BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1

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1 BTLA is lymphoyte inhiitory reeptor with similrities to CTLA-4 nd PD-1 Norihiko Wtne 1,5, My Gvrieli 1, John R Sedy 1, Jinfei Yng 1,5, Frnes Fllrino 2, Susn K Loftin 1, Mihelle A Hurhl 1, Ntlie Zimmermn 3, Juli Sim 3, Xingxing Zng 4, Theres L Murphy 1, John H Russell 3, Jmes P Allison 4 & Kenneth M Murphy 1 During tivtion, T ells express reeptors for reeiving positive nd negtive ostimultory signls. Here we identify the B nd T lymphoyte ttenutor (BTLA), n immunogloulin domin ontining glyoprotein with two immunoreeptor tyrosine-sed inhiitory motifs. BTLA is not expressed y nive T ells, ut it is indued during tivtion nd remins expressed on T helper type 1 (T H 1) ut not T H 2 ells. Crosslinking BTLA with ntigen reeptors indues its tyrosine phosphoryltion nd ssoition with the Sr homology domin 2 (SH2)-ontining protein tyrosine phosphtses SHP-1 nd SHP-2, nd ttenutes prodution of interleukin 2 (IL-2). BTLA-defiient T ells show inresed prolifertion, nd BTLA-defiient mie hve inresed speifi ntiody responses nd enhned sensitivity to experimentl utoimmune enephlomyelitis. B7x, peripherl homolog of B7, is lignd of BTLA. Thus, BTLA is third inhiitory reeptor on T lymphoytes with similrities to ytotoxi T lymphoyte ssoited ntigen 4 (CTLA-4) nd progrmmed deth 1 (PD-1). T ells reeive ostimultory signls from ntigen-presenting ells (APCs) tht n e positive nd negtive. During the initition of nive T ells, the primry positive ostimultory signl is medited through the reeptor CD28, wheres signling through CTLA-4 hs negtive effet on tivtion 1. Both CD28 nd CTLA-4 intert with the sme set of lignds, B7-1 (lso known s CD80) nd B7-2 (CD86), expressed on APCs. Additionl memers of the CD28-B7 fmily hve een identified. A seond tivting reeptor on T ells is induile ostimultor (ICOS) 2, nd its lignd B7h 3 (lso known s B7RP-1 (ref. 4), GL50 (ref. 5), B7H2 (ref. 6) nd LICOS 7 ) is expressed on oth lymphoid nd nonlymphoid ells 8,9. A seond inhiitory reeptor, PD-1 (refs. 10,11), inds to pir of B7-relted lignds PD-L1 (ref. 12), lso known s B7-H1 (ref. 13), nd PD-L2 (ref. 14), lso known s B7-DC (ref. 15). B7-H3, nother B7 homolog 16,17, inds n unidentified reeptor on tivted T ells. Here we report the loning nd hrteriztion of BTLA, n inhiitory reeptor expressed y T lymphoytes. BTLA is indued on tivtion of nive T ells nd is expressed y developing T H 1 nd T H 2 ells. Expression of BTLA is susequently lost on highly polrized T H 2 ells ut remins on T H 1 ells. BTLA ontins two ytoplsmi immunoreeptor tyrosine-sed inhiitory motifs (ITIMs) nd undergoes induile tyrosine phosphoryltion nd ssoition with SHP-1 nd SHP-2. We show tht oligtion of BTLA prtilly inhiits CD3-indued seretion of IL-2 nd tht BTLA-defiient T ells hve inresed prolifertion to ntigen presented y dendriti ells (DCs), suggesting tht BTLA exerts n inhiitory rther thn tivting influene on T ells. BTLA-defiient mie show moderte inrese in speifi ntiody response nd n inresed suseptiility to peptide ntigen indued experimentl utoimmune enephlomyelitis (EAE), further suggesting n inhiitory role for BTLA. We lso show tht BTLA is reognized y n orphn B7 homolog, B7x. Thus, BTLA shres severl struturl nd funtionl similrities with CTLA-4 nd PD-1, the other two inhiitory reeptors expressed on T lymphoytes. RESULTS Expression of BTLA mrna In previous sreen using Affymetrix rrys 18, we identified n nonymous T H 1-speifi expressed sequene tg (EST). The fulllength omplementry DNA of this EST, loned from murine omplementry DNA lirry (Fig. 1 nd Supplementry Tle 1 online), predits protein with signl sequene, extrellulr vrile (V)-like immunogloulin (Ig) domin, trnsmemrne region nd intrellulr domin of out 100 mino ids (Fig. 1). By homology serhing, we identified single humn gene homolog (Fig. 1,) with similr domin struture. Notly, three tyrosine residues in the ytoplsmi domin re ontined in sequene 1 Deprtment of Pthology & Immunology, Howrd Hughes Medil Institute, Wshington University Shool of Mediine, Box 8118, 660 South Eulid Avenue, St. Louis, Missouri 63110, USA. 2 Deprtment of Experimentl Mediine, University of Perugi, Vi del Giohetto, 06122, Perugi, Itly. 3 Deprtment of Moleulr Biology & Phrmology, Wshington University Shool of Mediine, Box 8103, 660 South Eulid Avenue, St. Louis, Missouri, 63110, USA. 4 Howrd Hughes Medil Institute, Deprtment of Moleulr nd Cell Biology, Cner Reserh Lortory, University of Cliforni t Berkeley, Berkeley, Cliforni 94720, USA. 5 Present ddresses: The Seond Deprtment of Internl Mediine, Chi University Shool of Mediine, J1-8-1 Inohn, Chuo-ku, Chi , Jpn (N.W.); Boehringer Ingelheim Phrmeutils, 900 Ridgeury Rod, Ridgefield, Connetiut 06877, USA (J.Y.). Correspondene should e ddressed to K.M.M. (murphy@immunology.wustl.edu). 670 VOLUME 4 NUMBER 7 JULY 2003 NATURE IMMUNOLOGY

2 Figure 1 BTLA sequene nd genomi struture. () Alignment of mouse nd humn BTLA. The signl peptide nd the trnsmemrne region re underlined. Potentil N-linked glyosyltion sites ( ) nd ysteine residues ( ) predited to prtiipte in disulfide onds in the Ig domin re indited. Spes hve een introdued for optiml omprison. Boxed sequenes re tyrosine-sed signling motifs onserved etween humn nd mouse BTLA, nd re lso onserved in the rt homolog (EST AI235902) expressed in normlized rt ovry. The mouse sequene is from the 129SvEv strin nd the humn sequene ws loned from the Rmos T ell line (Methods). () Exon nd intron orgniztion of mouse nd humn BTLA gene. Filled oxes indite oding sequene in exons, nd unfilled oxes indite 3 nd 5 untrnslted regions. The mino id numer enoded y eh exon is indited elow. () Predited struturl regions of mouse BTLA. Shown re full-length BTLA nd minor splie vrint (BTLAs) tht lks exon 2 nd thus the Ig domin. Romn numerls indite the exon from whih the predited region is derived. The moleulr weight of the predited protein without posttrnsltionl modifitions is indited in prentheses. motifs tht re onserved etween mouse nd humn: the first is in potentil Gr2 intertion site 19 nd the other two re in ITIM sequenes 20. In ddition to BTLA, minor lterntively splied trnsript, BTLAs, ws deteted y reverse trnsription polymerse hin retion (RT-PCR). BTLAs lks exon 2 nd thus the Ig