OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis

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1 OPGL is key regultor of osteolstogenesis, lymphoyte evelopment n lymph-noe orgnogenesis Young-Yun Kong*, Hiroki Yoshi*, Iliko Srosi², Hong-Lin Tn², Emm Timms³, Csey Cpprelli², Sen Morony², Antonio J. Oliveir-os-Sntos*, Gwyneth Vn², Annik Itie*, Wilson Khoo*, Anrew Wkehm*, Colin R. unstn², vi L. Ley³, Tk W. Mk*, Willim J. Boyle³ & Josef M. Penninger* * Amgen Institute, Ontrio Cner Institute, n the eprtments of Meil Biophysis n Immunology, University of Toronto, 62 University Avenue, Toronto, Ontrio M5G 2C1, Cn eprtments of ² Pthology n ³ Cell Biology, Amgen In., One Amgen Center rive, Thousn Oks, Cliforni , USA... The tumour-nerosis-ftor-fmily moleule osteoprotegerin lign (OPGL; lso known s TRANCE, RANKL n OF) hs een ienti e s potentil osteolst ifferentition ftor n regultor of intertions etween T ells n enriti ells in vitro. Mie with isrupte opgl gene show severe osteopetrosis n efet in tooth eruption, n ompletely lk osteolsts s result of n inility of osteolsts to support osteolstogenesis. Although enriti ells pper norml, opgl-e ient mie exhiit efets in erly ifferentition of T n B lymphoytes. Surprisingly, opgl-e ient mie lk ll lymph noes ut hve norml spleni struture n Peyer's pthes. Thus OPGL is new regultor of lymph-noe orgnogenesis n lymphoyte evelopment n is n essentil osteolst ifferentition ftor in vivo. Morphogenesis n remoelling of one is physiologilly ontrolle proess tht involves the synthesis of one mtrix y osteolsts n the oorinte resorption of one y osteolsts 1,2. Osteolsts n osteolsts rise from istint ell lineges n mturtion proessesðosteolsts rise from mesenhyml stem ells, wheres osteolsts ifferentite from hemtopoieti monoyte/mrophge preursors 3±5. Imlnes etween osteolst n osteolst tivities n rise from vriety of hormonl hnges or perturtions of in mmtory n growth ftors, resulting in skeletl normlities hrterize y erese (osteoporosis) or inrese (osteopetrosis) one mss 6,7. Inrese osteolst tivity is seen in mny osteopeni isorers, inluing postmenopusl osteoporosis, Pget's isese, lyti one metstses or rheumtoi rthritis; this inrese osteolst tivity les to inrese one resorption n rippling one mge 8±1. Severl ftors ffet osteolstogenesis t istint stges of evelopment, inluing olony-stimulting ftor-1 (CSF-1 or M- CSF), interleukin (IL)-1, trnsforming growth ftor (TGF)-, TGF-, tumour-nerosis ftor (TNF)-, TNF-, IL-6, vitmin 3, IL-11, litonin, prostglnin E 2 (PGE 2 ), n prthyroi hormone (PTH) 2. However, geneti ltion experiments hve shown tht these ftors re not essentil for osteolst evelopment in vivo. Only sf-1 mutnt osteopetroti mie exhiit n rrest of osteolstogenesis t the pre-osteolst stge 11,12. As the osteolst efet in sf-1 mutnt osteopetroti mie n e overome y overexpression of the nti-poptoti protein Bl-2 in the osteolst/monoyte linege n spontneously resue 13,14, it ppers tht CSF-1 expression is not essentil for osteolst evelopment. The essentil ftor for osteolst evelopment in vivo hs not yet een ienti e. It hs een shown, using in vitro ulture systems, tht the new TNF-fmily moleule osteoprotegerin lign (OPGL; lso known s osteolst ifferentition ftor, OF) n oth tivte mture osteolsts n meite osteolstogenesis in the presene of CSF-1 (refs 15, 16). OPGL is highly expresse in osteolst/stroml ells n OPGL expression n e upregulte y the one-resoring ftors vitmin 3, IL-11, PGE 2 n PTH 16, initing tht OPGL might e the elusive osteolst ifferentition ftor. Surprisingly, OPGL is ientil to the TNF-relte ytokine TNF-relte tivtion-inue ytokine (TRANCE)/reeptor tivtor of NFkB (RANK) lign (RANKL), whih hs een implite in intertions etween T ells n enriti ells in the immune system 17±19. OPGL expression in T ells is inue y ntigenreeptor enggement n is regulte y lineurin-regulte trnsription ftors 18. The TNF-reeptor (TNF-R)-fmily memer RANK, whih is expresse on enriti ells, T ells n hemtopoieti preursors, hs een ienti e s the reeptor for OPGL 15,17. These oservtions inite tht, like the intertions etween C4 lign (C4L) n C4, or etween C28 n C8/C86, the ining of OPGL to RANK my regulte enriti-ell funtions n T-ell tivtion 17,19. However, it is not known whether OPGL is essentil for osteolstogenesis n one remoelling, n whether it hs itionl funtions suh s moulting immune responses. Genertion of opgl mie The opgl gene ws lte in murine emryoni stem (ES) ells y the use of trgeting vetor in whih the exon ontining nuleoties 45±531 (enoing mino is () 136±177, portion of the TNF-homology omin) ws isrupte (Fig. 1). Seven G418- resistnt olonies were heterozygous for the muttion t the opgl lous. We use two heterozygous mutnt ES ells to generte himeri mie n krosse himers to C57BL/6 mie. Heterozygous opgl +/- mie, whih re helthy n fertile, were then interrosse to generte homozygous opgl mie (Fig. 1). The null muttion of opgl ws on rme y the sene of opgl expression, s etermine y northern lot nlysis (Fig. 1). Homozygous opgl-knokout mie were orn t the expete menelin frequeny n exhiite norml growth until wening t 3 weeks fter irth. After wening, growth of opgl mie ws severely retre (Fig. 1, Tle 1), presumly euse of poor nutrition seonry to filure of tooth eruption (Fig. 1e; see elow). NATURE VOL JANUARY

