T cell and B cell independent adaptive immunity mediated by natural killer cells

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1 26 Nture Pulishing Group T cell nd B cell independent dptive immunity medited y nturl killer cells Jcqueline G O Lery 1 3, Mhmoud Goodrzi 1,3, Dnielle L Dryton 1,3 & Ulrich H von Andrin 1 It is commonly elieved tht only T lymphocytes nd B lymphocytes expressing recomintion-dependent ntigen-specific receptors medite contct hypersensitivity responses to hptens. Here we found tht mice devoid of T cells nd B cells demonstrted sustntil contct hypersensitivity responses to 2,4-dinitrofluoroenzene nd oxzolone. Those responses were dptive in nture, s they persisted for t lest 4 weeks nd were elicited only y hptens to which mice were previously sensitized. No contct hypersensitivity ws induced in mice lcking ll lymphocytes, including nturl killer cells. Contct hypersensitivity responses were cquired y such mice fter doptive trnsfer of nturl killer cells from sensitized donors. Trnsferle hpten-specific memory resided in Ly49C-I + nturl killer supopultion loclized specificlly in donor livers. These oservtions indicte tht nturl killer cells cn medite long-lived, ntigen-specific dptive recll responses independent of B cells nd T cells. Immune responses to infectious or dmging gents re commonly ctegorized s either innte or dptive. Innte immune responses involve oth solule nd cell surfce ssocited germline-encoded molecules tht recognize finite set of moleculr ptterns ssocited with tissue dmge nd certin pthogens. A hllmrk of innte immunity is tht repeted exposure to the sme chllenge does not sustntilly lter the nture of the ensuing response 1. In contrst, dptive immune responses to previously encountered chllenge re qulittively nd/or quntittively enhnced compred with the first encounter. Moreover, the verstility of dptive responses is lmost unlimited with regrd to ntigen specificity. A principl mechnism generting this diversity is the rndom recomintion of vrile, diversity nd joining gene segments during lymphocyte development, which depends on the synergistic ctivity of proteins encoded y recomintion-ctivting gene 1 (Rg1) nd Rg2 (ref. 2) nd gives rise to millions of nive T cells nd B cells, ech with unique ntigenic specificity 3. Priming of nive T cells to pthogen-ssocited ntigen requires the encounter of T cell with specilized ntigen-presenting cells in secondry lymphoid orgns nd triggers mssive T cell prolifertion followed y the cquisition of T cell effector functions. Effector T cells give rise to long-lived memory T cells tht confer enhnced protection ginst reinfection. As only T cells nd B cells re known to hve the mchinery required for somtic rerrngement of ntigen receptor genes, it is commonly elieved tht dptive immunity is strictly dependent on the presence of T cells nd B cells. Tht ide hs een chllenged sed on results otined with invertertes 4. However, there is no evidence so fr tht ny cells other thn B cells or T cells cn elicit n dptive immune response in mmmls. A clssic exmple of dptive immunity is the hpten-induced contct hypersensitivity (CHS) response, in which n epithelil surfce is exposed to orgnic or inorgnic molecules tht chemiclly modify proteins. The hptented molecules re recognized s foreign ntigens nd trigger the formtion of hpten-specific long-lived immune memory. Rechllenge fter sensitiztion precipittes locl recll response ssocited with tissue swelling cused y the recruitment of inflmmtory cells from the lood. A widely used model for studying CHS involves the sensitiztion of rodents y pinting of 2,4-dinitrofluoroenzene (DNFB) or oxzolone () on the niml s dorsl skin followed severl dys lter y rechllenge t previously unexposed site, such s the er 5,6. The hpten-specific recll response cn e mesured during susequent dys s incresed er thickness 6. Both nd gd T cells s well s B-1 B cells hve een linked to this phenomenon 7,8. Indeed, nive mice cn e pssively sensitized y doptive trnsfer of T cells from hpten-sensitized donors, nd locl injection of single memory T cell t the site of chllenge cn e sufficient to chieve tht effect 9. Moreover, the structures of severl hpten-specific T cell receptors hve een elucidted 1,11. Given tht evidence, it hs een ccepted tht T cells medite CHS responses, lthough those findings do not rigorously exclude the possiility of the prllel existence of T cell independent effectors. Here we demonstrte tht hpten-induced CHS responses re not strictly dependent on T cells or B cells ut cn e 1 The CBR Institute for Biomedicl Reserch nd Deprtment of Pthology, Hrvrd Medicl School, Boston, Msschusetts 2115, USA. 2 Deprtment of Medicine, Hrvrd Medicl School nd Gstrointestinl Unit, Msschusetts Generl Hospitl, Boston, Msschusetts 2114, USA. 3 These uthors contriuted eqully to this work. Correspondence should e ddressed to U.H.v.A. (uv@cr.med.hrvrd.edu). Received 27 Ferury; ccepted 9 Mrch; pulished online 16 April 26; doi:1.138/ni1332 NATURE IMMUNOLOGY VOLUME 7 NUMBER 5 MAY 26 57

2 26 Nture Pulishing Group Cells/ldder ( 1 4 ) Norml ldder DNFB + DNBS Norml ldder DNFB + DNBS NK cell αβ T cell PMN MΦ B cell DC Sensitized Chllenged Sensitized & chllenged independently medited y suset of nturl killer (NK) cells in the livers of sensitized T cell nd B cell deficient mice. RESULTS CHS in the mouse ldder The initil ojective of our study ws to explore CHS responses in hpten-sensitized mice in tissue other thn the skin. We chose mouse ldder s suitle orgn ecuse the urothelil rrier cn e chllenged reltively esily y intrvesicl injection of wter-solule regents. Moreover, the ldder in rodents is ccessile to intrvitl microscopy 12 nd the composition of tissue-infiltrting leukocytes cn e ssessed quntittively y histology nd flow cytometry (M.G. nd U.H.v.A., unpulished dt). We sensitized mice on two consecutive dys y epicutneous pinting with DNFB. We chllenged the urinry trct 5 d lter y intrvesicl injection of 2,4-dinitroenzene sulfonic cid (DNBS), wter-solule nlog of DNFB tht is recognized y DNFB-specific T cells 13. Histologicl exmintion showed considerle inflmmtory infiltrtion in the ldder