domin (Fig. 1). BTLA mrna is expressed strongly in spleen nd lymph node tissues ut very wekly or undetetly y severl somti tissues (Fig. 2). It is expressed y oth spleni B ell nd T ells, with slightly higher levels in the former (Fig. 2). By using n ntigen-speifi system to mesure the expression of BTLA mrna y northern nlysis during T ell tivtion, we found low BTLA expression on dy 2 fter primry T ell tivtion, with no differene in expression etween T H 1 nd T H 2 onditions (Fig. 2). On dy 7 fter primry T ell tivtion, expression of BTLA mrna ws inresed, with slightly higher expression in T H 1 thn in T H 2 ells (Fig. 2). After seond week of in vitro ulture under T H 1 or T H 2 polriztion onditions, BTLA expression remined high in T H 1 ells, ut y omprison ws redued in T H 2 ells (Fig. 2d). Thus, expression of BTLA mrna seems to e regulted during the tivtion nd differentition of T ells. BTLA expression is not stritly dependent on ny single T H 1-induing signl, euse it remined expressed in T H 1 ells defiient for either signl trnsduer nd tivtor of trnsription 1 (STAT1) or STAT4. The A20 B ell line, ut not mrophges or lymphokine tivted killer (LAK) ells, lso expressed BTLA mrna (Fig. 2). BTLA surfe expression nd phosphoryltion To determine whether BTLA is trnsmemrne protein, we expressed three forms of BTLA tgged with the My epitope in the Bj ell line (Fig. 3). Cell surfe expression of wild-type BTLA ws deteted s predited (Fig. 3, top). Notly, deleting either the ytoplsmi or the Ig domin inresed surfe expression, suggesting tht these domins hve roles in ontrolling the mount of surfe expression, similr to the regultion of surfe CTLA-4 y its ytoplsmi domin 21,22. Next, we onfirmed tht BTLA is glyoprotein (Fig. 3). Tretment with peptide N-glyosidse F (PNGse F) redued the pprent moleulr weight of oth humn nd murine BTLA, onsistent with the d Figure 2 Expression of BTLA in lymphoid ells. () Tissue distriution of BTLA mrna. Northern nlysis of 10 µg of tissue or ellulr RNA proed with full-length BTLA or GAPDH DNA. () Northern nlysis of totl RNA from the indited ells. Cytotoxi T ell type 1 (T1) nd T2 ells were prepred from in vitro polrized 2C 51 TCR trnsgeni T ells, LAK ells were prepred y ulturing C57BL/6 splenoytes with 1,000 U/ml of IL-2 for 9 d, nd mrophges were from BALB/ one mrrow derived with L-ell onditioned medi nd onfirmed s >95% M-1 +. Spleni B nd T ells were purified to >98% y ell sorting. () Totl RNA ws isolted from T ells on dys 2 nd 7 fter 3 h of inution with PMA plus ionomyin, nd northern lots were proed for BTLA nd GAPDH mrna. Dt for dys 2 nd 7 re from lots tht were hyridized nd exposed together. (d) Expression of BTLA in polrized T H 1 nd T H 2 ells on dy 14. Wild-type, STAT1-defiient nd STAT4-defiient DO11.10 trnsgeni T ells 18 ( per ml) were tivted y 0.3 µm OVA peptide in T H 1 or T H 2 onditions nd gin fter 7 d in the sme onditions. Cells were olleted on dy 14 nd stimulted with PMA plus ionomyin for 3 h, nd totl RNA ws nlyzed for BTLA, IFN-γ, IL-4 nd GAPDH mrna s desried 18. NATURE IMMUNOLOGY VOLUME 4 NUMBER 7 JULY

3 d N-linked glyosyltion sites predited in the extrellulr region (Fig. 1). The pprent moleulr weight of humn nd murine BTLA treted with PNGse F ws still higher thn tht predited y its ore mino id sequene, suggesting tht it hs dditionl modifitions suh s O-linked glyosyltion. We lso found tht pervndte tretment indued tyrosine phosphoryltion of BTLA. Single muttion of tyrosines 226, 257 nd 282 to phenyllnine hd little effet on pervndte-indued BTLA phosphoryltion, ut the triple tyrosine muttion loked phosphoryltion ompletely. The Y226F nd Y257F doule muttion severely redued pervndte-indued phosphoryltion, suggesting tht Y282 is either wekly phosphorylted or requires prior phosphoryltion t Y226 or Y257 (Fig. 3,d). Thus, BTLA is n Ig domin-ontining trnsmemrne glyoprotein tht n e phosphorylted on tyrosines loted in onserved ytoplsmi ITIM-like motifs. Induile SHP-1 nd SHP-2 ssoition Sequenes surrounding Y226 ontin potentil Gr2-inding motifs 23, Y257 seems to e n ITIM 20, nd Y282 is similr to the immunoreeptor tyrosine-sed swith motif (ITSM) in PD-1 (ref. 24) nd SLAM (lso lled CD150 or IPO-3) 25. To evlute suh potentil intertions, we developed system of induile BTLA phosphoryltion. Extrellulr My-tgged BTLAs ws expressed stly in the DO11.10 hyridom 26 (Fig. 4). By similr strtegy to tht used for rosslinking PD-1 with the B ell reeptor omplex 27, we rosslinked BTLA nd the T ell ntigen reeptor (TCR) with ntiodies to CD3 nd the My epitope, nd then rried out seondry rosslinking with got nti mouse IgG. Tyrosine phosphoryltion of BTLA (Fig. 4) ws speifi to BTLA-trnsdued ells, dependent on seondry rosslinking nd not indued with nti-cd3 or nti-my lone (Fig. 4,). In ddition, tyrosine phosphoryltion of BTLA showed time-dependent response: it ppered rpidly nd peked t 2 3 min, nd ws extinguished within 10 min of the seondry rosslinking (Fig. 4). We exmined vrious signling moleules for oimmunopreipittion with My-BTLA. Notly, SHP-2 ws strongly ssoited with Figure 3 BTLA is trnsmemrne, glyoslyted, nd tyrosine-phosphorylted on indution. () Trnsmemrne ell surfe expression of BTLA. Bj ells were infeted with mphotrophi retrovirus ontining empty vetor, My 3 - mbtla-rv (BTLA), yt-my 3 -mbtla-rv ( yt BTLA) nd My 3 -mbtlas-rv (BTLAs). Expression of the My epitope ws ssyed on GFP-positive ells y FACS. () Murine nd humn BTLA ontin N-linked oligoshrides. A20 murine B lymphom ells expressing ontrol GFP-RV (lnes 1 nd 2) or mbtla-my 2 -RV (lnes 3 nd 4), nd Bj humn B lymphom ells expressing ontrol GFP-RV (lnes 5 nd 6) or hbtla-my 3 -RV (lnes 7 nd 8) were prepred y infetion with mphotrophi retrovirus, sorted for GFP expression nd nlyzed y immunolotting (Methods). (,d) Tyrosine phosphoryltion of BTLA on stimultion with pervndte. () Bj ells infeted with wild-type or single tyrosine mutnts of hbtla-my 3 -RV were inuted in the sene (lnes 1, 3, 5, 7, 9) or presene (lnes 2, 4, 6, 8, 10) of pervndte, nd exmined for tyrosine phosphoryltion (Methods). (d) Bj ells infeted with doule or triple tyrosine mutnts of hbtla- My 3 -RV were nlyzed for tyrosine phosphoryltion (Methods). BTLA. Assoition of SHP-2 orrelted with phosphoryltion of BTLA nd ws dependent on orosslinking (Fig. 4, lnes 8 13). Pervndte tretment lso