2 Wil-type gene Trgeting vetor Mutnt gene Xh Flnking proe +/+ +/- +/+ +/- B 1.9k 1.7k S S WT MT OPGL β-tin e X X Severe osteopetrosis in opgl mie In vitro ssys hve shown tht OPGL n inue osteolstogenesis n tivte mture osteolsts 15,16. To etermine whether isruption of opgl les to tul hnges in one physiology in vivo, we stuie whole-oy riogrphs of 3±4-week-ol opgl +/+ n opgl littermtes (Fig. 2±h). The mutnt mie exhiite severe osteopetrosis, hrterize y rio-opque long ones, verterl oies n ris (Fig. 2,, e±h). Osteopetrosis ws lrey evient in riogrphs t two ys fter irth (t not shown). The long ones were shortene n h istint roening of the ens of the one (lu-shpe ones) ue to one-remoelling efet (Fig. 2e, f). In none of these mie h the inisor or molr teeth erupte into the orl vity (Fig. 1e, Fig. 2, ). Filure of tooth eruption is typil ning in osteopetrosis s one resorption is require to open n venue through the one of the jw for eruption of teeth. Osteopetroti hnges were lso present in the xil skeleton; these hnges were hrterize y gretly inrese rioensity of the verterl oies n ris (Fig. 2g, h). In ontrst, ones tht form y intrmemrnous one formtion, suh s the X N N N +/+ Figure 1 Trgeting of the opgl gene., Restrition mp of portion of the opgl gene n onstrution of the neomyin-resistne (neo r ) vetor. opgl exons re shown s lle oxes. The opgl proes use for Southern lotting n expete frgment sizes fter igestion of wil-type n mutnt genomi NA y rii re inite., rii; B, BmHI; X, XI; N, NotI; Xh, XhoI; S, SlI. Arrows inite neo r insert., Genomi NA ws isolte from opgl +/+, opgl +/- n opgl mie, igeste with rii n nlyse y Southern lotting. Wil-type (WT; 1.9 k) n mutnt (MT; 1.7 k) ns re inite., Asene of opgl mrna in opgl mie. Totl mrna (4 mg per lne) from thymi of 2-week-ol mie ws nlyse for opgl trnsripts using full-length opgl NA proe., Growth retrtion n, e, lk of tooth eruption in opgl mie. Four-week-ol opgl +/+ n opgl littermte mie re shown. +/+ skull, ppere riogrphilly norml (Fig. 2,, n t not shown). We qunti e one ensity in opgl mie n their heterozygous n wil-type littermtes y peripherl quntittive ompute tomogrphy (pqct) mesurement of tiil one. At 4 weeks of ge, treulr one ensity in the metphysis ( mg m 2 3 in opgl versus mg m 2 3È in opgl +/- n mg m 2 3È in opgl +/+ mie; P, :5) n totl one ensity in the iphysis ( mg m 2 3 in opgl versus mg m 2 3 in opgl +/- n mg m 2 3 in opgl +/+ littermte mie; P, :5) were signi ntly inrese in opgl mie s ompre with opgl +/- n opgl +/+ mie. espite the severe osteopetrosis, omprle serum levels of lium, phosphorus n lkline phosphtse (ALP) were oserve mong 4-week-ol opgl, opgl +/- n opgl +/+ littermte mie (t not shown). Histologil nlysis of long ones in opgl mie on rme the ourrene of severe osteopetrosis (Fig. 2i±l). The long ones were osteopetroti in pperne, with the epiphysis, metphysis n iphysis showing umultion of rtilge n one tht lmost lle the mrrow spes. The mi-iphysis one ensity ws similr to tht in the metphysis, initing tht little, if ny, resorption of one ws ourring. There ws no eviene of periostel one moelling jent to the growth pltes: these surfes ppere smooth, in ontrst to the eroe surfes t this site in the wil-type littermtes. Moreover, the olumnr struture of honroytes ws isorgnize t the epiphysil growth pltes (Fig. 2k, l). This is onsistent with the shortene femurs seen on riogrphs. Osteopetrosis ws lso evient in histologil setions of verterl oies (Fig. 2m, n). The lvri of opgl mie ws thinner thn in ontrols n the mrrow spes were reue (t not shown). Oler opgl mie (.6 weeks of ge) evelope roune fes, possily euse of osteopetroti hnges in the fil skeleton (t not shown). Stining of one setions for trtrte-resistnt i phosphtse (TRAP), n enzyme tht is highly expresse in immture n mture osteolsts, showe tht opgl mie ompletely lke TRAP-positive immture n mture multinulete osteolsts, wheres TRAP-positive ells were unnt in ones of wil-type mie (Fig. 2o, p). Moreover, in histologil setions there were no ells showing osteolst morphology in ny of the ones stuie. These results estlish the solute epenene of osteolst ifferentition on the expression of OPGL n show tht OPGL is n importnt ftor for osteolst evelopment in vivo. Norml hemtopoieti osteolst progenitors Filure of osteolst formtion n result either from impire funtion of essory ells (osteolst/stroml ells) or from n intrinsi efet in osteolst ifferentition 11,2±23. To istinguish etween these possiilities in opgl mie, we ssye osteolst ifferentition in vitro. TRAP-negtive hemtopoieti progenitors from the spleen or one mrrow n ifferentite into immture TRAP-positive osteolst preursors n mture, TRAP-positive, multinulete osteolsts 24,25. We ulture spleen ells from wiltype n opgl mie with CSF-1 (M-CSF) in the presene or sene of reominnt OPGL for 5 ys. Although mture TRAP-position ells were not oserve in ultures ontining CSF-1 lone, lrge multinulete TRAP-positive osteolsts were etete in ultures of oth wil-type n opgl spleen preursors in the presene of CSF-1 n OPGL (Fig. 3). Osteolst formtion from opgl spleen ells ws on rme y o-ulture of spleen ells with ST2 stroml ells in the presene of exmethsone, vitmin 3 n PGE 2 (t not shown). We ssesse the tivity of osteolsts erive from in vitro o-ultures y using the TRAP solution ssy. Both opgl +/+ n opgl osteolsts erive from spleen ells exhiite TRAP tivity tht epene on the onentrtion of reominnt OPGL (Fig. 3). In vitro-ifferentite osteolsts from oth opgl n opgl +/+ mie oul resor one when ulture 316 NATURE VOL JANUARY

3 +/+ i +/+ j k l m n o p e f g h Figure 2 Osteopetrosis n sene of osteolsts in opgl mie. X-ry nlysis of one ensity n one histology in 4-week ol opgl = (,, e, g, i, k, m, o) n opgl 2 = 2 (,, f, h, j, l, n, p) littermte mie. ±h, X-ry nlysis of one ensity.,, Totl skeleton sn.,, Fil regions. e, f, Femur n tii/ ulr long ones in hin legs. g, h, Lterl view of the vertere (rrow) n the ris (rrowhes). Note mssive inrese in overll one ensity, lk of inisor (rrowhes in, ) n molr (rrows in, ) tooth eruption, shortening of long ones n enlrgement of metphyses (rrows in e, f) inopgl mie. i±p, Histologil hnges in the one. i, j, The femur of opgl mie is shortene n lu-shpe. k, l, The morphology of the growth plte n the olumnr orgniztion of honroytes (sterix) re isture, n the shft of the femur is lle with rtilge n one (rrowhe). There is no eviene of periostel one moelling ourring next to the growth pltes. Hemotoxylin n eosin stining xis use. Originl mgni tions, 3. m, n, Osteopetrosis (lk rrowhe) of opgl verterl one. Note the hemtopoieti isln lolize long the verter of the opgl mouse (white rrowhe). Hemotoxylin n eosin stining. Originl mgni tions 1. o, p, Complete sene of TRAP + osteolsts (re ells in opgl +/+ mie) in the opgl mie. Extensive histologil nlysis of one showe tht ells with lssil osteolst morphology were ompletely sent in opgl mie (t not shown). TRAP stining. Originl mgni tion, 6. on ortil one slies (t not shown). Moreover, ytometri nlysis of expression of the OPGL reeptor on spleen ells using uoresein isothioynte (FITC)-lelle OPGL showe similr osteolst-preursor frequenies mong opgl, opgl +/- n opgl +/+ mie (t not shown). These t show tht opgl mie ontin hemtopoieti preursors in the spleen tht n ifferentite into funtionlly mture osteolsts in the presene of exogenous OPGL. In opgl mie, osteolstogenesis is loke t the TRAPnegtive preosteolst stge of evelopment. The filure of osteolst formtion in opgl mie in vivo n the ourrene of norml osteolstogenesis from preursor ells in the presene of reominnt OPGL in vitro inites the filure of n essory ell (osteolst/stroml ells) to present OPGL to osteolst progenitors. To etermine iretly whether osteolsts from opgl mie n support osteolstogenesis from hemtopoieti preursors, we estlishe primry osteolst ultures from lvrie of neworn opgl n opgl +/- mie. Bone-mrrow ells from norml mie were o-ulture with osteolsts in mei ontining exmethsone, vitmin 3 n PGE 2. Osteolsts from opgl +/- mie supporte ifferentition of TRAP-positive multinulete osteolsts uner these onitions, ut TRAP-positive osteolsts were not etete in o-ultures of norml one-mrrow ells n opgl osteolsts (Fig. 3, ). The efet in the ility of opgl osteolsts to support osteolstogenesis ws resue y the ition of reominnt OPGL (t not shown), on rming the solute requirement of OPGL for osteolstogenesis. These t inite tht the lk of osteolsts in opgl mie results from the inility of osteolst/ stroml ells to support osteolstogenesis, n not from n intrinsi lok in osteolst evelopment. Extrmeullry hemtopoiesis Severe osteopetrosis promotes splenomegly n extrmeullry hemtopoiesis in the spleen n liver 11,2,22,23. opgl mie showe only mil mroyti hyperhromous nemi n no signi nt ltertions in lymphoyte, pltelet, grnuloyte or monoyte numers in peripherl loo (t not shown). Histologil nlysis revele the presene of extrmeullry hemtopoiesis in the spleen n, to lesser extent, in the liver of opgl mie (t not shown). We etete the formtion of etopilly orgnize hemtopoieti tissue lolize t the outer surfes of verterl oies (Fig. 2n, white rrowhe). This extrmeullry tissue long the verterl oies exhiite morphologil n phenotypi fetures hrteristi of hemtopoiesis n proliferting preursor ells (t not shown). Whether these hemtopoieti islns in opgl mie represent efet in the homing of preursors uring the swith from hepti to one-mrrow hemtopoiesis, or n event seonry to osteopetrosis, whih interferes with the seeing of one-mrrow vities, remins to e etermine. NATURE VOL JANUARY

4 Impire thymoyte evelopment Unexpetely, nlysis of primry lymphoi orgns showe tht thymi ellulrity n thymus size were signi ntly erese in 4- week-ol opgl mie (Tles 1, 2). Two geneti hekpoints regulte thymoyte ifferentition n ellulrity 26,27. The rst hekpoint, t the C44 - C25 + stge of thymoyte evelopment, epens on the expression of the pre-t-ell ntigen reeptor (TCR) on C4 - C8 - thymoyte preursors n regultes expnsion of these preursor ells. The seon hekpoint regultes progression from C4 + C8 + immture to mture C4 + or C8 + thymoytes n orreltes with positive thymoyte seletion 26. The reltive numers of C4 + C8 + immture thymoyte n mture C4 + n C8 + T-ell popultions ppere norml in opgl mie (Fig. 4, top). C4 + C8 + immture thymoytes n mture C4 + n C8 + thymoytes showe norml surfe expression of the TCR±C3 omplex n norml expression of ifferentition mrkers, inluing C5, C69 n het-stle ntigen (HSA; t not shown), initing tht the progression of C4 + C8 + immture thymoytes to mture C4 + or C8 + T ells is norml in opgl mie. In ition, the mount of C3- n C95 (Fs)- meite poptosis of C4 + C8 + thymoytes ws omprle mong opgl +/+, opgl +/-, n opgl mie, initing tht reue thymi ellulrity in opgl mie my not e ue to enhne suseptiility to ntigen-reeptor-meite ell eth (t not shown). Surprisingly, ifferentition of C4/C8 oule-negtive C44 - C25 + preursors to C44 - C25 - thymoytes ws loke (Fig. 4, mile). This lok in ifferentition is proly the use of the reue thymi ellulrity in opgl mie. OPGL is expresse in C4 - C8 - thymoyte preursors n we etete sttere expression of the OPGL reeptor (RANK) in the thymus y in situ hyriiztion (t not shown). Moreover, immunohistohemil stining of thymuses showe tht ortil n meullry strutures n the istriution of mrophges, enriti ells, n epithelil ells were similr mong opgl n opgl +/- mie (t not shown). Tle 1 Growth retrtion n splenomegly in opgl mie Prmeter opgl +/+ opgl... Totl oy weight (g) 13:5 6 1:76 8:5 6 :71 Length (m) 7:57 6 :15 5:87 6 :55 Liver weight (%) 6:9 6 :72 6:34 6 :38 Spleen weight (%) :46 6 :6 1:19 6 :14 Kiney weight (%) 1:6 6 :6 1:83 6 :21 Thymus weight (%) :7 6 :12 :39 6 :1... Four-week-ol opgl +/+ (n ˆ 8) n opgl (n ˆ 5) littermte mie were use. Perentge orgn weight ws lulte s proportion of totl oy weight. Bol numers inite sttistilly signi nt ifferenes etween opgl +/+ n opgl mie (Stuent's t-test; P, :5). Vlues re given s the men 6 s:: To test whether the efet in ifferentition is intrinsi to onemrrow-erive ells or whether it results from efet in the thymi strom, we trnsferre opgl, opgl +/- n opgl +/+ fetl liver ells from emryoni-y 14.5 (E14.5) mie into irrite mie tht were e ient in the reominse-tivting gene protein (RAG). Although thymoyte evelopment ppere norml in opgl +/- rg1 n opgl +/+ rg1 himeri mie, opgl rg himeri mie exhiite lok in the progression of C25 + C44 - to C25 - C44 - thymoytes n reue thymi ellulrity (Fig. 4, ottom; Tle 3). This evelopment lok ws lso oserve in osteoprotegerin (OPG) trnsgeni mie n norml mie injete with reominnt OPG (.5 mg per kg oy weight for 4 ys suutneously; t not shown), on rming tht OPGL hs ritil role in erly thymi evelopment. OPG ts s eoy reeptor n n inhiit OPGL funtions 15,16. opgl mie reonstitute with norml one-mrrow ells showe norml T-ell evelopment (t not shown), initing tht the efet in erly thymoyte evelopment oes not resie in the thymi environment ut is intrinsi to one-mrrow-erive ells. These t show tht the TNF-fmily ytokine OPGL is new regultor of erly thymoyte evelopment t the stge of pre-tcr expression. +/+ CSF-1 CSF A 45 +/+ +/- +/+ CSF-1+OPGL CSF-1+OPGL 1 +/ OPGL (ng ml 1 ) 1.5 +/- +/- PGE2 A PGE2 +/- Figure 3 opgl mie show norml osteolst preursors ut n inility of osteolsts to support osteolstogenesis.,, opgl mie ontin norml spleni osteolst preursors. opgl +/+ n opgl splenoytes ( ) were ulture in the presene of 3 ng ml -1 CSF-1 n the inite onentrtions of reominnt murine OPGL. After 5 ys in ulture, osteolst ifferentition ws etermine using,, in situ stining for the phenotypi mrker TRAP (re), or, the TRAP solution ssy initive of funtionl osteolsts. One result representtive of three experiments is shown.,, opgl osteolsts o not support osteolstogenesis in vitro. Wil-type non-herent mouse one-mrrow ells ( ) were ulture with osteolst ell lines (erive from the lvrie of neworn opgl +/- or opgl littermte mie) in the presene of exmethsone, vitmin 3, n PGE 2 (see Methos)., TRAP + osteolsts re present in ultures ontining opgl +/- osteolsts ut not in ultures ontining opgl osteolsts., TRAP solution ssy to etet osteolsts. No TRAP tivity is seen in ultures ontining opgl osteolsts. One result representtive of three experiments is shown. 318 NATURE VOL JANUARY

5 Tle 2 Hemtopoiesis in opgl ±/± mie Cell suset opgl +/+ opgl opgl = 2. rg1 2 = 2 opgl 2 = 2. rg1 2 = 2 Thymoytes ( 1 6 ) 25:8 6 22:63 69:4 6 23:83 97:6 6 2:19 43:3 6 9:94 C4 + C8 + thymoytes (%) 76:1 6 2:86 72:9 6 3:17 81:4 6 2:34 76:2 6 8:57 C4 + C8 - thymoytes (%) 15:2 6 1:87 13:4 6 2:4 1:7 6 1: 1:3 6 1:84 C4 - C8 + thymoytes (%) 4:7 6 :69 4:4 6 :75 3:7 6 :62 2:6 6 1:6 Splenoytes ( 1 6 ) 1:8 6 12:12 75: 6 3:83 179: 6 4:9 122:5 6 4:49 C4 + T ells (%) 16:8 6 2:62 35:6 6 2:88 19:3 6 4:11 19:5 6 3:37 C8 + T ells (%) 8:5 6 2:62 17:6 6 2:29 7: 6 1:34 8:8 6 1:64 B22 + IgM + ells (%) 44:9 6 5:24 22:2 6 3:1 N N IgM + Ig + ells (%) 22: 6 3:3 9:9 6 1:1 38:6 6 6:52 23:2 6 1:82 C11 + monoytes (%) 8:5 6 :85 1:6 6 2:4 N N Cells from thymi n spleens from opgl +/+ (n ˆ 6) n opgl (n ˆ 6) littermte mie n rg1 mie reonstitute with opgl +/- (n ˆ 3) E14.5 fetl liver ells were stine with ntioies ginst the inite moleules n popultions were etermine using FACSn. Totl ell numers ( 1 6 ) n perentges of supopultions re inite. Results were omprle etween opgl +/+ n opgl +/- mie n opgl +/+ rg1 n opgl +/- rg1 himers (t not shown). Bol numers inite sttistilly signi nt ifferenes etween opgl = n opgl 2 = 2 mie (Stuent's t-test; P, :5. Vlues re given s mens 6 s:e:m: N, not etermine. Impire B-ell evelopment Spleens of opgl mie were roughly two or three times lrger in size (spleen weight:oy weight rtio) thn spleens from ontrol littermtes (Tle 1), lthough totl ellulrity of the spleen ws omprle mong opgl, opgl +/- n opgl +/+ mie (Tle 2). This pprent isrepny in spleni size n ellulrity is ue to reue oy weight n spleni hemtopoiesis in opgl mie. opgl spleens ontine norml numers of C11 + F4/8 + mrophges n Gr-1 + grnuloytes, n inrese perentges of C4 + n C8 + T ells (Fig. 4 n Tle 2; n t not shown). opgl spleni T ells expresse wil-type levels of C4, C8, C3-e, TCR-, C28 n C45, ut i not express the tivtion mrkers C25 n C69 (t not shown), initing tht these T ells were in the resting stte. In ontrst to the numer of mture T ells, the totl n reltive numers of surfe(s) IgM + sig + or B22 + sigm + B ells were signi ntly reue (Tle 2). sigm + sig + B ells from opgl spleen expresse omprle levels of mjor histoomptiility omplex (MHC) lss II n C23 moleules on the ell surfe (t not shown). The reue ellulrity of spleni B ells oul either e the iret result of the opgl-null muttion, or oul inste e the result of n ltere miroenvironment or of hnges in the omposition of stroml ells outsie the one-mrrow vity tht ffet B-ell ifferentition. Reue numers of sigm + sig + (Fig. 4, ottom) B22 + n C19 + (t not shown) spleni B ells were lso oserve in rg1 mie reonstitute with opgl E14.5 fetl liver ells. Remrkly, opgl rg1 himeri mie h norml numers of B22 + C43 + n B22 + C25 - pro-b ells ut signi ntly reue numers of B22 + C43 -, B22 + C25 +, n B22 + sigm + B-ell preursors in the one mrrow (Tle 3) n rmti lok in the progression of B22 + C25 - pro-b ells to B22 + C25 + pre-b ells (Fig. 4). opgl mie reonstitute with norml one-mrrow ells exhiite norml B-ell evelopment n homing of B ells into the spleen of opgl mie (t not shown). Thus OPGL is importnt in the evelopment of B-ell preursors. enriti ells n T-ell tivtion Intertions etween OPGL n RANK my regulte enriti-ell survivl n the upregultion of o-stimultory moleules on enriti ells 17,19. Monoyte/grnuloyte progenitors give rise to enriti ells when ulture with pproprite ytokines suh s grnuloyte/mrophge olony-stimulting ftor (GM-CSF) n IL-4 (ref. 28). Moreover, monoytes/mrophges re losely relte to osteolsts, n monoyte ell lines n ifferentite into osteolsts if ulture with CSF-1 n OPGL 15,16. Thus, it is possile tht mrophges, enriti ells n osteolsts shre ommon ommitte preursor. As OPGL n t s enriti-ell survivl ftor y enhning Bl-X L expression in in vitro ellulture systems 17,19, we speulte tht OPGL±RANK intertions might in uene enriti-ell evelopment. However, we foun, using uoresene-tivte ell sorting (FACS) nlysis, tht the numers of spleni C11 + enriti ells were similr in opgl +/- n opgl littermtes (Fig. 4, mile). C11 + ells from opgl +/+, opgl +/- n opgl mie expresse omprle levels of MHC lss II moleules, C86, C8, C11, interellulr hesion moleule (ICAM)-1 n C4 (t not shown). Numers n istriutions of enriti ells n mrophges in opgl mie were lso similr to those in opgl +/- littermtes, s etermine y immunohistohemil nlyses of spleen, skin, n thymus setions with pnel of ifferent ntioies spei for enriti ells n mrophges (t not shown). To etermine whether the enriti ells funtion normlly, we stimulte norml llogenei T ells from BALB/ mie with puri e spleni C11 + enriti ells. C11 + enriti ells from oth opgl +/- n opgl mie were le to inue prolifertion (t not shown) n ytokine proution (Fig. 4) in llogenei T ells to similr extent, initing tht opgl C11 + enriti ells re funtionlly ompetent. These t show tht OPGL expression is not essentil for enriti-ell n mrophge evelopment. To stuy further the role of OPGL in T-ell tivtion y enriti ells, we ulture puri e T ells from opgl n opgl +/- littermte mie with llogenei BALB/ spleni C11 + enriti ells isolte Tle 3 Erly T- n B-ell supopultions in opgl ±/± > rg1 ±/± mie Cell suset opgl =. rg1 2 = 2 opgl = 2. rg1 2 = 2 opgl 2 = 2. rg1 2 = 2 Thymus N thymoytes (%) 4:24 6 1:33 3:96 6 1:22 7:42 6 2:21 C25 + C44 - (%) 3:61 6 3:74 32:22 6 4:64 54:77 6 3:6 C25 - C44 - (%) 54:67 6 4:11 52:3 6 3:48 34:43 6 6:41 Bone mrrow B22 + C43 + (%) 39:7 6 :78 4:87 6 1:27 3:85 6 :32 B22 + C43 - (%) 29:24 6 1:67 28:87 6 4:22 7:86 6 2:47 B22 + C25 + (%) 24:94 6 1:19 24:86 6 4:77 3:11 6 :1 B22 + C25 - (%) 8:11 6 1:21 8:67 6 :95 8:4 6 2:32 B22 + sigm - (%) 23:66 6 1:9 22:54 6 2:79 8:3 6 2:13 B22 + sigm + (%) 1:58 6 1:69 1:94 6 1:73 2:3 6 :3 Erly T- n B-ell supopultions in rg1 mie reonstitute with E14.5 opgl fetl liver ells. Numers inite men perentges n ˆ 3 6 s:e:m: of positive ells per totl ells. Only onor ells were inlue in the nlysis using Ly9.1 ntioy (see Methos). Bol numers inite sttistilly signi nt ifferenes etween opgl 2 = 2. rg1 2 = 2 n opgl =. rg1 2 = 2 mie. NATURE VOL JANUARY

6 y overnight ulture 29, or with one-mrrow-erive C11 + enriti ells ulture in the presene of GM-CSF n IL-4 for 6 ys 3. opgl T ells showe reue proution of the ytokines IL-2 n interferon (IFN)-g ompre with opgl +/- (Fig. 4e) n opgl +/+ (t not shown) T ells; the mount of ytokine proution epene on the numer of llogenei enriti ells use. In ontrst, opgl, opgl +/- n opgl +/+ T ells proliferte similrly following tivtion with spleni or one-mrrow-erive enriti ells (t not shown). Thus, lthough opgl enriti ells n inue ytokine proution in norml llogenei T ells, stimultion of opgl T ells y wil-type enriti ells is impire. To etermine whether opgl T ells hve inherent efets in ytokine proution, we stimulte highly puri e T ells from opgl +/- n opgl littermte mie with nti-c3 n nti-c28 rosslinking ntioies; this vois the ourrene of effets ttriutle to ntigen-presenting ells. Proution of the ytokines IL-2 (Fig. 4f), IFN-g, IL-4, IL-5 n IL-6 (t not shown) ws signi ntly reue in puri e opgl T ells. IL-2 proution ws lso impire in T ells puri e from opgl rg1 himeri mie s ompre with T ells from opgl +/- rg1 himers (Fig. 4g). T H 1 n T H 2 helper ells evelope in the sene of OPGL expression, ut proution of oth T H 1 n T H 2 ytokines ws signi ntly reue in opgl T ells (t not shown). These t inite tht OPGL hs no pprent role in the T H 1/T H 2 ihotomy ut is require for optiml ytokine proution following ntigen-reeptor tivtion. To test whether efetive ytokine proution ws ue to iret effet of OPGL on T ells or ws seonry to ltere thymoyte mturtion, we nlyse ytokine proution in the presene of reominnt OPGL n the reominnt eoy reeptor OPG. +/ / /- +/ C25 C25 C C8 +/ C C11 C C8 +/- > rg1 > rg1 +/- > rg1 > rg1 sigm +/ C B22 B22 B22 +/- > rg1 +/- > rg > rg1 > rg C44 sig C25 sigm IL-2 (pg ml 1 ) ,25 1, IFN-γ (pg ml 1 ) e IL-2 (pg ml 1 ) IFN-γ (pg ml 1 ) f IL-2 (pg ml 1 ) 6, 4, 2, +/- +/- + OPGL + OPGL g IL-2 (pg ml 1 ) 6, 4, 2, +/+>rg1 +/->rg1 >rg1 >rg1 + OPGL Numer of Cs Numer of Cs Anti-C3ε (µg ml 1 ) 1 Anti-C3ε (µg ml 1 ) Figure 4 Impire lymphoyte evelopment in opgl mie., Flow-ytometri nlysis of thymoytes from 4-week-ol opgl +/- n opgl mie n from rg1 mie reonstitute with opgl +/- ( = 2.rg1 2 = 2 )oropgl ( 2 = 2.rg1 2 = 2 ) fetl liver ells 8 weeks fter fetl liver-ell trnsfer. Thymoytes were stine for C4 n C8 expression (top) or for expression of C44 n C25 (mile n ottom) on gte C4 - C8 - C3 - B22 - C11 - TCRg - Gr-1 - (linege-negtive) thymoyte preursors. Popultions in himeri mie were nlyse using nti- Ly9.1-ntioy stining to exlue potentil ontriutions of rg1 ells in the nlysis. Numers represent the perentges of eh popultion in eh qurnt., Totl splenoytes from ove mie were stine for C4 n C8 (top) n slg n slgm (ottom). Spleni enriti ells prepre y overnight ulture were stine for C11 n C8 (mile). Numers represent the perentges of eh popultion in eh qurnt., Flow-ytometri nlysis of one-mrrow ells from 6-week-ol opgl +/- mie n from rg1 mie reonstitute with opgl +/- ( = 2.rg1 2 = 2 )oropgl ( 2 = 2.rg1 2 = 2 ) fetl liver ells 8 weeks fter ell trnsfer. Bone-mrrow ells were stine for B22 n C25 expression (left pnels) or for expression of B22 n sigm (right pnels). Popultions in himeri mie were nlyse using nti-ly9.1-ntioy stining to exlue potentil ontriutions of rg ells. ±f, Cytokine proution., Wil-type llogenei BALB/ T ells (H±2 / ) were tivte for 4 ys with ifferent numers (x-xis) of spleni C11 + C8 + enriti ells (Cs) (H±2 / ) isolte from opgl +/- (open rs) or opgl ( lle rs) littermtes. e, T ells (H±2 / ) from opgl +/- (open rs) n opgl ( lle rs) littermtes were tivte for 4 ys with ifferent numers (x-xis) of spleni C11 + C8 + Cs from wil-type llogenei (H±2 / ) BALB/ mie. f, Puri e T ells (purity.98%) from 4-week ol opgl +/- n opgl littermtes, n g, puri e T ells (purity.98%) from opgl +/+ rg1 ( =. rg1 2 = 2 ), opgl +/- rg1 ( = 2.rg1 2 = 2 ) n opgl rg1 ( 2 = 2.rg1 2 = 2 ) mie were tivte for 48 h with ifferent onentrtions of plte-oun nti-c3e ntioy (inite on the x-xis) plus solule nti-c28 ntioy (1 ng ml -1 ) in the sene or presene of reominnt OPGL (1 mgml -1 ). Cytokines were qunti e y ELISA. Men vlues of triplite ultures 6 stnr evitions re shown. 32 NATURE VOL JANUARY