wll of sensitized (ut not nive) mice tht ws concentrted in the lmin propri (Fig. 1). Collgense digestion of excised ldders llowed us to generte single-cell suspensions tht we nlyzed for leukocyte suset content y flow cytometry (Fig. 1). Although chllenged ldders in nive mice nd unchllenged ldders in sensitized mice contined few leukocytes, chllenged ldders in sensitized mice were hevily infiltrted with leukocytes. Compred with seline leukocyte counts in unchllenged ldders in sensitized mice, the reltive increse in leukocyte numers induced y DNBS chllenge ws highest for T cells followed y NK cells nd grnulocytes (Fig. 1c). The totl numer of CD45 + leukocytes recovered fter DNBS chllenge from ldders of sensitized mice ws more thn twice s high s the numer recovered from nive mice (Fig. 1d). To determine whether this cquired immune response in the ldder ws T cell dependent, we pplied the sme protocol to Rg2 / mice, which re completely devoid of T cells nd B cells 2,14.Unexpectedly, the sensitiztion-dependent recruitment of inflmmtory cells in chllenged ldders of Rg2 / mice ws identicl in mgnitude to our mesurements in wild-type mice (Fig. 1d). c Fold increse γδ T MΦ B PMN NK αβ T d CD45 + cells in ldder ( 1 5 ) Sensitized & chllenged P =.2 Chllenged P =.4 WT WT Rg2 / Rg2 / Figure 1 Hpten-induced DTH response in mouse ldder. Mice were sensitized trnsdermlly with DNFB on dy nd dy 1. On dy 5, mice were nesthetized nd chllenged y intrvesicl DNBS instilltion; 48 h fter chllenge, excised ldders were prepred for histology () or were digested in collgense nd nlyzed y flow cytometry ( d). () Hemtoxylin nd eosin stined sections of C57BL/6 ldders (tretment, ove imges). Originl mgnifiction, 2 (left) nd 4 (right). () Flow cytometry of ldder-infiltrting leukocytes. Infiltrting CD45 + leukocytes were chrcterized for suset composition with linege-specific ntiodies, nd the solute numer of T cells, B cells, NK cells, neutrophils (PMN), mcrophges (MF) nd dendritic cells (DC) in ldders of mice (tretment, horizontl xis) ws determined. n ¼ 4 mice per group. (c) Chllenge-induced increse in ech suset in reltive to tht in unchllenged sensitized ldders. (d) Totl numer of CD45 + leukocytes in DNBS-chllenged ldders of wild-type (WT) nd Rg2 / mice with nd without prior sensitiztion (unpired Student s t-test; n ¼ 3 mice per group). Intct cutneous CHS in Rg2 / mice Hving noted in recomintion-deficient mice wht seemed to e the induction of immunologicl memory, we sought to determine whether tht phenomenon ws unique to the ldder. Thus, we sought to determine whether mice lcking T cells nd B cells cn lso mount sensitiztion-dependent recll responses in the clssic er CHS model. We tested three geneticlly distinct strins of T cell nd B cell deficient mice (Rg2 / mice on either the C57BL/6 or C57BL/1 ckground nd severe comined immunodeficiency (SCID) mice on the BALB/c ckground). In greement with pulished oservtions 15, ll three strins developed er swelling responses to DNFB equivlent to those in wild-type control mice (Fig. 2 nd dt not shown). After thus confirming tht hpten exposure leds to full sensitiztion in the sence of lymphocytes expressing conventionl recomintion-dependent ntigen receptors, we sought to determine whether tht reflected hpten-specific dptive immune response or simple excerted innte defense rection to recently encountered irritnt. We mesured the response of mice to nother well chrcterized hpten,, which is chemiclly distinct from DNFB. Rg2 / C57BL/1 mice showed significnt inflmmtory rection when sensitized nd chllenged with, ut they responded only to hptens to which they hd een previously exposed; chllenge of DNFB-sensitized Rg2 / mice with hd no effect nd vice vers (Fig. 2). This finding indictes tht immune responses to hpten chllenge in Rg2 / mice re inducile nd hpten specific. Next we sought to determine whether hpten sensitiztion of Rg2 / mice cn induce long-lived immunologicl memory. We llowed DNFB-sensitized Rg2 / mice to rest for 4 weeks efore chllenge. The resulting CHS response ws equivlent to tht in wildtypemice(fig. 2c), indicting tht mice cn mount T cell nd B cell independent immune responses hving ll the functionl hllmrks of dptive immunity, given tht this CHS response required prior sensitiztion, ws ntigen specific nd resulted in persistent memory. NK cell ccumultion in hpten-chllenged tissues Which leukocytes might confer hpten-specific memory in the sence of T cells nd B cells? We focused on NK cells, ecuse 58 VOLUME 7 NUMBER 5 MAY 26 NATURE IMMUNOLOGY

3 26 Nture Pulishing Group Figure 2 Hpten-induced CHS responses in ers of wild-type nd Rg2 / mice. Mice were sensitized on two consecutive dys y pinting of.5% DNFB or 1% on the shved ck; 5 d or 4 weeks lter, the right er ws chllenged with.25% DNFB or 1% nd the left er ws pinted with vehicle (cetone), nd er thickness ws mesured 24 h lter. To ccount for cute hpten-induced tissue irrittion, ckground swelling ws mesured in prllel with tht of nonsensitized mice. Hpten-specific er swelling ws clculted s follows: [treted er thickness control er thickness] ckground swelling. () Hemtoxylin nd eosin stined tissue sections of ers from DNFB-sensitized, chllenged mice (left mrgins). Originl mgnifiction, 1. () CHS responses in wild-type nd Rg2 / mice re hpten specific. Mice were sensitized with either DNFB or nd were chllenged with either the sme or the other hpten (elow grph; n ¼ 4 mice per group). (c) CHS responses in wild-type nd Rg2 / mice re long lived. Mice were sensitized to DNFB nd were llowed to rest for 5 d or 4 weeks efore chllenge. P ¼.4663 (ANOVA); n ¼ 8 1 mice per group from two independent experiments. our ldder delyed-type hypersensitivity (DTH) model showed tht other thn T cells, NK cells hd the gretest reltive increse t the site of hpten chllenge (Fig. 1c). By immunofluorescence microscopy, we lso detected extrvsculr cells expressing NK1.1 fter sensitiztion nd chllenge in sections of inflmed ers from oth wild-type nd Rg2 / mice, wheres NK1.1 + cells were sprse in the contrlterl ers tht did not receive chllenge (dt not shown) nd in chllenged ers of mice tht hd not een sensitized