indued the ssoition of SHP-2 with BTLA (Fig. 4, lne 14), nd this ondition ws used to exmine further the ssoition of BTLA with SHP-1 nd SHP-2 (Fig. 4 e). SHP- 2 opreipitted with pervndte-treted BTLA (Fig. 4,d, ompre lnes 3 nd 4). Copreipittion of SHP-1 ws lso dependent on phosphoryltion of BTLA (dt not shown). Induile ssoition of SHP-2 ws lso oserved with humn BTLA (Fig. 4e). My-tgged humn BTLA ws expressed in the humn T ell line Jurkt. My-hBTLA opreipitted with SHP-2 only in pervndte-treted ells nd ws speifi to expression of My-hBTLA (Fig. 4e). Likewise, only nti SHP-2 immunopreipittes from pervndtetreted ells ontined My-BTLA (Fig. 4e, lne 8). We lso onfirmed tht SHP-1 ssoited with phosphorylted humn BTLA (dt not shown). The induile ssoition of BTLA with SHP-1 nd SHP-2, hrteristi shred y CTLA-4 nd PD-1, suggests tht BTLA my hve n inhiitory funtion in lymphoytes. To explore the downstrem effets of rosslinking BTLA with TCR, we exmined the prodution of IL-2 in T ell hyridoms (Fig. 4f). My-tgged BTLA nd BTLAs were stly expressed in DO11.10 hyridom T ells 26. Control DO11.10 hyridoms infeted with the empty green fluoresent protein (GFP) retrovirl vetor, GFP-RV, showed nti-cd3 indued prodution of IL-2 tht ws unffeted y plte-ound nti-my (Fig. 4f). In ontrst, prodution of IL-2 y DO11.10 ells expressing My-BTLA nd My-BTLAs ws dosedependently inhiited y plte-ound nti-my (Fig. 4f). No differenes in IL-2 prodution indued y phorol 12-myristte 13-ette (PMA) plus ionomyin were oserved (Fig. 4g). Thus, it seems tht BTLA n modestly inhiit ntigen-indued, ut not hemilly indued, prodution of IL-2 y DO11.10 hyridom ells. Immune response in BTLA-defiient mie To test whether BTLA hs n inhiitory funtion in vivo, we trgeted the Btl gene (Fig. 5) to produe mie lking BTLA (Fig. 5). BTLAdefiient mie on 129SvEv kground lked expression of BTLA 672 VOLUME 4 NUMBER 7 JULY 2003 NATURE IMMUNOLOGY

4 f d e Figure 4 Induile ssoition of BTLA with SHP-2. ()Tyrosine phosphoryltion of BTLA on TCR rosslinking. DO11.10 hyridom T ells were infeted with empty vetor (GFP-RV) 48 or My 3 -mbtlas-rv (My-BTLAs) nd purified. After rosslinking (Methods), nti-my immunopreipittes were nlyzed with ntiptyr (top), reproed with polylonl rit nti-my (middle), nd then reproed with rit nti SHP-2 ntiody (ottom). Arrowheds indite the prinipl glyosylted forms of BTLAs. () BTLA tyrosine phosphoryltion requires orosslinking. Cells in were treted s indited nd nlyzed for ptyr (top) or My (ottom). () Cells in were inuted in the sene ( ) or presene (+) of pervndte for 2 min t 37 C nd lysed in 1% NP-40 lysis uffer. Anti-My immunopreipittes nd whole-ell lystes ( ells) were nlyzed sequentilly for SHP-1 (dt not shown), SHP-2, ptyr nd My. (d) Cells were treted nd lysed s in. Anti SHP-2 immunopreipittes nd whole-ell lystes were nlyzed with nti-my (top), nd reproed with nti SHP-2 (ottom). (e) Jurkt T ells were infeted with GFP-RV or My 3 -hbtla-rv (hbtla), sorted to >95% high surfe expression of My-hBTLA, inuted in the sene ( ) or presene (+) of pervndte for 4 min t 37 C, nd lysed in 1% Triton X-100 lysis uffer. Anti-My (left) nd nti SHP-2 (right) immunopreipittes were nlyzed for My, SHP-2 nd ptyr. (f,g) Crosslinking BTLA with TCR ttenutes prodution of IL-2. DO11.10 ells expressing ontrol vetor (GFP-RV), My 3 -mbtlas-rv (My-BTLAs) or My 3 -mbtla-rv (My-BTLA) were stimulted with nti-cd3ε plus the indited onentrtions of nti-my (f) or with PMA plus ionomyin (g). The IL-2 onentrtion ws determined y ELISA. In f, the IL-2 titer is normlized y the IL-2 onentrtion indued y stimultion with nti-cd3 lone. g mrna in peripherl lymphoytes (Fig. 5). No developmentl defets in T or B ells in thymus or one mrrow were oserved in these mie (Supplementry Fig. 1 online). We produed BTLA-defiient DO11.10 TCR trnsgeni mie on mixed 129SvEv nd BALB/ kground for the in vitro nlysis of T ells (Fig. 5d). Fully polrized BTLA-defiient T H 1 ells showed roughly twofold inrese in prolifertion in response to 0.3 µμ OVA peptide presented y either CD8 + or CD8 CD11 + DCs (Fig. 5d). We lso exmined T nd B ells from wild-type nd BTLA-defiient T ells for mitogen- nd TCR-indued prolifertion in vitro (Fig. 6). The prolifertive response of wild-type nd BTLA-defiient T ells to onnvlin A were omprle, ut BTLA-defiient T ells showed heightened response to stimultion with nti-cd3. Lipopolyshride (LPS)-indued prolifertion ws similr in wild-type nd BTLA-defiient B ells, ut BTLA-defiient B ells showed slightly greter responses to stimultion with nti-igm. The inresed in vitro responses, lthough not mrked, were similr in mgnitude to the inreses in prolifertion seen in PD-1-defiient T ells 11 nd in CTLA-4-defiient CD8 + T ells 28 in vitro. In ft, CTLA-4-defiient CD4 + T ells show no enhnement of primry ntigen-driven responses nd only twofold inrese in seondry responses 29,30. These results suggest tht BTLA hs n inhiitory, rther thn tivting, influene on T lymphoyte responses. Consistent with this, 4 weeks fter immuniztion with nitrophenol-onjugted keyhole limpet hemoynin (NP-KLH), BTLA-defiient mie showed threefold inrese in the mount of NP-KLH-speifi IgG1, IgG2 nd IgG2 isotypes s ompred with ontrol littermte 129/SvEv mie (Tle 1). EAE suseptiility in BTLA-defiient mie As our dt suggested tht BTLA might e inhiitory, we used model of EAE to detet potentilly enhned T ell responses in BTLA-defiient mie. Initilly, we rried out n ntigen dose titrtion of the myelin oligodendroyte glyoprotein (MOG) peptide on the pure 129SvEv kground to determine dose tht would generte suoptiml disese ple of showing ugmenttion (Fig. 7). We found tht 10 µg nd 50 µg of peptide indued severe disese in 129SvEv mie, wheres 2 µg indued milder disese with delyed onset (Fig. 7). At this ntigen dose, BTLA-defiient mie showed higher inidene, inresed linil sore, erlier onset nd prolonged durtion of disese s ompred with wild-type littermte ontrols (Fig. 7). To sertin ny potentil differenes etween the nture of MOGindued EAE in BTLA-defiient mie nd tht in C57BL/6 ontrol mie, we exmined the lumr spinl ords of ffeted mie (Fig. 8). There ws n undnt ellulr infiltrte in the lumr spinl region of BTLA-defiient mie with linil sore of 4, whih ws similr in severity to the infiltrte in C57BL/6 ontrol mie indued with high NATURE IMMUNOLOGY VOLUME 4 NUMBER 7 JULY