7 Aition of OPG t ifferent onentrtions i not impir the kinetis n extent of prolifertion n IL-2 n INF-g proution in norml T ells (t not shown). Reominnt OPGL t onentrtions of.1±1 mgml -1 i not restore ytokine proution y opgl T ells to norml levels n i not enhne ytokine proution y norml T ells (Fig. 4f, g; n t not shown). OPGL-riven osteolstogenesis ours lrey t 1 ng ml -1 OPGL 15,16. Moreover, wheres the TNFR-fmily moleule 41BB n funtion s o-stimultory moleule in 28 T ells 31,32, ition of exogenous OPGL i not inue IL-2 proution in the sene of the o-stimultory reeptor C28 (t not shown). Expression of the OPGL reeptor RANK ws omprle mong C3-tivte opgl +/+, opgl +/-, opgl n 28 T ells (t not shown). These results inite tht impire ytokine proution y opgl T ells is ue to evelopmentl efet of these T ells n not to iret effet of OPGL on mture T ells. However, our results o not exlue the possiility tht, like C4L±C4 intertions, OPGL expresse y T ells hs role in T-ell± enriti-ell ommunition. Our t inite tht muttions ffeting thymoyte evelopment n hve profoun effet on the funtion of mture T ells. e g Figure 5 OPGL regultes lymph-noe orgnogenesis.,, Asene of mesenteri lymph noes in opgl mie (); mrosopi view of mesenteri lymph noes from 3-week ol opgl +/+ littermte mouse is shown s ontrol (). Lymph noes re inite y the rrowhes in. Arrowhes in inite mesenteri loo vessels. ±h, Presene of Peyer's pthes in opgl +/+ (, e, g) n opgl (, f, h) mie.,, Hemotoxylin n eosin stining to visulize morphology of Peyer's pthes. e, f, Anti-C3e immunostining to visulize T ells. g, h, Anti-B22 immunostining to visulize B ells in Peyer's pthes. Smll intestines were ollete from 4-week-ol opgl +/+ n opgl littermtes, setione n nlyse for the presene of Peyer's pthes. Originl mgni tions, 1. f h Complete lk of lymph noes Most surprisingly, ntomil nlysis of seonry lymphoi orgns revele tht opgl mie h efet in lymph-noe orgnogenesis n ompletely lke mesenteri (Fig. 5, ), ervil, mniulr, inguinl, xillry, pr-orti n poplitel (t not shown) lymph noes. Extensive seril histologil setioning through the ove regions on rme the omplete sene of lymph noes n the sene of ny tissues resemling erly lymph-noe nlgen. Trnsfer of opgl fetl liver ells into rg1 mie showe tht opgl T n B ells n populte lymph noes (t not shown), initing tht the lk of lymph noes is not ue to efet in ellulr homing. Trnsfer of norml one-mrrow ells into neworn opgl mie i not restore lymph-noe formtion (t not shown). It hs een reporte tht mutnt mie lking lymph noes hve extr efets in the formtion of intestinl Peyer's pthes n show isorgnize spleni rhiteture, initing tht these efets my e genetilly or funtionlly linke 33±38. However, espite the omplete e ieny of ll lymph noes, Peyer's pthes, lthough reue in size, were reily oserve in opgl mie (Fig. 5, ). Peyer's pthes from opgl mie lso ontine e ne T-ell (Fig. 5e, f) n B-ell (Fig. 5g, h) res. opgl mie lso showe intt spleni rhiteture, inluing norml istriution of re n white pulp, norml T- n B-ell segregtion, n norml primry follile struture, inluing T- n B-ell res, mrginl zones n folliulr n retiulr enriti-ell networks (t not shown). These t show tht the sene of OPGL hs no pprent effet on spleni rhiteture or formtion of Peyer's pthes, ut tht OPGL expression is essentil for lymph-noe formtion. Conlusions Our results show tht OPGL is n importnt ftor for osteolst mturtion in vivo. Surprisingly, the sme moleule tht regultes osteolstogenesis is lso key ftor in erly ifferentition of thymoytes n B-ell preursors. Moreover, opgl mie ompletely lk lymph noes. OPGL is n essentil osteolst ifferentition ftor. In in vitro ulture systems, OPGL n oth tivte mture osteolsts n meite osteolstogenesis in the presene of CSF-1 (refs 15, 16). opgl mie exhiit severe osteopetrosis, stunte growth n efet in tooth eruption, n opgl osteolsts nnot support osteolstogenesis. However, these mie ontin hemtopoieti preursors tht n ifferentite into phenotypilly n funtionlly mture osteolsts in vitro in the presene of reominnt OPGL n CSF-1. As osteolst ell lines erive from opgl mie o not support osteolst formtion, the efet in osteolstogenesis oserve in opgl mie must e ue to n intrinsi efet in osteolsti strom. Wheres sf-1 mutnt osteopetroti mie show evelopmentl rrest in oth monoyte/mrophge n osteolst lineges, opgl mie hve norml ifferentition of monoytes/mrophges n enriti ells. Thus, OPGL is spei n essentil ifferentition ftor for osteolst preursors. RANK hs een ienti e s the reeptor for OPGL 17. Our results inite tht intertion etween OPGL expresse y stroml ells/osteolsts n its reeptor, RANK, expresse on osteolst preursors is essentil for osteolstogenesis. It remins to e etermine whether RANK is the only reeptor for OPGL in vivo. Our results estlish the solute epeneny of osteolst ifferentition on the expression of OPGL. Lymph-noe orgnogenesis. Stuies of mie e ient in lymphotoxin- 33, lymphotoxin- 34,35, TNFR1 (TNFRp55) 36, TNF- 37 or the lymphotoxin- reeptor 38,39 hve revele importnt roles of eh of these moleules in the evelopment n orgniztion of seonry lymphoi tissues. Mie with isrupte lymphotoxin-, lymphotoxin-, or lymphotoxin--reeptor gene lk lymph noes, Peyer's pthes, n folliulr enriti ells, n show ltere spleni rhiteture 33±35,39. Ativtion of TNFR1 y TNF- is neessry for the formtion of spleni B-lymphoyte folliles, folliulr enriti NATURE VOL JANUARY