previously (Fig. 3). WT Rg2 / NK1.1 CD31 DAPI NK Chllenged only Sensitized only WT vehicle WT DNFB Rg2 / DNFB CD45 Wild-type Sensitized & chllenged Sensitized & chllenged Chllenged only c CD45 + cells in er ( 1 5 ) d NK1.1 cells in er ( 1 3 ) Rg2 / Sensitized only Sensitiztion: Chllenge: c Hpten-specific er swelling (µm) Hpten-specific er swelling (µm) P <.1 P <.1 DNFB DNFB DNFB P <.1 P <.1 DNFB DNFB DNFB DNFB DNFB WT Rg2 / WT Rg2 / WT Rg2 / Time to chllenge: 5 d 4 weeks P <.1 P <.1 P <.1 P <.1 P <.1 Given the inherent limittions of immunofluorescence microscopy, it is difficult to detect nd ccurtely count smll numers of tissueinfiltrting leukocytes. Thus, we used n dditionl pproch to investigte the ccumultion of NK cells in hpten-chllenged skin. We removed ers from sensitized wild-type nd Rg2 / mice 24 h fter chllenge. We then split the ers nd trnsferred them to chemottrctnt-contining medi. After overnight culture, we recovered lrge numers of CD45 + leukocytes tht included.5 1.8% NK cells from hpten-chllenged ers, wheres few cells emigrted from Sensitized & chllenged P <.1 P =.2 WT Rg2 / P =.2 Sensitized & chllenged P =.3 WT Rg2 / Figure 3 NK cell ccumultion in ers of DNFBsensitized mice versus DNFB-sensitized nd chllenged mice. () Immunofluorescence stining of frozen er sections from DNFBchllenged wild-type nd Rg2 / mice with nd without DNFB sensitiztion. Sections re stined for NK1.1 (red) nd the pn-endothelil mrker CD31 (green) nd re counterstined with the nucler dye DAPI (4,6-dimindino-2-phenylindole; lue). Originl mgnifiction, 1. Microgrphs re representtive of four experiments. ( d) DNFB- nd vehicle-chllenged ers were removed from sensitized wild-type nd Rg2 / mice, were sterilized, were split long the crtilge nd were plced in medi contining CCL21, leukotriene B 4 nd interleukin 2. After 14 h, emigrted cells were counted with hemocytometer nd nlyzed y flow cytometry. () Flow cytometry of NK1.1 expression on CD45 + emigrted leukocytes; dt re representtive of four experiments. (c,d) Numer of CD45 + leukocytes (c) nd CD45 + NK1.1 + cells (d) recovered from cultured ers of wild-type nd Rg2 / sensitized-only versus sensitized-ndchllenged ers (unpired Student s t-test; n ¼ 4 mice per group). NATURE IMMUNOLOGY VOLUME 7 NUMBER 5 MAY 26 59

4 26 Nture Pulishing Group P <.5 P <.1 P <.1 P <.1 WT B/c SCID SCID eige WT B1 Rg2 / Rg2 / Il2rg / WT WT Rg2 / Rg2 / Rg2 / Il2rg / Antiody: Control GM1 Control GM1 None Antiody: vehicle-exposed control ers (Fig. 3 d). Similr to our results in the ldder model, we recovered nerly equivlent numers of CD45 + leukocytes from the hpten-chllenged ers of wild-type nd Rg2 / mice (Fig. 3c). These dt demonstrte tht hpten chllenge fter sensitiztion induces rpid nd sustntil influx of NK cells to the inflmed tissue. NK cells in T cell independent CHS The consistent ccumultion of NK cells in hpten-chllenged tissues prompted us to exmine the function of NK cells in DNFB-induced CHS using two seprte pproches. First we tested two strins of T cell nd B cell deficient mice tht either contined dysfunctionl NK cells (SCID eige 16 ) or completely lcked NK cells (Rg2 / mice lcking the common cytokine receptor g-chin (Il2rg / ) 17,18 ). Both doule-mutnt strins filed to mount CHS response to DNFB (Fig. 4). Although these oservtions were suggestive of function for NK cells, neither the lysosoml storge defect cused y the eige muttion nor deficiency in the common cytokine receptor g-chin in Rg2 / Il2rg / mice re selective for NK cells, leving open the possiility tht nother leukocyte suset could e involved. Therefore, we used second pproch y depleting NK cells from wild-type nd Rg2 / mice with ntiody to silo-gm1 (nti-silo-gm1) 19 or nti-nk1.1 (ref. 2). As expected, NK cell depletion did not olish CHS responses in wild-type mice, which cn develop T cell dependent memory specific for DNFB. In contrst, Rg2 / mice treted with nti-silo-gm1 completely lost the ility to respond to DNFB chllenge (Fig. 4). We otined equivlent results when we depleted mice of NK cells using nti-nk1.1 (Fig. 4c) P <.1 P <.1 P <.1 P <.1 P <.1 P <.5 P <.5 c WT Control WT NK1.1 P <.5 P <.1 Figure 4 NK cells re needed to medite CHS responses in T cell nd B cell deficient mice. () CHS response to DNFB in wild-type mice (BALB/c (WT B/c) nd C57BL/1 (WT B1)); T cell nd B cell deficient SCID (BALB/c) nd Rg2 / (C57BL/1) mice; nd T cell, B cell nd NK cell deficient SCID eige (BALB/c) nd Rg2 / Il2rg / (C57BL/1) mice (n ¼ 6 1 mice per group). (,c) Effect of tretment with NK cell depleting niodies on the DNFB-induced CHS response in wild-type nd Rg2 / mice. At 24 h efore DNFB chllenge, sensitized mice were depleted of NK cells with nti-silo-gm1 (GM1; n ¼ 5 6 mice per group; ) or with nti-nk1.1 (NK1.1; n ¼ 3 mice per group; c). Control mice received rit IgG () or mouse IgG2 (c), respectively. Rg2 / Control Rg2 / NK1.1 We lso compred the ility of wild-type nd mutnt mice lcking only T cells nd B cells (SCID nd Rg2 / )ordditionlly defective in NK cells (SCID eige nd Rg2 / Il2rg / ) to mount CHS responses to two dditionl hptens ( nd picryl chloride (2,4,6-trinitrochloroenzene)). Consistent with our findings using DNFB, Rg2 / mice responded vigorously when sensitized nd chllenged with, wheres Rg2 / Il2rg / mice filed to mount CHS response to (Fig. 5). We were unle to elicit significnt CHS response to in sensitized SCID mice on the BALB/c genetic ckground (Fig. 5), lthough this strin developed vigorous CHS fter sensitiztion nd chllenge with DNFB. This result is in line with pulished reports on the effects of DNFB nd in SCID BALB/c mice 15,21. Conversely, the third hpten, picryl chloride, induced mild ut sttisticlly significnt CHS response in SCID BALB/c mice ut not in Rg2 / C57BL/1 mice (Fig. 5c,d), indicting tht there my e strin-specific genetic modifiers tht determine whether given hpten cn elicit T cell independent CHS. Sensitized NK cells confer CHS The results reported ove demonstrted tht NK cells were required for T cell nd B cell independent CHS responses, ut they did not exclude the possiility of involvement of other leukocytes. To ddress tht issue, we isolted nd pooled NK cells nd non NK cells from livers nd spleens of DNFB-sensitized nd nive Rg2 / donors nd doptively trnsferred the cells into nive Rg2 / Il2rg / recipients. Susequent chllenge with DNFB elicited vigorous CHS response in recipients of sensitized NK cells, wheres recipients of nive NK cells 2 25 P <.1 c P <.1 P <.1 P < NS Picryl chloride Picryl chloride WT Rg2 / Rg2 / Il2rg / WT SCID WT Rg2 / Rg2 / Il2rg / SCID eige WT SCID SCID eige 5 P <.5 NS d P <.1 P <.1 P <.5 Figure 5 Differentil CHS responses of mutnt mouse strins to nd picryl chloride. The effect of (,) nd picryl chloride (c,d) ws determined in vrious mice (horizontl xes). Rg2 / nd Rg2 / Il2rg / mice were on C57BL/1 ckground; SCID nd SCID eige mice were BALB/c. Results represent four to seven mice per group (ANOVA with Newmn-Keuls multiple-comprison test). NS, not significnt. 51 VOLUME 7 NUMBER 5 MAY 26 NATURE IMMUNOLOGY

