmonry omprtment to eome ompetent to indue AHR. Although the speifi signls provided y V α 14i NKT ells to the llergen-sensitized CD4 + αβ-tcr T ells re unknown, our model is unique in tht V α 14i NKT ell funtion n e indued without the dministrtion of α-glcer; this ontrsts with most other models of utoimmunity or infetion, in whih the role of NKT ells requires their tivtion y α-glcer 31, In our system, we propose tht ntigen enountered in the lung lters the muosl environment of the respirtory trt, exposing self glyolipid ntigens tht re reognized y self gonist seleted NKT ells 8,37. Although the glyolipid ntigen in our system, whih tivtes NKT ells in vivo when OVA, BSA or Aspergillus ntigen is dministered into the lungs, hs not een identified, gonist-seleted T ells in the muus memrnes re thought to look for ells tht express enhned quntities of self ntigens in response to stress indued y environmentl stimuli. Suh gonist-seleted T ells, s prt of the innte immune system, then respond rpidly y produing ytokines 37. Our studies therefore suggest tht ntigens other thn α-glcer n indeed tivte V α 14i NKT ells, nd tht there re innte immune mehnisms involving V α 14i NKT ells in the lung tht respond rpidly to inhled ntigens. V α 14i NKT ells indued AHR through mehnisms tht involved the prodution y NKT ells of oth IL-4 nd IL-13, euse NKT ells defiient in IL-4 nd IL-13 prodution ould not reonstitute AHR in J281 / mie. We onfirmed tht NKT ells produe oth IL-4 nd IL-13 (refs. 38, 39) nd showed tht oth IL-4 defiient nd IL-13 defiient NKT ells ould prtilly reonstitute AHR in J281 / mie, suggesting tht IL-4 nd IL-13 possess prtly overlpping funtions in this proess. IFN-γ defiient NKT ells were s effetive s wild-type NKT ells in reonstituting AHR in J281 / mie, inditing tht NKT ell prodution of IFN-γ hs no role in the development of AHR, lthough IFN-γ prodution y NKT ells is importnt in the lerne of Pseudomons eruginos from the lung 31. We suggest tht IL-4 nd IL-13 produed y V α 14i NKT ells potentite the development nd tion of Th2-ised OVA-speifi T ells in the lungs, whih drives the development of sthm. There is no ler onsensus, however, on how NKT ells funtion in the prevention or use of ny disese; preisely how the IL-4 nd IL-13 relesed into the respirtory trt y NKT ells enle Th2 ells to indue AHR is not known. NATURE MEDICINE ADVANCE ONLINE PUBLICATION

6 23 Nture Pulishing Group In summry, we hve identified role for V α 14i NKT ells in the regultion of llergen-indued AHR nd hve shown tht AHR does not develop in the sene of V α 14i NKT ells. Our studies indite tht V α 14i NKT ells tht produe IL-4 nd IL-13 tively regulte Th2-ised immune responses in the respirtory trt when ntigen is enountered in the lungs, ut not when ntigen is dministered suutneously t non-muosl sites. Administrtion of nominl ntigen into the lungs seems to tivte NKT ells, whih then liense Th2 ells tht enter the lung to indue AHR through mehnisms tht re independent of α-glcer. Our studies indite tht V α 14i NKT ells in the respirtory trt my hve ruil role in the development of sthm, nd tht therpies trgeted t limiting or ltering NKT ell funtion my seletively restrit the development of AHR nd sthm. Methods Mie nd ntigens. BALB/ wild-type, Il4 / mie were purhsed from The Jkson Lortory (Br Hror, Mine). CD1.1 (Cd1d1 / ) mie 26 (krossed with BALB/) were gift from M. Grusy (Hrvrd University, Boston, Msshusetts) nd J281 / mie 36 (krossed with BALB/) were gift from M. Tniguhi (Chi University, Chi, Jpn) nd S. Blk (Brighm nd Women s Hospitl, Boston, Msshusetts). Il13 / nd Il4 / Il13 / (krossed with BALB/) were gift from A. MKenzie (MRC Lortory of Moleulr Biology, Cmridge, UK) 28,4. We immunized the mie with OVA (ICN, Auror, Ohio), BSA (Sigm, St. Louis, Missouri) nd A. fumigtus ntigen (Ntionl Institutes of Helth, Bethesd, Mrylnd). The Stnford University Committee on Animl Welfre (Administrtion