8 networks, n the germinl entre 36,37. Surprisingly, opgl mie lk ll lymph noes ut exhiit intt spleni rhiteture n evelop Peyer's pthes, initing tht OPGL my hve spei n essentil role in lymph-noe orgnogenesis. The role of OPGL ppers to e istint from those of the lymphotoxin--reeptor n TNFR1 in regulting morphogenesis of lymphoi orgns. Interestingly, TNFR1-e ient mie exhiit retine, ut smll, Peyer's pthes 36,37, initing tht oth TNFR1 n OPGL might hve role, leit n inessentil one, in the formtion of Peyer's pthes n might ooperte in this proess. The onerte tivity of severl ell lineges, inluing rolsts, mrophges, retiulr ells n enothelil ells, is require for the formtion of primoril lymph noes. The primoril lymph noes re susequently seee y lymphoytes to form mture ompt noes 4. In situ hyriiztion of norml lymph noes hs shown tht OPGL- n RANK-expressing ells re present in lymph noes, lote minly in the ortil res next to supsulr sinuses (t not shown). However, s RANK n OPGL re lso expresse in the spleen n Peyer's pthes (t not shown), restrite OPGL±RANK expression nnot ount for the seletive lk of lymph noes. We hve exlue the possiility tht efet homing of opgl lymphoytes might e the use of efetive lymph-noe formtion, n norml one-mrrow ells nnot resue the lymph-noe efet in opgl mie in himeri trnsfer experiments. Thus, OPGL proly ts s growth n/or survivl ftor on lymph-noe-orgnizing ell uring emryoni evelopment. The ellulr n moleulr mehnisms of lymphnoe morphogenesis n the link etween lymph-noe evelopment, Peyer's pth formtion n spleni rhiteture re not unerstoo, ut OPGL isruption is the rst muttion tht seprtes the evelopment of lymph noes from tht of Peyers's pthes. Lymphoyte evelopment. An unexpete ning ws tht OPGL expression is require for T- n B-ell mturtion. Our results show tht OPGL is importnt for the progression of C25 + C44 - preursors to C25 - C44 - thymoytes t the stge of pre-tcr expression. Moreover, OPGL hs role in the mturtion of B22 + C43 + C25 - pro-b ells to B22 + C43 - C25 + pre-b ells in the one mrrow, initing tht the OPGL is new regultor of erly B-lymphoyte evelopment. This efet in erly thymoyte n B-ell evelopment is intrinsi to one-mrrow-erive ells ut oes not resie in the mesenhyml or epithelil miroenvironment. Severl muttions tht rrest thymoyte evelopment t the stge of pre-tcr expression, n B-ell evelopment t the stge of pro-b ells, hve een reporte 41±45. These muttions either ffet expression of the pre-tcr n pre-b-ell ntigen-reeptor omplexes iretly or ffet signlling moleules thought to e ownstrem of these erly ntigen reeptors. In ition, muttions tht regulte ell survivl, suh s muttions of the IL-7 reeptor 46,47, n ffet T- n B-ell evelopment. It is not known whether OPGL ts s survivl ftor require for erly thymoyte n B-ell evelopment or whether it ffets lymphoyte mturtion iretly. T ells n osteolsts. One interesting implition of our stuy is tht OPGL serete from tivte T ells my iretly moulte osteolstogenesis n the tivity of mture osteolsts. As mutnt mie tht lk T ells still hve norml one vities n tooth eruption (t not shown) 48, T ells re proly not require for norml one homeostsis. However, lol in mmtion within the one, s result of metstsis, infetions n frtures, or joint in mmtion in rthritis ttrts T ells whih oul then tively prtiipte in one remoelling though proution of OPGL. The role of OPGL-prouing T ells in one resorption remins to e eluite. Inhiition of OPGL funtion might llow the tretment T-ell-meite rthritis, onition tht les ultimtely to one estrution n rippling. Our results show tht lymph-noe orgnogenesis, T- n B-lymphoyte evelopment n osteolst ifferentition re ll regulte y the TNF-fmily ytokine OPGL. M... Methos Genertion of opgl mie. Murine opgl genomi NA frgments were otine from 129/SVJ mouse genomi lirry y sreening with riolelle NA frgment orresponing to nuleoties 39±7 of the murine opgl omplementry NA (GenBnk ession numer AF53713). We onstrute trgeting vetor tht ontine 785-se-pir short rm n 5.5-kilose (k) long rm of homology nking pgk-neo ssette. The trgeting vetor ws eletroporte into E14K ES ells, whih were selete in G418 (2 mgml -1 ). Southern lot nlysis using genomi NA prepre from polymerse hin retion (PCR)-positive ES-ell olonies ienti e seven reominnt ES lones with single trgete llele. Trgete ells were injete into fertilize lstoysts from C57Bl/6 femle mie. Chimeri mle mie were rosse with C57Bl/6 femles for germline trnsmission. Following heterozygous mtings, homozygotes were ienti e n istinguishe from heterozygous n wil-type mie y Southern lot nlysis of rii-igeste genomi NA hyriize to nking proe. To on rm the sene of opgl expression, we extrte totl RNA from thymuses of 2-week-ol mie n use this RNA for northern lotting. opgl messenger RNA n -tin (ontrol) mrna were etete using full-length opgl NA n -tin NA proes. C57Bl/6 n BALB/ mie were purhse from Tonis. All opgl mie use were on 129/C57Bl/6 kgroun (expressing MHC moleule H±2 / ). All mie were mintine in miroisoltor ges t the niml filities of the Ontrio Cner Institute uner spei pthogen-free onitions. Histology n one-ensity mesurements. Groups of opgl +/+, opgl +/- n opgl mie were neropsie on y 21 or 28 fter irth. Riogrphy ws performe on Fxitron X-ry system (moel 43855A, Fxitron X-ry Corp.). Peripherl loo ws nlyse for linil hemistry n hemtology. Totl oy n mjor orgns were weighe n ll tissues were xe in 1% formlin. Prts of selete orgns were frozen for immunohistology. Bone tissue ws eli e using formi i solution n emee in prf n. The expression of TRAP tivity ws etermine using metho of enzyme histohemistry tht spei lly stins osteolsts re 49. Two.5-mm rosssetions of one tken t 1.5 n 2. mm from the proximl en of the tii were nlyse to etermine totl n treulr one minerl ensity in the metphysis (XMICE 5.2, Strte). One.5-mm ross-setion of one tken 4. mm from the proximl en of the tii ws nlyse to etermine the ortil one ensity in the tiil iphysis. In the metphysel mesurement, totl one ensity n treulr one ensity (e ne s the innermost 2% of the one ross-setion) were etermine. In vitro osteolst formtion. Spleen ells were ulture overnight in MEM meium ontining 