5 26 Nture Pulishing Group Trnsferred cells: Donor orgn: Numer of cells: Nive NK Pooled P <.1 P <.1 Sen non-nk Pooled Sen NK Pooled Nive NK Liver P <.5 P <.5 Sen NK Spleen Sen NK Liver or of pooled leukocytes from sensitized donors tht hd een depleted of NK cells showed no response (Fig. 6). Thus, NK cells re oth necessry nd sufficient for the trnsfer of hpten-specific memory from sensitized Rg2 / mice to nive recipients. Memory NK cells in the liver To explore the mechnism(s) underlying tht unexpected function of NK cells, we sought to determine whether hpten-specific memory is induced y ll NK cells in sensitized mice or if only distinct supopultion of NK cells is cple of conferring hpten-specific memory. The ltter seemed plusile, s pulished work hs demonstrted tht mmmlin NK cells re composed of multiple susets sed on surfce phenotype, immunologicl ctivity nd ntomic distriution. CD Thy c Cell numer Thy-1.2 For exmple, liver NK cells exert more potent ntitumor ctivity nd hve distinct trnscriptionl profiles compred with circulting NK cells 22. Thus, we purified NK cells from DNFB-sensitized Rg2 / livers nd spleens nd compred their ility to induce CHS in DNFBchllenged Rg2 / Il2rg / recipients. Only recipients of sensitized heptic NK cells mounted detectle CHS responses (Fig. 6), wheres splenic NK cells from the sme donors were devoid of trnsferle hpten-specific memory ctivity. Thus, hpten-specific NK memory cells in sensitized Rg2 / mice re loclized in the liver. Heptic memory NK cell surfce phenotype In n effort to phenotypiclly define hpten-specific NK cells, we stined Rg2 / heptic NK cells for CD11 nd Thy-1, which re d P <.1 Control Thy-1.2 Figure 6 A suset of heptic NK cells crries DNFB-specific memory in sensitized Rg2 / mice. () CHS responses fter doptive trnsfer. Sorted cells pooled from multiple orgns (left three rs) or sorted cells from individul orgns (elow grph; right three rs) were doptively trnsferred into nive Rg2 / Il2rg / mice. Recipient ers were chllenged with DNFB 24 h lter (n ¼ 3 4 mice/group); CHS responses were mesured 24 h fter chllenge. Sen, sensitized. ( d) Anti-Thy-1 rogtes CHS in Rg2 / mice. () Contour plot of Thy-1.2 nd CD11 expression on liver NK1.1 + cells from DNFBsensitized Rg2 / mice. Numers in qudrnts indicte suset frequency. (c) Thy-1.2 stining (detected y secondry phycoerythrin-conjugted got nti-rt IgG) on NK cells in Rg2 / liver 48 h fter tretment with nti-thy-1.2 (roken line) or isotype control ma (solid line). Gry histogrm, ckground stining of secondry ntiody on NK cells in control mice. Mice were given 1 mg of ech ma 2 d efore sensitiztion nd 1 d efore chllenge. (d) Depletion of Thy-1 + NK cells in nti-thy-1-treted, ut not in isotype control ma treted (Control), sensitized Rg2 / mice results in complete inhiition of CHS fter DNFB chllenge (unpired Student s t-test; n ¼ 6 mice/group). Ly49G Ly49D Ly49F Ly49C-I Ly49A KLRG Hpten-specific er swelling (µm) P =.4 Ly49C Ly49C + Rg2 / Rg2 / Il2rg / c Thy-1.2 WT Rg2 / Ly49C-I Figure 7 NK receptors in CHS. () Survey of NK receptor expression on NK cells in lymph node (LN), liver nd spleen efore nd fter sensitiztion with DNFB or. On dy, Rg2 / mice were injected with B16-Flt3L tumor cells to induce systemic prolifertion of NK cells; on dys 9 nd 1, mice were sensitized with DNFB, or vehicle; on dy 12, NK receptor expression on NK1.1 + cells in vrious tissues ws nlyzed y flow cytometry (n ¼ 3 mice per group). Sensitiztion (horizontl xis): N, none; D, DNFB; OX, oxzolone. () CHS response fter doptive trnsfer. Flow cytometry sorted LY49C-I + or Ly49C-I heptic DX5 + NK cells from DNFBsensitized Rg2 / donors were doptively trnsferred into Rg2 / or Rg2 / Il2rg / mice ( to cells per mouse); recipients were chllenged with DNFB 24 h lter nd the CHS response ws mesured the next dy (unpired Student s t-test). Ech symol represents one mouse. (c) Thy-1.2 nd Ly49C-I expression on NK1.1 + cells in wildtype nd Rg2 / livers. Numers in qudrnts indicte the frequency of cells in ech. Dt in c re representtive of two experiments NATURE IMMUNOLOGY VOLUME 7 NUMBER 5 MAY

6 26 Nture Pulishing Group Reltive chnge in er thickness (%) Antiody: Control L-selectin CD18 P-Eselectin NKG2D Figure 8 Function of dhesion molecules nd NKG2D in CHS responses in Rg2 / mice. Rg2 / mice were treted intrvenously with mas specific for L-selectin (MEL-14), CD18 (GAME-46), P-selectin (RB4.34), E-selectin (9A9) nd NKG2D (C7). All mas were given t dose of 1 mg/mouse; nti-l-selectin ws given 24 h efore sensitiztion nd ll other mas were given 2 h efore chllenge. The hpten-specific er swelling in ech tretment group is expressed s percentge of tht of the isotype control group. P o.1, inhiition of CHS for control versus ll ma-treted groups (n ¼ 4 6 mice per group). P-E-selectin, P- nd E-selectin. upregulted on ctivted NK susets 23. We noted considerle heterogeneity in CD11 nd Thy-1 expression, with some NK cell susets expressing neither, one or oth mrkers (Fig. 6). Systemic tretment with monoclonl ntiody (ma) specific for Thy-1 hs een shown to rogte CHS responses in wild-type mice 24. Tht effect ws ttriuted to ntiody-induced depletion of T cells, which re uniformly Thy-1 +. However, given our oservtion tht Thy-1 ws