Pnel of Lortory Animl Cre) pproved ll mouse protools used in this study. Adoptive trnsfer. Spleen or NKT ells (3 1 6 ), purified from spleen using α-glcer loded CD1d tetrmers (purity ws out 76% y flow ytometry for tetrmer-positive ells doule-stined with monolonl ntiody ginst TCR β-hin (lone H7-97; Phrmingen, Sn Diego, Cliforni), were doptively trnsferred intrvenously into J281 / or SCID reipients (on BALB/ kground) tht hd een sensitized 8 d erlier y intrperitonel dministrtion of OVA in lum. The donors of spleen ells were wildtype BALB/, Il13 /, Il4 / or Il4 / Il13 / mie. Restimultion of lymph node ells in vitro. Cells isolted from lymph nodes of OVA-primed mie were restimulted in vitro (. 1 ells per well in 96-well plte) with 62. µg/ml of OVA. We olleted the superntnts fter 4 d nd ssyed them for IL-4, IL-13 nd IFN-γ y ELISA. Cytokine ELISAs nd OVA-speifi IgE ssy. ELISAs were done s desried 41. We used the following pirs of monolonl ntiodies for pture nd iotinylted detetion, respetively: R4-6A2 nd XMG1.2 for IFN-γ nd 11B11 nd BVD6-24G2 for IL-4. Regents for deteting mouse IL-13 were purhsed from R&D Systems (Minnepolis, Minnesot). For the OVA-speifi IgE ssy, mie were led nd OVA-speifi IgE ntiodies were mesured in serum using modified OVA-speifi ELISA s desried 41. Identifition of NKT ells. Splenoytes or lymph nodes ells were pre-inuted with monolonl ntiodies ginst Fγ reeptor (2.4G2 ulture superntnt), wshed nd inuted with α-glcer loded CD1d tetrmers or ontrol tetrmers, prepred with phyoerythrin (PE)-streptvidin s desried 42, for 3 min in 1 µl of PBS ontining 1% FCS. The ells were wshed nd nlyzed on FACSCliur flow ytometer (Beton Dikinson, Frnklin Lkes, New Jersey). Indution of AHR nd mesurement of irwy responsiveness. To indue AHR, we sensitized mie with 1 µg of OVA, 1 µg of BSA or 2 µg of A. fumigtus ntigen in lum dministered intrperitonelly. After 8 d, mie were exposed to intrnsl ntigen ( µg/d for OVA nd BSA; 2 µg/d for A. fumigtus) or PBS on 3 onseutive dys, s desried 41,43. AHR responses were ssessed y methholine-indued irflow ostrution in onsious mie pled in whole-ody plethysmogrph (Buxo Eletronis, Troy, New York) s desried 41. In some experiments we ssessed AHR y invsive mesurement of irwy resistne, in whih nesthetized nd trheostomized mie were mehnilly ventilted using modified version of desried method 44. Aerosolized methholine ws dministered for 2 reths in inresing onentrtions (1.2, 2., nd 1 mg/ml of methholine). We ontinuously omputed R L nd C dyn y fitting flow, volume nd pressure to n eqution of motion. In other experiments, nive Cd1d1 / mie were treted with 2 µg of reominnt IL-13 ( gift from the Genetis Institute) intrnslly for 3 onseutive dys in µl of norml sline efore mesurement of AHR. Suutneous immuniztion of mie. J281 / or wild-type BALB/ mie were immunized suutneously with OVA in lum one in the footpds. After 8 d, the drining lymph nodes were olleted, nd the T ells were ultured in vitro with OVA. Superntnts were ssessed for ytokines 4 d lter y ELISA. Colletion nd nlysis of BAL fluid. A lethl dose of phenoritl (4 mg per kg ody weight) ws dministered intrperitonelly to mie. After the trhe ws nnulted, the lungs were lvged 3 times with.3 ml of PBS, nd the fluid ws pooled. We ounted nd nlyzed ells in BAL fluid s desried 41. The reltive numer of different types of leukoyte (lung ell differentil) ws determined from slide preprtions of BAL fluid stined with H&E. Flow ytometry nd fluoresene-tivted ell sorting (FACS) nlysis. Anlytil flow ytometry ws done on FACSn instrument (Beton Dikinson). We olleted ells from lymph nodes, spleen or lungs nd inuted them on ie with CD1d tetrmer nd monolonl ntiody ginst Vβ (PhrMingen) using stndrd proedures. Flow ytometri mesurements of ytokine prodution in NKT ells ws done ording to modified protool 4,46. In rief, we stimulted NKT ells with phorol 12-myristte 13-ette (PMA; 2 ng/ml) nd