1% het-intivte fetl ovine serum supplemente with murine CSF-1 (3 ng ml -1 ). Following this inution, non-herent ells were ollete n sujete to grient puri tion puri e nonherent ells were ulture with vrious onentrtions (.16±5 ng ml -1 )of murine OPGL (158±316) in the presene of murine CSF-1 (3 ng ml -1 ). To evlute the ility of osteolsts from opgl +/- n opgl mie to support osteolstogenesis, we isolte osteolsts from lvrie of 3-y-ol mie using sequentil ollgense/protese igestion proeure s esrie 5. Osteolsts ( per ml) were o-ulture with non-herent mouse onemrrow ells ( per ml) from C57Bl/6 mie in MEM supplemente with vitmin 3 (1 nm), exmethsone (1 nm), n vrious onentrtions (.8±25 nm) of PGE 2 in the presene or sene of murine OPGL (1 ng ml -1 ) for 7 ys. Reominnt murine OPGL ws prepre s esrie 15. Osteolst ifferentition ws evlute y TRAP solution ssy, ytohemil stining n one resorption on one slies s esrie 49. Cytometry n immunohistohemistry. Single-ell suspensions of thymi n spleens were stine with FITC-, phyoerythrin- or iotin-onjugte ntioies (Phrmingen) retive to C3-e, TCR-, C4, C8, C25, C44, C5, C28, C45, HSA, C69, C11 (M-1), ICAM-1, C11, C8, C86, C4, MHC lss II (I±A ), B22, C43, sigm, sig, C19 or Gr-1. For the nlysis of thymoyte preursors, single-ell suspensions were stine with phyoerythrin-onjugte nti-c4, nti-c8, nti-c3e, nti- B22, nti-c11, nti-gr1 n nti-tcrg, FITC-onjugte nti-c25, n iotin-onjugte nti-c44 ntioies. Phyoerythrin-negtive ells were nlyse for their expression of C25 n C44. Biotinylte ntioies were visulize using streptviin±re67 (Life Tehnologies). All smples 322 NATURE VOL JANUARY

9 were nlyse y ow ytometry using FACSn (Beton ikinson). For immunoytohemil stining, frozen or eprf nize setions were preinute in 3% H 2 O 2 solution to lok enogenous peroxise tivity. After loking of nonspei ntioy-ining sites, setions were wshe n susequently inute with primry ntioies in PBS stining uffer (ph 7.4) for 3 min t room temperture. The following primry ntioies were use: nti-c3e (ko) n nti-b22 (PhrMingen). Biotinylte got ntirt (BioGenex), nti-rit (Vetor) n nti-hmster (Vetor) immunogloulins were use s seonry regents. Biotinylte ntioies were visulize using ABC regent (Vetor) n AB (iminoenziine tetrhyrohlorie) enzyme sustrtes (Reserh Genetis). Setions were ounterstine with hemtoxylin. In ll experiments, non-immune speies-mthe immunogloulins were use s negtive ontrols for eh primry ntioy. Lymphoyte prolifertion ssys n puri tion of enriti ells. Spleni enriti ells were prepre y overnight ulture of freshly isolte spleen ells from syngenei C57Bl/6 n llogenei BALB/ mie s esrie 29. Bone-mrrow-erive enriti ells were generte using GM-CSF (Enogen) n IL-4 (Genzyme) s esrie 3 n were use on y 6 of ulture. C11 + enriti ells were puri e using ell sorting. The purity of sorte enriti ells ws 95% C11 +. Spleni T ells from opgl +/+, opgl +/- n opgl mie were puri e (.98% C3 + T ells) using mgneti es. Puri e T ells ( ells per well) were o-ulture with serilly ilute n irrite (2 Gy) enriti ells in 96-well roun-ottome pltes for 4 ys n pulse for 8 h with 1 mci 3 H-thymiine per well. To test whether opgl enriti ells funtione normlly, we ulture puri e llogenei T ells ( per well) from BALB/ mie (H±2 / ) with puri e spleni C11 + enriti ells from opgl +/- n opgl mie (H±2 / ). For ntioy-meite T-ell-tivtion ssys, we ple puri e T ells ( ells per well) into 96-well rounottome pltes n tivte them with immoilize nti-c3e (lone 145-2C11; PhrMingen) n solule nti-c28 (lone 37.51; PhrMingen) ntioies in the sene or presene of murine reominnt OPG (.1, 1, n 1 mgml -1 ) n murine reominnt OPGL (.1, 1, n 1 mgml -1 ) 15,16.To inue T H 1 n T H 2 ells, we ulture splenoytes ( per ml) with immoilize nti-c3e ntioy (1 mgml -1 ) in the presene of IL-12 or IL- 4, respetively, for 7 ys, n restimulte them with nti-c3e ntioy (1 mgml -1 ) for 24 h. Culture superntnts were ollete t 24 n 48 h n ytokine proution ws etermine y enzyme-linke immunosorent ssy (ELISA; Genzyme). Fetl liver-ell n one-mrrow himers. For genertion of himeri mie, livers were ollete from E14.5 opgl +/+, opgl +/- n opgl Ly9.1 + emryos. Single-ell suspensions of totl liver ells ( ells per :2 ml) were injete into the til vein of irrite (3 Gy) Ly week-ol rg1 host mie. To onstrut one-mrrow himers, we isolte one-mrrow ells from 8-week-ol Ly9.2 + opgl +/+ (H±2 / ) mie, n trnsferre one-mrrow ells intrperitonelly into neontl (3 ys fter irth), irrite (9 Gy) Ly9.1 + opgl +/+ n Ly9.1 + opgl hosts. Rition himers were mintine uner pthogen-free onitions. The evelopment of T ells n B ells from fetl liver preursors or one-mrrow ells ws etermine t 6±8 weeks fter trnsfer using linege-spei ntioies. Chimerism of hemtopoieti ells ws etermine using n ntioy retive to the surfe ntigen Ly9.1 (PhrMingen). In ll himers nlyse,.95% of hemtopoeiti ells were erive from onor ells (t not shown). Reeive 1 Otoer; epte 4 eemer Felix, R., Hofstetter, W. & Cehini, M. G. Reent evelopments in the unerstning of the pthophysiology of osteopetrosis. Eur. J. Enorinol. 134, 143±156 (1996). 2. Roomn, G.. Avnes in one iology: the osteolst. Enor. Hev. 17, 38±332 (1996). 3. Mnolgs, S. C. & Jilk, R. L. Bone mrrow, ytokines, n one remoeling. 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Simonet, W. S. et l. Osteoprotegerin: novel serete protein involve in the regultion of one ensity. Cell 89, 39±319 (1997). 5. Tkhshi, N. et l. e ieny of osteolsts in osteopetroti mie is ue to efet in the lol miroenvironment provie y osteolsti ells. Enorinology 128, 1792±1796 (1991). Aknowlegements. We thnk. Bouhr for ell sorting;. urye n C. Burgh for tehnil ssistne; R. Boy, M. Bhmnn, G. Wik, n N. Romni for regents; M. Suners for eiting this mnusript; n R. Yoshi, K. Bhmier, A. Hkem, I. Kozierzki, T. Sski n M. Nghiem for omments. Corresponene n requests for mterils shoul e resse to J.M.P. (e-mil: jpenning@mgen.om). NATURE VOL JANUARY