expressed on out 5% of heptic NK cells, it seemed plusile tht nti-thy-1 might lso remove liver-resident Thy-1 + NK cells. Indeed, tretment with nti-thy-1 produced depletion of heptic Thy-1 + NK cells (Fig. 6c) nd olished CHS responses to DNFB in sensitized Rg2 / mice (Fig. 6d). Thus, it is likely tht most or ll of the hpten-responsive NK cells in sensitized Rg2 / mice were contined in the Thy-1 + suset. To further chrcterize the heptic NK cells in Rg2 / mice, we nlyzed the expression of vrious ctivting nd inhiitory NK cell receptors. Mouse NK cells express vrile comintions of Ly49 receptors, nd the solute numer nd reltive frequency of NK cells expressing ech Ly49 receptor comintion depends on genetic ckground 25. Flow cytometry demonstrted distinct expression ptterns of Ly49 receptors in liver, lymph nodes nd spleen, ut those expression ptterns were not ltered fter hpten sensitiztion (Fig. 7). A study of C57BL/6 nd C57BL/1 mice hs shown tht NK cells expressing Ly49C, cognte inhiitory receptor for self mjor histocomptiility complex (MHC) clss I molecules, re uniquely le, reltive to other NK cells, to exert effector ctivity in response to vriety of stimuli 26. To ssess whether this self MHC rective NK cell suset cquired hpten-specific memory, we sorted heptic NK cells from DNFB-sensitized Rg2 / C57BL/1 donors sed on their rectivity with ma specific for Ly49C nd nother inhiitory receptor, Ly49I. Both receptors recognize MHC clss I molecules expressed in C57BL/1 mice 27. After doptive trnsfer into nive Rg2 / or Rg2 / Il2rg / recipients, sensitized Ly49C-I + cells conferred significntly greter CHS responsiveness thn did sensitized Ly49C-I NK cells (Fig. 7). We next nlyzed expression of Thy-1 nd Ly49C-I on heptic NK cells in wild-type nd Rg2 / mice (Fig. 7c). Only minority (out 13 2%) of Thy-1 + heptic NK cells expressed Ly49C-I, nd pproximtely hlf of the Ly49C-I + NK cells in Rg2 / nd wild-type livers did not express Thy-1, indicting tht the hpten-specific Thy-1 + Ly49C-I + NK cells represent only smll suset (less thn 1%) of heptic NK cells. Adhesion molecules in NK cell medited CHS Severl studies hve ddressed the function of dhesion molecules during the sensitiztion nd chllenge phse of CHS. For exmple, L-selectin is criticl for homing of nive T cells to skin-drining lymph nodes, where T cells re primed y hpten-presenting dendritic cells 28. When Rg2 / mice were treted with nti-l-selectin efore sensitiztion with DNFB, the hpten-specific CHS response ws ttenuted (Fig. 8), suggesting tht NK cells my lso e primed in lymph nodes. Susequently, primed cells must enter the hpten-exposed inflmed skin. Recruitment of effector T cells to inflmed mouse ers requires P-selectin nd E-selectin, which medite slow rolling in derml venules 29. Rolling cells must then engge 2 -integrins (CD11 CD18) to rrest nd emigrte into inflmed tissues 3. Those trffic rules lso seem to pply to NK cell medited CHS responses in DNFBsensitized Rg2 / mice, s locking ntiodies specific for either the endothelil selectins or CD18 rogted CHS responses (Fig. 8). Activting receptor NKG2D in NK cell medited CHS Once NK cells hve emigrted into the er, they must detect nd respond to the locl hpten chllenge. As hptens cuse tissue irrittion, the exposed prenchym is likely to generte innte distress signls. NK cells hve ctivting receptors tht detect mrkers of cellulr injury cused y infection, mlignnt trnsformtion nd other dmging fctors 25. Possily the est chrcterized of these sensors is NKG2D, which recognizes group of MHC clss I relted surfce proteins tht re induced on distressed cells y certin noxious stimuli 31,32. Ligtion of NKG2D triggers functionl ctivtion of NK cells, which in the sence of inhiitory signls my then secrete cytokines nd exert cytotoxic effects, leding to locl excertion of inflmmtion. Tretment with ma specific for NKG2D efore chllenge of sensitized Rg2 / mice suppressed the ensuing CHS response (Fig. 8). This effect ws not due to depletion of NK cells, s ma-treted mice hd norml numers of NK cells in their livers nd spleens (dt not shown). These results suggest tht NKG2D might contriute to NK cell medited CHS owing to its ility to detect certin stress-induced lignds. DISCUSSION The findings presented here hve demonstrted previously unknown function for NK cells: the ility to cquire selective memory of t lest three chemiclly distinct foreign moleculr entities (DNFB, nd picryl chloride). Using cross-chllenge pproch with DNFB nd, we hve shown tht this memory ws hpten specific (for exmple, mice exclusively responded to the hpten to which they were previously sensitized). Further experiments in the DNFB model demonstrted tht NK cell memory ws long lived nd could e pssively trnsferred to nive mice y trnsfusion of smll hptenspecific suset of primed heptic NK cells. The impetus for this work ws the unexpected oservtion tht DNFB-immunized Rg2 / mice demonstrted norml hpteninduced inflmmtion fter chllenge of either the ldder or the uriculr skin. The lst oservtion is consistent with report tht noted norml DNFB-induced er swelling responses in sensitized nude nd SCID mice s well s in mice lcking RAG1 or RAG2 (ref. 15). Our phenotypic nlysis of inflmmtory cells in chllenged ldders nd er explnts showed sustntil ccumultion of NK cells in oth wild-type nd Rg2 / mice. Those dt re in line with studies 512 VOLUME 7 NUMBER 5 MAY 26 NATURE IMMUNOLOGY