5 Figure 5 Genertion nd nlysis of BTLAdefiient mie. () The Btl lous nd trgeting onstrut. Exons III VI, enoding the extrellulr, trnsmemrne nd ytoplsmi regions, re indited. BglII digestion of the germline lous genertes restrition frgment of 14.2 k tht hyridizes to proes A nd B, nd frgment of 8.4 k in orretly trgeted lones. B, BmHI; Bg, BglII; E, EoRI; Sl, SlI; X, XI; Xh, XhoI. TK, thymidine kinse gene; neo, neomyin resistne ssette. () Southern nlysis. BglII-digested til DNA hyridized with proe B. () Northern nlysis. RNA ws prepred diretly from splenoytes of mie of the indited genotype. Blots were hyridized to full-length mouse BTLA DNA proe, stripped nd reproed for GAPDH. (d) Prolifertive responses of polrized T H 1 ells indued y inution with ntigen-pulsed DCs. Nive CD4 + T ells from DO11.10 BTLA wild-type or BTLA-defiient mie were tivted in vitro nd pssed iweekly in T H 1 onditions. Resting T H 1 ells ( ) were inuted with BALB/-derived CD8 + or CD8 DCs (top, ; ottom, ), with or without 300 nm OVA peptide in volume of 200 µl. Cell prolifertion ws mesured y pulsing with [ 3 H]thymidine for 16 h. dose of MOG peptide nd lso with linil sore of 4 (ompre Fig. 8 nd ). Less undnt ellulr infiltrtes were oserved in BTLAdefiient mie tht were killed when the linil sore ws 1.5 (Fig. 8). Consistent with the MOG peptide indued infiltrtes oserved in the C57BL/6 kground 31, ellulr infiltrtes in BTLA-defiient mie onsisted of oth CD4 + nd CD11 + ells (Fig. 8), whih were more frequent in mie with linil sore of 4 thn in those with sore of 1.5 (ompre Fig. 8f,g nd d,e). Thus, loss of BTLA in 129SvEV mie inreses the sensitivity to ntigen-indued EAE, ut the disese remins hrterized y similr types of ellulr infiltrte into the entrl nervous system (CNS). BTLA intertion with B7 homolog The proposed similrity of BTLA to PD-1 nd CTLA-4 is sed primrily on the overll domin struture of the proteins, s eh possesses single extrellulr Ig-like domin, trnsmemrne region d Figure 6 In vitro responses of BTLA-defiient lymphoytes. T nd B ell from wild-type (WT) or BTLA-defiient (KO) mie were purified y ell sorting using nti-cd4 FITC, nti-cd8α FITC or nti-b220 PE. Cells were stimulted with the indited finl onentrtions of plte-ound nti-igm, LPS, onnvlin A or plte-ound nti-cd3e. Cell prolifertion ws mesured y pulsing with [ 3 H]thymidine for 16 h. Figure 7 Inresed EAE suseptiility in BTLA-defiient mie. () Titrtion of MOG peptide in 129SvEv mie. Mie were injeted suutneously with MOG peptide t 2 µg, 10 µg nd 50 µg (n = 5) in inomplete Freund s djuvnt nd 500 µg of myoterium on dy 0. Pertussis toxin (300 ng) of ws injeted intrvenously on dys 1 nd 3. C57BL/6 (B6) mie injeted with 10 µg of MOG were used s positive ontrols. Mie were monitored dily for symptoms. Clinil sores: 0, norml, no overt signs of disese; 1, limp til or hindlim wekness, ut not oth; 2, limp til nd hindlim wekness; 3, prtil hind lim prlysis; 4, omplete hindlim prlysis; 5, moriund stte, deth y EAE, killed for humne resons. () Ative indution of EAE y suoptiml dose of MOG peptide in BTLA-defiient mie. BTLA-defiient (KO) or wild-type (WT) littermte ontrol 129SvEv mie ged 6 8 weeks (n = 5) were injeted with 2 µg of MOG peptide s in. Men linil sores: wild type, 0.6 ± 0.9; BTLA-defiient, 2.4 ± 1.7. Men pek linil sore; wild type, 1.5 ± 0.7; BTLA-defiient 3.0 ± VOLUME 4 NUMBER 7 JULY 2003 NATURE IMMUNOLOGY

6 Tle 1 Augmented IgG responses of BTLA-defiient mie to Td ntigen Anti-NP Btl +/+ (%) Btl +/ (%) Btl / (%) IgG ± ± ± 82.6 IgG ± ± ± IgG ± ± ± d Dt re presented s the perentge of men isotype prodution s ompred with wild-type BTLA serum, whih ws used s uniform stndrd serum to determine reltive titers of nti-np. Dt re the men ± s.d. of mesurements from five mie. P = 0.029, P = 0.014, d P = versus wild-type BTLA. nd onserved intrellulr ITIM motifs tht intert with SHP-1 nd SHP-2 phosphtses, nd eh hs inhiitory rther thn tivting effets on lymphoytes. Both CTLA-4 nd PD-1 intert with memers of the B7 fmily 32, nd we therefore tested whether BTLA lso shres this feture. We identified onserved B7 homolog, B7x, with high similrity etween the mouse nd humn sequenes. An lignment of humn nd murine B7x (Supplementry Fig. 2 online) identified predited signl peptide nd two Ig-like domins followed y potentil trnsmemrne region. We generted fusion protein of Ig nd murine B7x nd otined dditionl Ig fusion proteins of B7h, PD-L1 nd PD-L2. The B7h-Ig fusion protein showed no differene in inding etween wild-type nd BTLA-defiient T ells; this ws expeted, euse B7h inds ICOS 3, whih presumly should e expressed y oth wild-type nd BTLA-defiient T ells. In ontrst, the B7x-Ig fusion protein ound to wild-type ut not to BTLA-defiient T ells (Fig. 9), suggesting tht BTLA is responsile for most of the inding of B7x-Ig to T ells. PD-1 ws expressed y oth wild-type nd BTLA-defiient T ells, ut it ws deteted more strongly y nti PD-1 thn y the PD-L1 or PD-L2 Ig fusion proteins (Fig. 9,). In ontrst, the B7-1 nd B7-2 Ig fusion proteins generted stronger signls (Fig. 9) on wild-type nd BTLA-defiient T ells, onsistent with the high ffinity of CTLA-4 for B7-1 nd B7-2. Tken together, these results indiretly suggest tht B7x is lignd, lthough not neessrily the only lignd, for BTLA, therey highlighting nother similrity etween BTLA nd the other inhiitory reeptors, CTLA-4 nd PD-1, known to e expressed on T ells. DISCUSSION BTLA seems to e third inhiitory reeptor expressed y T ells tht, like PD-1, is indued on T ell tivtion ut remins expressed more Figure 9 BTLA interts with n orphn B7 B7x. () Spleen nd lymph node ells from BTLA wild-type nd BTLA-defiient DO TCR trnsgeni mie were olleted nd stimulted with 0.3 µm OVA peptide, 10 U/ml of IL-12 nd neutrlizing ntiodies to IL-4, nd ssyed for Ig fusion inding fter 4 d. Cells were stined with nti-cd4 FITC. Left, ells were stined with humn IgG1 ntiody s negtive ontrol (filled) or with B7x-Ig fusion protein (open), followed y got nti humn IgG PE. Right, ells were unstined (filled) or stined with B7h-Ig (open), followed y iotinylted nti-my (murine IgG1 isotype) nd streptvidin-pe. Anti-My ws used s negtive ontrol for the B7h-Ig fusion protein. (,) T H 1 ell lines derived from BTLA wild-type nd BTLA-defiient DO mie were stimulted s ove, olleted on dy 3, nd ssyed for inding