ionomyin ( ng/ml) for 3 h. Fixtion nd permeiliztion ws done on olleted ells using Cytofix/Cytoperm nd Perm/Wsh (PhrMingen) ording to the mnufturer s instrutions. Cytoplsmi IL-4 ws stined with FITC-onjugted leled monolonl ntiody (PhrMingen) nd ytoplsmi IL-13 ws stined with mouse monolonl ntiody ginst IL-13 (R&D Systems). Lung ell isoltion. The ort nd the inferior ven v were setioned nd the lungs were perfused with PBS through the right ventrile until white. We slied the loes of the lungs into smll ues nd inuted them in 1 ml of solution ontining.1% DNse I (frtion IX; Sigm) nd 1.6 mg/ml of ollgense (CLS4; Worthington Biohemils, Lkewood, New Jersey) t 37 C for 2 h. Mononuler ells were seprted y entrifugtion on disontinuous Peroll grdients (7%, %, 3% nd 2% Peroll; Phrmi, Uppsl, Sweden). We isolted NKT ells y positive seletion using mgneti ell sorting (MACS) fter inuting the ells first with PE-leled CD1d tetrmer nd then with mgneti eds onjugted to ntiodies ginst PE. The Auto-MACS (Miltenyi Bioteh, Bergish Gldh, Germny) system ws used in ordne with the mnufturer s instrutions. Aknowledgments We thnk R. Blumerg for disussions, A. MKenzie for regents, V.P. Yeung for tehnil support, nd J. Ful, S. Glli nd M. Tsi for help with the invsive mesurement of AHR. These studies were supported y Ntionl Institutes of Helth Puli Helth Servie Grnts RO1 AI26322 (D.T.U.), RO1 HL62348 (D.T.U.), RO1 CA211 (M.K), AI4171 (M.J.G) nd GM6213 (M.J.G); grnt from the Amerin Lung Assoition of Cliforni (O.A.); fellowship STO 467/2-1 from the Deutshe Forshungsgemeinshft (P.S.); nd trining grnt T32AI729 (E.M.). Note: Supplementry informtion is ville on the Nture Mediine wesite. Competing interests sttement The uthors delre tht they hve no ompeting finnil interests. 6 NATURE MEDICINE ADVANCE ONLINE PUBLICATION

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Interleukin 4, ut not interleukin or eosinophils, is required in murine model of ute irwy hyperretivity. J. Exp. Med. 183, (1996). 3. Lekie, M. et l. Effets of n interleukin- loking monolonl ntiody on eosinophils, irwy hyper-responsiveness, nd the lte sthmti response. Lnet 36, (2). 31. Nieuwenhuis, E.E. et l. CD1d-dependent mrophge-medited lerne of Pseudomons eruginos from lung. Nt. Med. 8, (22). 32. Mtsud, J. et l. Homeostsis of Vα14i NKT ells. Nt. Immunol. 3, (22). 33. Wng, B., Geng, Y. & Wng, C. CD1-restrited NK T ells protet nonoese dieti mie from developing dietes. J. Exp. Med. 194, (21). 34. Hong, S. et l. The nturl killer T-ell lignd α-gltosylermide prevents utoimmune dietes in non-oese dieti mie. Nt. Med. 7, (21). 3. Shrif, S. et l. Ativtion of nturl killer T ells y α-gltosylermide tretment prevents the onset nd reurrene of utoimmune Type 1 dietes. Nt. Med. 7, (21). 36. Cui, J. et l. Requirement for Vα14 NKT ells in IL-12 medited rejetion of tumors. Siene 278, (1997). 37. Leishmn, A. et l. Preursors of funtionl MHC lss I or lss II restrited CD8αα + T ells re positively seleted in the thymus y gonist self-peptides. Immunity 16, (22). 38. Tere, M. et l. NKT ell-medited repression of tumor immunosurveillne y IL- 13 nd the IL-4R STAT6 pthwy. Nt. Immunol. 1, 1 2 (2). 39. Heller, F., Fuss, I., Nieuwenhuis, E., Blumerg, R. & Stroer, W. Oxzolone olitis, Th2 olitis model resemling ulertive olitis, is medited y IL-13-produing NK- T ells. Immunity 17, (22). 4. MKenzie, G.J., Fllon, P.G., Emson, C.L., Grenis, R.K. & MKenzie, A.N. Simultneous disruption of interleukin (IL)-4 nd IL-13 defines individul roles in T helper ell type 2 medited responses. J. Exp. Med. 189, (1999). 41. Hnsen, G., Berry, G., DeKruyff, R.H. & Umetsu, D.T. Allergen-speifi Th1 ells fil to ounterlne Th2 ell indued irwy hyperretivity ut use severe irwy inflmmtion. J. Clin. Invest. 13, (1999). 42. Mtsud, J. et l. Trking the response of nturl killer T ells to glyolipid ntigen using CD1d tetrmers. 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