7 26 Nture Pulishing Group in humn CHS in which derml nd epiderml inflmmtory infiltrtes re mrked y n influx of NK cells 33. NK cells re lso present, leit t low frequency, in norml mouse nd humn skin (refs. 33,34 nd reported here). Moreover, NK cells represent pproximtely 4% of leukocytes in norml humn skin-drining lymph fluid 34. These dt suggest tht NK cells prticipte in the physiologicl immune surveillnce of mmmlin skin nd tht fter contct sensitiztion nd renewed exposure to hpten, NK cells ccumulte rpidly in chllenged tissues regrdless of whether other lymphocytes re present. Given those oservtions, we hypothesized tht NK cells might e involved in T cell nd B cell independent CHS. Indeed, doulemutnt mice lcking oth T cells nd B cells s well s functionl NK cells were unresponsive to hpten chllenge, nd NK cell depletion in Rg2 / ut not wild-type mice olished DNFB-induced CHS. Moreover, nonsensitized mice could e pssively immunized to DNFB y doptive trnsfer of purified NK cells from DNFB-sensitized RAG2- deficient donors. In contrst, doptive trnsfer of NK cells from nive mice or of non NK leukocytes from sensitized donors did not confer hpten sensitivity. These experiments estlish tht NK cells re oth required nd sufficient for mediting potent CHS response in the sence of other lymphocytes. Where nd how might NK cells ecome first educted out sensitizing hpten? There is mounting evidence tht homing of lymphocytes to secondry lymphoid tissues is criticl for immune surveillnce nd the development of dptive immunity to regionl ntigens 35. To respond to cutneous hpten chllenge, nive lymphocytes require L-selectin to home to skin-drining lymph nodes, where they re ctivted y ntigen-presenting dendritic cells, prticulrly Lngerhns cells 36. Consequently, T cell priming y sensitizing hpten nd the ensuing CHS response re compromised in L-selectin-deficient mice owing to specific defect in homing of lymphocytes to lymph nodes 28. Moreover, mice devoid of lymph nodes fil to mount CHS responses 37. Becuse tretment of Rg2 / mice with nti-l-selectin efore sensitiztion impired CHS responses, it is likely tht lymph nodes re lso the min sites of NK cell priming for CHS. Indeed, NK cells re effectively recruited to ntigenchllenged lymph nodes, nd NK cells hve multiple mens for communicting with dendritic cells 38,39. Once the hpten-primed effector cells hve returned to the lood, they require different set of trfficking molecules for entry into hpten-exposed inflmed skin. L-selectin is proly not involved in this migrtion step, s CHS responses re not ffected y the sence of L-selectin fter sensitiztion 28. Insted, leukocyte recruitment to inflmed skin requires P-selectin nd E-selectin, which re constitutively expressed in the lumen of skin venules 29,4. A hllmrk of skinhoming leukocytes is their expression of selectin lignds tht medite slow rolling interctions in selectin-expressing venules. Susequently, the rolling cells must ctivte 2 -integrins (CD11 CD18), which ind endothelil intercellulr dhesion molecule 1 to llow the cells to rrest nd emigrte 3. NK cells express selectin lignds s well s 2 -integrins 41, nd our results hve shown tht oth pthwys were required for the induction of CHS response in DNFB-sensitized Rg2 / mice. Thus, NK cells seem to use the sme migrtory routes nd dhesion pthwys tht conventionl T cells use during the priming nd effector phses of CHS 42. Unexpectedly, hpten-specific memory NK cells nd T cells differed in orgn distriution in sensitized mice. Although mny studies hve shown tht memory T cells re undnt in the spleen, we were unle to trnsfer hpten rectivity from sensitized Rg2 / donors with spleen-derived NK cells. In contrst, trnsfer of liver NK cells ws very effective in eliciting contct sensitivity in nive hosts. The mechnisms resulting in the ccumultion of intrinsic memory NK cells in the liver re still uncler. Notly, unique function for the CXCL16-CXCR6 pthwy in the retention nd survivl of NKT cells in the liver hs een demonstrted 43. It will e interesting to explore whether this or other chemokine pthwys hve similr function in NK cell migrtion or homeostsis in the liver. Experiments of ma depletion nd doptive trnsfer using sorted liver NK cell susets hve indicted tht the trnsferle memory ctivity is concentrted in the Thy-1 + Ly49C-I + frction, which represents less thn 1% of heptic NK cells. However, this suset is lso present in the spleen (D.L.D. nd U.H.v.A., unpulished dt), indicting tht these mrkers lone re not sufficient to define hpten-specific NK cell memory. Indeed, lthough sorted Ly49C-I + NK cells in the liver were more efficient t conferring hpten sensitivity thn were their Ly49C-I counterprts, there ws no sustntil difference in the numer or frequency of heptic Ly49C-I + NK cells efore nd fter hpten sensitiztion. In fct, the reltive frequency of the Ly49C-I + suset mong ll tissue-resident NK cells ws lwys sustntilly lower in the liver thn in the spleen or lymph nodes. A similrly ised distriution pttern ws lso pprent in the Ly49F + NK popultion, suggesting the possiility tht phenotypiclly distinct NK cell susets my e preferentilly recruited or retined in certin ntomic comprtments sed on their individul NK cell receptor repertoire. It will e necessry to identify the moleculr mechnisms y which NK cells discriminte etween distinct hptens or hpten-modified mcromolecules in the sence of RAG-medited receptor rerrngement. As NK cells re ctegorized into mny susets, ech chrcterized y distinct pttern of ctivting nd inhiitory receptors 25,44,it is possile tht hpten-specific NK cell memory is rooted in this popultion diversity. Moreover, there re sustntil differences in the composition of NK cell receptor loci mong different strins of mice 45, which might explin the differentil hpten effects in BALB/c versus C57BL/1 mice. Ly49C is of prticulr interest in this context. As mentioned ove, the Ly49C-I + popultion of heptic NK cells ws more efficient thn the Ly49C-I heptic NK cell popultion in trnsferring hpten sensitivity to Rg2 / Il2rg / mice. In contrst, Ly49C-I + NK cells were more undnt in the spleen thn in the liver, yet splenic NK cells did not confer hpten sensitivity. Thus, it is unlikely tht Ly49C-I lone could ccount for hpten recognition y NK cells. Moreover, hpten sensitiztion did not detectly lter the composition of other NK susets s defined y pnel of ntiodies specific for Ly49 fmily memers nd killer cell lectin-like receptor G1 (KLRG1). A chnge in suset frequency nd/or distriution might e expected if one or more of these receptors were driving hptenrective NK cells to undergo clonl prolifertion. Similrly, lthough NK cell medited CHS could e locked with nti-nkg2d, the fct tht this ctivting receptor is expressed on lmost ll NK cells 46, wheres only suset of NK cells crry hpten specificity, mkes it unlikely tht NKG2D is directly responsile for hpten recognition. Its function in NK cell medited CHS is perhps more similr to tht of costimultory molecules on T cells. Given the considertions discussed ove, the possiility tht NK cells hve other ctivting receptors tht specificlly recognize given hpten nd/or hpten-modified mcromolecule(s) cnnot e ruled out. Lymphocytes in gnthn vertertes cn generte vrile lymphocyte receptors through gene rerrngements tht do not involve RAG proteins 47. However, whether similr system for RAG-independent somtic diversifiction exists in NK cells or in ny other cell type in jwed vertertes hs not een determined. NATURE IMMUNOLOGY VOLUME 7 NUMBER 5 MAY