to Ig fusion proteins. All ells were stined with nti-cd4 FITC. In, Cells were stined with humn IgG1 ntiody (filled) or with B7.1-Ig, B7.2-Ig, PD-L1 Ig nd PD-L2 Ig fusion proteins (open), followed y got nti humn Fγ F( ) 2 PE. In, ells were stined with hmster IgG2 PE s negtive ontrol (filled) or with nti PD-1 PE. Histogrms re gted on CD4 + ells. strongly on polrized T H 1 ells. Similr to PD-1 nd CTLA-4 (ref. 27), BTLA n e indued to undergo phosphoryltion nd to ssoite with the SHP-1 nd SHP-2 phosphtses y orosslinking with the TCR on T ells. The ssoition with these phosphtses, the inresed in vivo sensitivity to MOG peptide indued EAE, nd the inresed rther thn deresed in vitro prolifertive responses suggest tht d e Figure 8 Infiltrtion of the CNS in MOG-indued EAE in BTLA-defiient mie. C57BL/6 ontrol () or BTLA-defiient ( g) mie immunized with MOG peptide were killed when showing the indited linil sore (CS): () C57BL/6, CS 4; () BTLA-defiient, CS 4; (-e) BTLA-defiient, CS 1.5; (f,g) BTLA-defiient, CS 4. Hemtoxylin nd eosin ( ) or immunofluoresene stining for CD4 (left) nd CD11 (right) (d g) ws done on frozen setions s desried 31. Immunofluoresent setions were ounterstined with 4,6- dimidino-2-phenylindole dihydrohloride (DAPI, lue). f g NATURE IMMUNOLOGY VOLUME 4 NUMBER 7 JULY

7 BTLA my hve n inhiitory funtion similr to tht of PD-1 nd CTLA-4, nd my hve role in ontrolling lte phses of immune responses nd possily utoimmunity. The phenotypes generted y defiienies in CTLA-4, PD-1 nd BTLA suggest tht PD-1 nd BTLA my hve prtilly redundnt inhiitory signls. Of these three inhiitory reeptors, defiieny in CTLA-4 produes y fr the most mrked in vivo phenotype 33. In ft, the sis for spontneous lymphoprolifertive disese in CTLA-4-defiient mie is not fully understood, ut it requires properties of the norml T ell repertoire in vivo, s it is ted or prevented when CTLA-4-defiient mie re rossed onto TCR trnsgeni kground 29 nd delyed when rossed onto FoxP3 trnsgeni kground 34. Antigen-speifi prolifertion or ytokine prodution in CTLA-4- defiient T ells is not enhned during the primry response nd shows modest, if ny, inreses during seondry responses 29,30. This result indites tht the importnt inhiitory tion of CTLA-4 is unlikely to e pplied during the ostimultion-dependent tivtion of T ells. Indeed, the gretest in vitro differene etween wild-type nd CTLA-4-defiient T ells ours fter the in vivo intrvenous delivery of solule ntigen to indue nergy 30. CTLA-4 my exert t lest prt of its regultory tivity y non ell-utonomous mehnism 35 37, ontrry to the expeted inhiitory tion of SHP-1 or SHP-2 in ells expressing surfe CTLA-4. A non ell-utonomous inhiitory role for CTLA-4 might, for exmple, result from the enggement nd rosslinking of B7 expressed y APCs 38, leding to n indiret regultory tion 39. Thus, lthough CTLA-4 lerly hs n importnt in vivo regultory role, it is not yet totlly ler how, when or where this tion is exerted, or why no mrked in vitro effet is seen. In ontrst to CTLA-4 defiieny, loss of PD-1 expression in vivo results in phenotype tht is vrile nd dependent on geneti kground, eing mild in 129SvEv mie 11 nd more severe in BALB/ mie 40. How the geneti kground ffets the requirement for PD-1 in regulting self-tolerne is not understood. In vitro nlysis of PD- 1-dependent T ell responses suggests tht CD28-dependent, ut not ICOS-dependent, ostimultion overrides PD-1-dependent ellulr inhiition 41. In ddition, inhiition y PD-1 of CD3-CD28 driven T ell tivtion is restrited to very nrrow rnge of tivtion; for exmple, it ours t 1 µg/ml, ut not t 2 µg/ml, of nti-cd3 in vitro 12 nd is overome y IL-2 (ref. 42). Thus, for oth CTLA-4 nd PD-1, the very modest effets seen in in vitro ssys do not reflet the more mrked nd importnt tions of these inhiitory reeptors oserved in vivo. In summry, wheres CTLA-4 defiieny produes mrked lymphoprolifertive phenotype, PD-1 nd BTLA defiienies (on the 129SvEv kground) individully produe sutle phenotype hrterized y enhned propensity to utoimmunity. Although CTLA-4 n regulte oth erly events in T ell tivtion 43 nd the lter unfolding of utoimmunity 44,45, it seems tht PD-1 nd BTLA my funtion lter in regulting effetor responses, on the sis of their delyed expression in T ells. Coneivly, then, PD-1 nd BTLA my hve inhiitory tions tht provide redundnt inhiitory system gered towrd mintining peripherl tolerne. An exmintion of mie defiient for oth PD-1 nd BTLA will e required to test this hypothesis. CTLA-4 nd PD-1 eh intert with two known lignds, B7-1 nd B7-2, nd PD-L1 nd PD-L2, respetively 32. So fr, we hve identified only one ndidte protein ontining n Ig domin tht my intert with BTLA. We my infer tht n intertion ours etween BTLA nd B7x from the loss of inding of the B7x-Ig fusion protein to BTLAdefiient T ells; however, it is oneivle tht BTLA does not intert diretly with the B7x-Ig fusion protein, ut insted ontrols the expression of nother, diretly interting, prtner. On the sis of our interprettion tht BTLA funtions s n inhiitory reeptor like PD-1 nd CTLA-4, rther thn s regultor of protein trnsport, we fvor the interprettion tht there is diret intertion etween BTLA nd B7x. Our nlysis of potentil BTLA-interting ndidtes is in its erly stges. So fr, our serh hs een restrited to using Ig fusions of vrious B7 fmily memers, whih my limit the sensitivity of deteting intertions. For exmple, the limited sensitivity of Ig fusion proteins eme evident in our ontrol nlysis of PD-1 expression. Anti PD-1 deteted muh higher signl tht did the PD-L1 or PD-L2 Ig fusion proteins, proly result of the lower ffinities of the PD-L1 or PD-L2 Ig fusion proteins, s ompred with the ffinity of the PD-1 ntiody, for PD-1. Thus, deteting dditionl BTLA-interting proteins my require tehniques with higher sensitivity. The existene of humn homolog of BTLA tht is very losely relted to the mouse sequene, omined with the oservtion tht murine nd humn BTLA ytoplsmi domins ssoite with the phosphtses SHP-1 nd SHP-2, rgue for onserved inhiitory funtion. Polymorphisms in CTLA-4 my e linked to utoimmune disorders in humns 46,47. Given the vrile PD-1-defiient phenotype on different geneti kgrounds, nd the quntittive nture of the BTLA-defiient phenotype, we propose tht polymorphisms ffeting the funtion of either of these inhiitory reeptors