8 26 Nture Pulishing Group Finlly, given the decdes of intense reserch on hpten-induced CHS, it is surprising tht the function of NK cells hs een overlooked for so long. This my hve een due in prt to the fct tht T cells nd NK cells re y themselves sufficient to mintin norml CHS responses, t lest to DNFB. Consequently, NK cell depletion of wild-type mice cused only mild, sttisticlly nonsignificnt reduction in CHS. Similrly, eige mice re known to mount norml CHS responses despite lysosoml defect in these mice tht renders NK cells dysfunctionl 48. A distinct phenotype ecme pprent only when we exmined doule-mutnt mice crrying oth the eige nd SCID muttion. Conversely, wheres there is undnt evidence tht T cells y themselves cn medite formidle CHS responses 7,thereis insufficient pulished informtion to rigorously exclude the possiility of prllel existence of T cell independent effector popultions. Studies spnning more thn three decdes hve shown tht tretment of nimls with depleting ntiser or mas specific for ntigens expressed on T cells cn eliminte hpten-specific immune responses, including CHS 24, However, the T cell ssocited ntigens trgeted in those experiments my e shred y other cell types, such s susets of NK cells (for exmple, Thy-1) or dendritic cells. For exmple, depletion with nti-cd8 olishes CHS responses to DNFB 51. However, the so-clled lymphoid suset of CD8 + dendritic cells is known to interct with NK cells 52, nd depletion of CD8 + dendritic cells in nude mice using ma hs een shown to compromise NK cell medited nti-tumor immunity 53. Mny studies hve used gene-knockout mice to exmine the function of immune receptors nd leukocyte trffic molecules in CHS responses to severl hptens 8. Mny mutnt strins show n incomplete reduction (or n increse) in CHS, consistent with contriution of other effector pthwys. However, to identify mechnisms criticl for ll custive gents of CHS, it is most informtive to focus on studies showing complete sence of CHS in specific mutnt mice. A review of the literture hs identified three exmples in which CHS is olished in mice in which T cells or ntigenpresenting cells re specificlly compromised: SCID mice (BALB/c) responding to 15,21 ; T cell receptor--deficient mice (129/J) responding to picryl chloride 54 ;nd 2 -microgloulin-deficient mice (C57BL/6) responding to DNFB 51. Those findings re not necessrily in conflict with our study here. Our oservtions hve indicted tht there re strin-dependent differences in the ility of mice to respond to, nd the modest T cell independent response to picryl chloride could e esily missed nd is pprently lso influenced y strin-dependent genetic modifiers. The filure of 2 -microgloulindeficient mice to mount CHS response to DNFB hd een ttriuted to the sence of CD8 + T cells in these mice 51. However, pulished work hs shown tht self MHC clss I recognition y developing Ly49C + cells is necessry to enle these cells to chieve functionl competence 26. Thus, oth MHC clss I on unidentified licensing cells nd Ly49C on NK cells re necessry for norml NK cell function, t lest in some settings. However, the issue of whether MHC clss I expression is merely necessry to license NK cells during their development 26 or hs lso direct function in the priming nd/or effector phse of hpten-induced CHS (s is the cse for T cell medited immunity) will require further dissection of the moleculr mechnisms of hpten recognition y NK cells. In conclusion, our findings hve identified previously unrecognized function of NK cells s meditors of hpten-specific immunity. This unexpected ctivity of NK cells represents lerned response tht persists for severl weeks nd cn exquisitely discriminte etween different hptens. Thus, NK cell medited CHS fetures three essentil hllmrks of dptive immunity: cquired ctivity, long-lived memory nd ntigen specificity. Although severl studies hve suggested tht the innte immune system cn mount dptive responses in lower nimls 4, to our knowledge this is the first oservtion of T cell nd B cell independent cquired immune response in higher vertertes. METHODS Mice. Rg2 / mice (C57BL/6 or C57BL/1 ckground), Rg2 / Il2rg / mice (C57BL/1), nd SCID nd SCID eige mice (oth on BALB/c ckground), s well s ge-, ckground- nd sex-mtched wild-type mice were purchsed from Tconic or were red in-house. Mice were used t 5 1 weeks of ge in ccordnce with Ntionl Institutes of Helth guidelines. All procedures were pproved y the institutionl niml committees t oth the CBR Institute for Biomedicl Reserch nd Hrvrd Medicl School (Boston, Msschusetts). Regents. DNFB, DNBS, picryl chloride nd were purchsed from Sigm Fine Chemicls. Anti-silo-GM1 nd rit IgG were from Wko Pure Chemicl Industries; nti-nkg2d ws from BioLegend; phycoerythrinconjugted got nti-rt IgG ws from ImmunoReserch; nd nti-e-selectin (9A9) ws purified from culture superntnt of hyridom cells. All other ntiodies were from BD Biosciences. Bldder DTH protocol. On dy nd dy 1, femle mice were pinted with 25 ml DNFB (.5% in cetone) on shved dorsl skin. On dy 5, mice were nesthetized, the hir surrounding the genitli ws removed with epilting creme (Nir; Crter Products) nd the exposed skin ws clened with lcohol. With the help of dissecting microscope, polyethylene ctheter 1 cm in length (.28-mm internl dimeter nd.61-mm outer dimeter; Becton Dickinson) ws introduced trnsurethrlly into the ldder nd ws dvnced until resistnce ws pprent. To minimize dilution nd reflux of inocul, urine ws drined y ppliction of gentle suprpuic