ould e sis for vrily penetrnt suseptiility to utoimmunity in the humn popultion. In this regrd, we hve noted vritions mong murine strins of the BTLA sequene (dt not shown). Vritions in reeptor-lignd ffinity, lignd expression or inherent reeptor signling ould ontriute to vrile utoimmune suseptiility mong individuls, prtiulrly if these two inhiitory reeptors provide redundnt shield for peripherl tolerne. METHODS Isoltion of BTLA. We used n EST (839766) expressed y T H 1, ut not T H 2, ells to sreen T H 1 DNA phge lirry mde in the Lmd ZAP vetor (Strtgene) nd isolted prtil lone, BTLAs, tht lked n Ig domin. Fulllength BTLA DNA, mplified from WEHI ell RNA y RT-PCR with primers J10-3K (5 -TTTGGCCTAAGATGCTGCTA-3 ) nd J10-7F (5 -CACA- GATTGGGTACGACATG-3 ), ws inserted into the GEM-T Esy Vetor (Promeg) to produe mj11w1. We otined dditionl full-length BTLA DNA isoltes y sreening seond mouse splenoyte DNA lirry (Strtgene) using the 5 region of mj11w1 s proe. Coding sequene nd intron-exon oundries were further determined y sequening 129SvEv strin teril rtifiil hromosome lones ontining the BTLA region (Genome Systems). Some Ig domin sequene polymorphisms our mong mouse strins. Humn BTLA DNA, mplified from Rmos B lymphom RNA y RT- PCR with primers hj10 (5 -TTTTCCATCACTGATATGTGCAGG-3 ) nd hj10 AS (5 -GGTCCCTGTTGGAGTCAGAAAC-3 ) sed on the Celer humn genome ssemly, ws inserted into the GEM-T Esy Vetor to produe hj11#14u. Plsmid onstrutions. My-tgged BTLA onstruts were prepred s follows. The open reding frme of mbtlas ws mplified from olony otined from sreening DO11.10 T H 1 DNA lirry with primers J10-RV1-Bgl2 (5 - AGCTCTGAAGATCTCTAGGGAGGAAG-3 ) nd J10-Xho1 (5 -CATGCTC- GAGGAAGGTCCAGACAGAGGTATTG-3 ). The produt ws digested with BglII nd XhoI nd loned into the IRES-GFP-RV retrovirus 48 t the BglII nd XhoI sites to produe mbtlas-rv. The N-terminl My-tgged version of mbtlas (My 3 -mbtlas-rv) ontins triple My tg inserted downstrem of the signl peptide. To produe this onstrut, PCR produt ontining the mbtla signl sequene nd 3 overhng homologous to the My tg ws prepred with mbtlas-rv s the templte nd primers J10-RV1-Bgl2 nd J10-A2 (5 GTTCAGATCCAAGGAT- GCTCCAGAGGCCC-3 ). This PCR produt ws nneled to seond PCR 676 VOLUME 4 NUMBER 7 JULY 2003 NATURE IMMUNOLOGY

8 produt omprising three opies of the My epitope with 5 nd 3 overhngs homologous to the N- nd C-terminl portions of BTLA, respetively, whih hd een mplified from the triple My/Bluesript templte with primers J10- A3 (5 -GAGCATCCTTGGATCTGAACAAAAGCTGATTA-3 ) nd J10-A4 (5 - CTTTCTCACAGAGCTCGTACAGGTCCTCT-3 ). The triple My/Bluesript templte ontins nhor sequenes 5 (GS) nd 3 (YEL) to the My 3 oding sequene, whih re inluded in the finl My-tgged mbtla protein. We then mplified the two nneled piees with primers J10-RV1-Bgl2 nd J10-A4. This produt ws nneled to third PCR produt ontining 5 My homologous til nd the C-terminl portion of BTLA mplified from the templte mbtlas- RV with primers J10-A5 (5 -GTACGAGCTCTGTGAGAAAGCTACTAA- GAGG-3 ) nd J10-Xho1, nd the full-length himeri DNA ws mplified with primers J10-RV1-Bgl2 nd J10 Xho1. The resulting produt ws digested with BglII nd XhoI nd ligted into the BglII nd XhoI sites of IRES-GFP-RV to yield My 3 -mbtlas-rv. To produe the N-terminl My-tgged version of mbtla (My 3 -mbtla- RV), primers J10-RV1-Bgl2 nd J10-A4 were used to mplify the signl sequene linked to the triple My epitope from templte My 3 -mbtlas-rv. A seond PCR produt ws mplified with primers J10-A5 nd J10 Xho1 nd the templte mj11w1. The two PCR produts were nneled nd mplified with primers J10-RV1-Bgl2 nd J10 Xho1, digested, nd ligted into the retrovirl vetor to produe My 3 -mbtla-rv. A further modifition ws mde y using the Quik Chnge mutgenesis kit (Strtgene) to onvert ysteine downstrem of the My tg to lnine to mimi more urtely the predited signl sequene proessing in whih this ysteine would e removed (SignlP V2.0). yt-my 3 -mbtla-rv ws generted using Quik Chnge mutgenesis of My 3 -mbtla-rv with the primers mj11 trun top (5 -TGATATTCCATAAAC CTGCCACTGAGCCAG-3 ) nd mj11 trun ottom (5 -TGGCAGGTTTATG GAATATCAACCAGGTTAGTG-3 ). mbtla-my 2 -RV, whih expresses mbtla with two C-terminl My epitopes, ws generted y spliing y overlp extension (SOEing) together two PCR produts (generted from primers J10-RV1- Bgl2 nd 3 mj11 My til (5 -GCTTTTGTTCACTTCTCACACAAATGGA TGC-3 ) with templte mj11w1, nd primers 5 mjll My til (5 -TGAGGAGTG AACAAAAGCTGATTAGCGAAG-3 ) nd new 3 Xho My til (5 -CCGCTCG AGCTCCTACAGGTCCTCTTC-3 ) with templte triple My/Bluesript) with primers J10-RV1-Bgl2 nd new 3 XhoI My til nd Pfu polymerse. After digestion with BglII nd XhoI, the PCR produt ws ligted into the retrovirl expression vetor T-lym-GFP RV 49, whih hd een digested with BglII nd XhoI, to generte mbtla-my 2 -RV. The N-terminl My-tgged version of hbtla ontining triple My tg inserted downstrem of the signl peptide (My 3 -hbtla-rv) ws prepred similrly. Three seprte PCR produts were generted using the following primers nd templtes: 5 Bgl2 hj11 (5 -GAAGATCTGCAGGAAATGAAGACATTGCCT- 3 ) nd 3 My/hj11 ottom (5 -TCAGCTTTTGTTCCCCATGGATGTTCCA- GATGTCC-3 ) with hj11#14u; 5 hj11/my top (5 -CATCCATGGGGAACAAA AGCTGATTAGCGAAGAG-3 ) nd 3 hj11/my ottom (5 -CACATGATTCTT TCAGGTCCTCTTCGCTAATCAGC-3 ) with triple My/Bluesript; nd 5 My/hj11 top (5 -GAGGACCTGAAAGAATCATGTGATGTACAGCTTTA-3 ) nd 3 Xho hj11 (5 -CCGCTCGAGTTGGAGTCAGAAACAGACTTAAC-3 ) with hj11#14u. These PCR produts were sequentilly nneled nd mplified, nd loned into t-lym-gfp-rv, whih hd een digested with BglII nd XhoI. hbtla ontining three roxy-terminl My epitopes (hbtla-my 3 -RV) ws generted y SOEing together two PCR produts (from primers 5 Bgl2 hj11 nd 3 hj11 My til (5 -TGAGGAGTGAACAAAAGCTGATTAGCGAAG-3 ) with templte hj11#14u, nd primers 5 hj11 My til (5 -TGAGGAGTGAA- CAAAAGCTGATTAGCGAAG-3 ) nd new 3 Xho My til with templte triple My/Bluesript) with primers 5 Bgl2 hj11 nd new 3 Xho My til nd Pfu polymerse. After digestion with BglII nd XhoI, the PCR produt ws ligted into retrovirl expression vetor T-lym-GFP-RV 49, whih hd een digested with BglII nd XhoI, to generte hbtla-my 3 -RV. Emyroni stem ells (MC50) were gift from R. Shreier (Wshington University Shool of Mediine, St. Louis, Missouri). Tyrosine muttions. Single tyrosine-to-phenyllnine muttions of hbtla- My 3 -RV were produed using Quik Chnge mutgenesis nd Pfu polymerse (Strtgene) with the following oligonuleotide pirs: Y226F top2 (5 -GA AACTGGAATTTATGATAATGACCCTGACCTTTG-3 ) nd Y226F ot (5 -GG GTCATTATCAAAAATTCCAGTTTCTGATAGCAG-3 ); Y257F top2 (5 -ACCA GGCATTGTTTATGCTTCCCTGAACCATTCTG-3 ) nd Y257F ot (5 -AGG GAAGCAAAAACAATGCCTGGTTTGT-3 ); Y282F top2 (5 -GCACCAACAG AATATGCATCCATATGTGTGAGG-3 ) nd Y282F ot (5 -ATATGGATGCAA ATTCTGTTGGTGCTTCTTTTA-3 ). We produed doule nd triple tyrosine-to-phenyllnine muttions of hbtla-my 3 -RV y using the oligonuleotide pir Y257F top2 nd Y257F ot first with the Y226F-mutted hbtla-my3-rv templte to produe Y226F/Y257F nd then with the Y282F-mutted templte to produe Y257F/Y282F. The oligonuleotide pir Y282F top2 nd Y282F ot ws used with the Y226F-mutted templte to produe Y226F/Y282F, nd with the Y226F/Y257F-mutted templte to produe Y226F/Y257F/Y282F. Cell ulture nd expression nlysis. Ativtion of DO11.10 TCR trnsgeni T ells 50 nd retrovirl infetions, northern nlysis nd immunolotting 49 were done s desried. We prepred tissue nd ellulr RNA with the RNesy Midi kit (Qigen). A 20 stok of pervndte ws prepred 5 min efore use y diluting 12.5 µl of 1 M NVO 4 nd 4 µl of 30% H 2 O 2 to 600 µl in distilled wter. The Optei Mouse IL-2 set (PhrMingen) ws used to mesure for IL-2 y enzyme-linked immunosorent ssy (ELISA). Immunolotting nd nlysis of N-linked glyosyltion. To nlyze the glyosyltion sttus (Fig. 3), ells ( per ml) were lysed in Triton X-100 lysis uffer (25 mm HEPES (ph 7.5), 0.15 M NCl, 1% Triton (v/v), 1 mm pervndte, 1 µg/ml of leupeptin, 1 µg/ml of pepsttin, 1 µg/ml of protinin nd 1 mm phenyl methylsulfonyl fluoride) for 30 min t 4 C nd entrifuged t 14,000g for 10 min. Extrts from ells were immunopreipitted with 1 µg of monolonl ntiodies to My (lone 9E10; Snt Cruz) nd 20 µl of 1:1 slurry of protein G Sephrose (PGS) (Phrmi). After eing wshed three times in Triton lysis uffer, the pellets were oiled for 10 min in 10 µl of PNGse denturing uffer (NEB). After entrifugtion to remove PGS, eluted proteins were trnsferred to PCR tues ontining 1 µl of 10% Nonidet P-40 (NP-40) nd 1 µl of 10 G7 uffer (NEB), divided into two 6-µl liquots, nd treted without or with 1 µl of PNGse F (NEB) for 1 h t 37 C. We oiled smples with 6 µl of 2 SDS-PAGE smple uffer nd resolved them on 10% polyrylmide gels. The proteins were trnsferred to nitroellulose, loked in 3% ovine serum lumin (BSA) in TBS-T uffer, lotted with rit nti-my (Snt Cruz) nd horserdish peroxidse (HRP)-onjugted got nti rit IgG (Jkson), nd nlyzed y enhned hemiluminesene (ECL). To nlyze the phosphoryltion sttus (Fig. 3,d), ells were treted with 1 mm pervndte for 2 min t 37 C, pled on ie for 1 min, lysed in n equl volume of 2 1% Triton X-100 lysis uffer for 30 min nd entrifuged for 10 min t 8,000g. Extrts from ells were immunopreipitted using 1 µg of nti-my (lone 9E10) nd PGS. Blots were first nlyzed for phosphotyrosine (ptyr) using HRP-onjugted (lone 4G10, Upstte Biotehnology), nd then stripped nd renlyzed using rit nti-my nd HRP-onjugted got nti-rit IgG. TCR rosslinking. To nlyze the indution of tyrosine phosphoryltion nd ssoition with SHP-1 nd SHP-2 on TCR rosslinking, we infeted DO11.10 hyridom T ells with GFP-RV 48 or My 3 -mbtlas-rv nd purified them y sorting. Cells were inuted with 4 µg/ml of hmster nti-cd3ε (lone 145-2C11, PhrMingen) nd 2 µg/ml of nti-my for 30 min t 4 C, nd rosslinked with 100 µg/ml of prewrmed got nti mouse IgG (GαM; Cltg) for vrious times, s indited (Fig. 4). We used fluoresene-tivted ell sorting (FACS) to onfirm the ross-retivity of got nti mouse IgG with hmster nti-cd3ε. As positive ontrol for phosphoryltion, some ells were inuted with 1 mm pervndte for 2 min t 37 C. Cells were lysed in RIPA uffer, nd 1 ml of lystes from ells were immunopreipitted with 2 µg of nti-my (9E10). We used the following ntiodies to nlyze the immunopreipittes: nti-ptyr (RC20H, Trnsdution Lortories), polylonl rit nti-my (A-14, Snt Cruz), rit nti SHP-2 (C-18, Snt Cruz), rit nti SHP-1 ntiody (C-19, Snt Cruz) nd nti-my (9E10). To mesure the effet of rosslinking on IL-2 prodution, DO11.10 ells expressing GFP-RV, My 3 -mbtlas-rv or My 3 -mbtla-rv were stimulted with 1 µg/ml of immoilized nti-cd3ε in omintion with vrious onentrtions of immoilized polylonl rit nti-my or 50 ng/ml of PMA NATURE IMMUNOLOGY VOLUME 4 NUMBER 7 JULY

9 plus 1 µm ionomyin. Culture superntnts of triplite ultures were olleted fter 24 h, nd the IL-2 onentrtion ws determined y ELISA. Antiody responses. Eight-week-old littermte wild-type, Btl +/ nd Btl / mie on pure 129SvEv kground (n = 5) were injeted intrperitonelly with 100 µg of NP 17 -KLH (Bioserh Tehnologies) in lum (Piere) on dys 0 nd 14. Ser ws olleted on dy 28, nd the titers of nti-np were determined y ELISA using NP 25 -BSA (Bioserh Tehnologies) for ntiody pture nd the SBA Clonotyping system/hrp kit for IgG sulss-speifi ELISA (Southern Bioteh). In vitro responses of BTLA-defiient lymphoytes. T nd B ells from wildtype or BTLA-defiient mie were purified y ell sorting using fluoresein isothioynte (FITC)-onjugted nti-cd4 (Cltg), FITC-onjugted nti- CD8α (PhrMingen) or phyoerythrin (PE)-onjugted nti-b220 (PhrMingen). Cells ( per ml) were stimulted with vrious onentrtions of plte-ound nti-igm (Affinipure F( ) 2 frgment got nti mouse IgM , Jkson ImmunoReserh), LPS (serotype 055:B5, Sigm), onnvlin A or plte-ound nti-cd3e (PhrMingen, 145-2C11). Cell prolifertion ws mesured fter 48 h y pulsing with [ 3 H]thymidine for 16 h. Prodution nd intertion of B7x-Ig. In the puli dtses we identified B7 homolog, B7x, tht ws onserved in mouse (ession ode XP_ nd AAH ), rt (ession ode XP_ ) nd humn (ession ode NP_ ) nd ws highly onserved in sequene. B7x-Ig ws prepred y fusing the oding region of the extrellulr domin of B7x to the CH2-CH3 domin of mouse IgG1 nd My-His tg in pdna4 ( gift of W. Sh, Univ. Cliforni Berkeley, Berkeley, Cliforni, USA). The onstrut ws linerized with BglII nd trnsfeted into 293T ells with FuGENE 6 (Rohe). Stle trnsfetnts were seleted in 1 mg/ml of Zeoin (Invitrogen). To otin fusion protein, we ultured stle trnsfetnts in serum-free Duleo s modified Egle s medium for 72 h, olleted the superntnt nd purified B7x-Ig y ffinity olumn hromtogrphy over His-Bind resin (Novgen). The purity of the fusion