pressure. Mice were then chllenged y instilltion of freshly prepred DNBS (2 mg in 5 ml sterile.9% NCl) into the ldder. To ensure consistent contct of the compound with the urothelium, the ldder ws drined 3 min lter nd the injection ws repeted. Controls included sensitized mice treted with intrvesicl instilltion of sterile sline nd nonsensitized mice chllenged with DNBS. At 48 h fter chllenge, the ldder ws removed nd ws used either for stndrd histology or for nlysis of leukocyte content. For the ltter, ldders were dissected longitudinlly nd were digested with mild gittion for 3 min t 37 1C with 4 U/ml of collgense-d (Roche) plus 1 mg/ml of DNAseI (Roche). Bldders were then plced on 7-mm nylon mesh cell striner (BD Bioscience) nd were dissocited into single-cell suspensions. Wshed cells were counted with hemocytometer nd were stined for flow cytometry. Er CHS protocol. On dy nd dy 1, shved dorsl skins of mice were pinted with.5% DNFB or 1% in 25 ml cetone.alterntively,mice were immunized on dy with 7% picryl chloride (VeZerf Lorsynthesen) in 1 ml cetone. On dy 5, the right er ws chllenged with.25% DNFB or 1% or 1% picryl chloride (1 ml to ech side) nd the left er ws pinted with vehicle (cetone). Er thickness to the first crtilge ridge ws mesured 24 h lter with micrometer (Mitutoyo). To ccount for cute hpten-induced irrittion, ckground swelling ws mesured in prllel with tht of nonsensitized mice. Hpten-specific er swelling ws clculted s follows: (treted er thickness control er thickness) ckground swelling. Results were highly reproducile when mesurements mde y three independent investigtors (who in most experiments were linded to tretment protocol) were compred. Culture of er skin sheets. DNFB-chllenged nd vehicle-exposed ers of DNFB sensitized mice were removed 24 h fter chllenge, were sterilized with 7% ethnol nd were seprted long the crtilge with forceps. The skin sheets were then plced for 14 h on top of 1 ml medi (RPMI medium with 1% FCS nd stndrd supplements) contining 1 ng/ml of CCL21 (R&D Systems), 1 nm leukotriene B 4 (Biomol) nd 5 U/ml of recominnt humn IL-2 (Roche). Emigrted leukocytes in the medi were counted with hemocytometer nd their phenotype ws nlyzed y flow cytometry. NK cell purifiction nd doptive trnsfer. Rg2 / mice were injected sucutneously with B16 melnom cells tht secrete the lignd for the receptor 514 VOLUME 7 NUMBER 5 MAY 26 NATURE IMMUNOLOGY

9 26 Nture Pulishing Group tyrosine kinse Flt3 (Flt3L), which cused NK cell prolifertion 55,56.After9d, mice were ssigned to one of two groups: one group ws sensitized with DNFB (.5% in 5 ml) on dy 9 nd dy 1; the second group remined unsensitized. On dy 12, NK cells were purified from splenocyte smples fter red lood cell lysis (using ACK lysis uffer) nd from the liver with digestion with 4 U/ml of collgense D (Roche Dignostics; 37 1C for 3 min) followed y pssge through 4-mm filter, ACK lysis nd 3 min of centrifugtion over Percoll grdient (4 6%; Amershm Biosciences). Smples were further enriched for NK cells y negtive selection with Dyneds (Dynl Biotech ASA) with depleting mas to Gr-1, I-A nd Ter119 or to CD4, CD8 nd Ter119. The negtive selection step ws omitted when liver NK cells were sorted seprtely. NK cells were then stined with phycoerythrin-leled DX5 ma nd/or nti- Ly49C-I (5E6) nd were sorted to more thn 95% purity on FACSVntge DiV cell sorter (Becton Dickinson). Sorted NK cells were doptively trnsferred into recipient mice y til vein injection. Immunofluorescence. Er tissue ws collected nd ws immeditely frozen in optimum cutting temperture compound (Tissue-Tek). Sections 7 mm in thickness were cut onto poly-l-lysine-coted glss slides (Sigm Dignostics), were fixed in 1% cold cetone for 1 min, were ir-dried t 25 1C ndwere stored t 7 1C. For stining, slides were ir-dried t 25 1C nd were locked with 5% mouse serum contining 3% BSA in PBS, ph 7.4. Phycoerythrinconjugted nti-nk1.1 (PK136; Phrmingen) nd purified nti-cd31 (MEC13.3; Phrmingen) were diluted in locking solution nd were used t concentrtion of 1 mg/ml or 2.5 mg/ml, respectively. Sections were stined for 1 h t 25 1C with primry ntiodies. CD31 ws detected y incution with crocynine-conjugted got nti-rt IgG (Jckson ImmunoReserch). As negtive control, species-specific isotype-mtched irrelevnt ntiodies were used. Sections were counterstined with 3 nm 4,6-dimindino-2-phenylindole (Moleculr Proes). Sttisticl nlysis. All dt re presented s men ± s.e.m. nd were nlyzed y unpired, one-wy nlysis of vrince (ANOVA) with the Bonferroni multiple-comprison test unless specified otherwise. Significnce ws set t P vlue of less thn.5. ACKNOWLEDGMENTS We thnk G. Cheng nd B. Reinhrdt for technicl ssistnce; P. Scherli for ssistnce in the design of er sheet culture experiments; J. Lieermn, K. Rjewsky, F. Alt, D. Mthis nd D. Podolsky for criticl reding of the mnuscript; nd F. Alt for providing some of the Rg2 / mice used. Supported y the Americn Assocition for the Study of Liver Diseses (J.G.O.) nd the Ntionl Institutes of Helth (AI61663, HL56949 nd AR42689 to U.H.v.A.; T32 DK7191 to J.G.O.; T32 HL66987 to M.G.; nd T32 AR to D.L.D.). AUTHOR CONTRIBUTIONS J.G.O. designed nd did the CHS er experiments nd doptive trnsfers of sorted NK cells; M.G. designed the mouse ldder DTH model nd did the experiments; nd D.L.D. designed nd did the nlysis of NK cells in CHS er experiments nd doptive trnsfers of sorted Ly49C + nd Ly49C NK cells. COMPETING INTERESTS STATEMENT The uthors declre tht they hve no competing finncil interests. Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions/ 1. Jnewy, C.A., Jr. & Medzhitov, R. Innte immune recognition. Annu. Rev. Immunol. 2, (22). 2. Jung, D. & Alt, F.W. Unrveling V(D)J recomintion; insights into gene regultion. Cell 116, (24). 3. Bssing, C.H., Swt, W. & Alt, F.W. The mechnism nd regultion of chromosoml V(D)J recomintion. Cell 19, S45 S55 (22). 4. Kurtz, J. Specific memory within innte immune systems. Trends Immunol. 26, (25). 5. Bloch, B. The role of idiosyncrcy nd llergy in dermtology. Arch. Dermt. Syph. 19, (1929). 6. Asherson, G.L. & Ptk, W. 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