protein ws onfirmed y SDS-PAGE nd y immunolotting with ntiodies ginst My nd mouse IgG. The following regents were used to mesure reeptor nd B7 lignd intertions (Fig. 9): nti-cd4 FITC (Cltg); humn IgG1 ntiody (Sigm); iotinylted nti-my (Snt Cruz); streptvidin-pe (PhrMingen); B7.1-Ig, B7.2-Ig, PD-L1-Ig nd PD-L2-Ig fusion proteins (F portion; humn IgG1 isotype; R&D Systems); got nti humn Fγ F( ) 2 PE (Jkson ImmunoReserh); nd nti PD-1 PE (PhrMingen). FACS nlysis. Humn IgG1 nd got nti humn PE were gifts of M. Cell (Wshington Univ., St. Louis, Missouri, USA). The onstrut for the B7h-Ig fusion protein 3, gift of W. Sh (Univ. Cliforni Berkeley), nd the DNA enoding the fusion protein were inserted into the GFP-RV retrovirl vetor 48, nd the retrovirus ws used to infet J558 ells. We purified fusion protein from infeted J558 superntnt with His-Bind resin (Novgen). B7.1-Ig, B7.2-Ig, PD-L1-Ig nd PD-L2-Ig fusion proteins (F portion; humn IgG1 isotype) were otined from R&D Systems. All nlyses were done on FACSCliur. To mesure the surfe expression of BTLA, Bj ells were infeted with mphotrophi retrovirus prepred in Phoenix A pkging ells to express empty vetor, My 3 -mbtla-rv, yt-my 3 -mbtla-rv nd My 3 -mbtlas- RV. Expression of the My epitope on GFP-positive ells ws ssyed on FACSliur with rit nti-my polylonl serum (Snt Cruz) nd PE-onjugted got F( ) 2 nti rit IgG (Jkson Reserh Lortories). Histologil nlysis. CNS tissues were removed from mie nd frozen nd 10- µm setions were prepred. Setions shown re from lumr spinl ord. We prepred setions stined with hemtoxylin nd eosin, nd mesured the expression of CD4 nd CD11 y immunofluoresene mirosopy s desried 31. Aession odes. Murine BTLA, AY293285; humn BTLA, AY293286; murine B7x, XP_ nd AAH ; rt B7x, XP_ ; humn B7x, NP_ ( From the NCBI Protein dtse. Note: Supplementry informtion is ville on the Nture Immunology wesite. ACKNOWLEDGMENTS We thnk B. Slekmn for help with gene trgeting; M. White for generting himeri mie; M. Gimenez for help with immunohistohemistry; nd W. Sh for disussions. This work ws supported in prt y grnts from the Ntionl Institutes of Helth. J.P.A. nd K.M.M. re Investigtors of the Howrd Hughes Medil Institute. COMPETING INTERESTS STATEMENT The uthors delre tht they hve no ompeting finnil interests. Reeived 13 Jnury; epted 13 My 2003 Pulished online 8 June 2003; doi: /ni Coyle, A.J. & Gutierrez-Rmos, J.C. The expnding B7 superfmily: inresing omplexity in ostimultory signls regulting T ell funtion. Nt. Immunol. 2, (2001). 2. Hutloff, A. et l. ICOS is n induile T-ell o-stimultor struturlly nd funtionlly relted to CD28. Nture 397, (1999). 3. Swllow, M.M., Wllin, J.J. & Sh, W.C. B7h, novel ostimultory homolog of B7.1 nd B7.2, is indued y TNFα. Immunity 11, (1999). 4. Yoshing, S.K. et l. T-ell o-stimultion through B7RP-1 nd ICOS. Nture 402, (1999). 5. Ling, V. et l. Cutting edge: identifition of GL50, novel B7-like protein tht funtionlly inds to ICOS reeptor. J. Immunol. 164, (2000). 6. Wng, S. et l. Costimultion of T ells y B7-H2, B7-like moleule tht inds ICOS. Blood 96, (2000). 7. Brodie, D. et l. LICOS, primordil ostimultory lignd? Curr. Biol. 10, (2000). 8. Ling, L. & Sh, W.C. The right ple t the right time: novel B7 fmily memers regulte effetor T ell responses. Curr. Opin. Immunol. 14, (2002). 9. Ling, L., Porter, E.M. & Sh, W.C. Constitutive expression of the B7h lignd for induile ostimultor on nive B ells is extinguished fter tivtion y distint B ell reeptor nd interleukin 4 reeptor medited pthwys nd n e resued y CD40 signling. J. Exp. Med. 196, (2002). 10. Ishid, Y. et l. Indued expression of PD-1, novel memer of the immunogloulin gene superfmily, upon progrmmed ell deth. EMBO J. 11, (1992). 11. Nishimur, H. et l. Immunologil studies on PD-1 defiient mie: implition of PD- 1 s negtive regultor for B ell responses. Int. Immunol. 10, (1998). 12. Freemn, G.J. et l. Enggement of the PD-1 immunoinhiitory reeptor y novel B7 fmily memer leds to negtive regultion of lymphoyte tivtion. J. Exp. Med. 192, (2000). 13. Dong, H. et l. B7-H1, third memer of the B7 fmily, o-stimultes T-ell prolifertion nd interleukin-10 seretion. Nt. Med. 5, (1999). 14. Lthmn, Y. et l. PD-L2 is seond lignd for PD-I nd inhiits T ell tivtion. Nt. Immunol. 2, (2001). 15. Tseng, S.Y. et l. B7-DC, new dendriti ell moleule with potent ostimultory properties for T ells. J. Exp. Med. 193, (2001). 16. Chpovl, A.I. et l. B7-H3: ostimultory moleule for T ell tivtion nd IFN-γ prodution. Nt. Immunol. 2, (2001). 17. Sun, M. et l. Chrteriztion of mouse nd humn B7-H3 genes. J. Immunol. 168, (2002). 18. Yng, J. et l. IL-18-stimulted GADD45β required in ytokine-indued, ut not TCRindued, IFN-γ prodution. Nt. Immunol. 2, (2001). 19. Tomsello, E. et l. Signling pthwys engged y NK ell reeptors: doule onerto for tivting reeptors, inhiitory reeptors nd NK ells. Semin. Immunol. 12, (2000). 20. Bollnd, S. & Rveth, J.V. Inhiitory pthwys triggered y ITIM-ontining reeptors. Adv. Immunol. 72, (1999). 21. Shirtori, T. et l. Tyrosine phosphoryltion ontrols internliztion of CTLA-4 y regulting its intertion with lthrin-ssoited dptor omplex AP-2. Immunity 6, (1997). 22. Zhng, Y. & Allison, J.P. Intertion of CTLA-4 with AP50, lthrin-oted pit dptor protein. Pro. Ntl. Ad. Si. USA 94, (1997). 23. Songyng, Z. et l. Speifi motifs reognized y the SH2 domins of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, nd Vv. Mol. Cell. Biol. 14, (1994). 24. Nishimur, H. et l. Development of lupus-like utoimmune diseses y disruption of the PD-1 gene enoding n ITIM motif-rrying immunoreeptor. Immunity 11, (1999). 25. Shlptsk, L.M. et l. CD150 ssoition with either the SH2-ontining inositol phosphtse or the SH2-ontining protein tyrosine phosphtse is regulted y the dptor protein SH2D1A. J. Immunol. 166, (2001). 26. Hskins, K. et l. The mjor histoomptiility omplex restrited ntigen reeptor on T ells. I. Isoltion with monolonl ntiody. J. Exp. Med. 157, (1983). 27. Okzki, T. et l. PD-1 immunoreeptor inhiits B ell reeptor medited signling y reruiting Sr homology 2-domin-ontining tyrosine phosphtse 2 to phosphotyrosine. Pro. Ntl. Ad. Si. USA 98, (2001). 28. Chmers, C.A. et l. Seondry ut not primry T ell responses re enhned in CTLA-4-defiient CD8 + T ells. Eur. J. Immunol. 28, (1998). 678 VOLUME 4 NUMBER 7 JULY 2003 NATURE IMMUNOLOGY