ANGPTL binding ANGPTL binding ANGPTL4 GST ANGPTL5 ANGPTL6 ANGPTL7. A5+LILRB2 Fc. A5+Tie-2 Fc 10,000 7,500. Tie-2 Fc LILRB2 Fc. 5,000 K d = 5.

Transcription

1 doi:1.138/nture1195 ; Inhiitory reeptors ind ANGPTLs nd support < lood stem ells nd leukemi development Junke Zheng 1,2, Msto Umikw 1,3, Chngho Cui 1, Jiyun Li 1, Xioli Chen 1, Chozheng Zhng 1, HongDinh Hyunh 1, Xunlei Kng 1, Roert Silvny 1, Xun Wn 1, Jingxio Ye 1, Alerto Puig Cntó 4, Shu-Hsi Chen 5, Hun-You Wng 6, E. Slly Wrd 4 & Cheng Cheng Zhng 1 How environmentl ues regulte dult stem ell nd ner ell tivity through surfe reeptors is poorly understood. Angiopoietin-like proteins (ANGPTLs), fmily of seven sereted glyoproteins, re known to support the tivity of hemtopoieti stem ells (HSCs) in vitro nd in vivo 1 1. ANGPTLs lso hve importnt roles in lipid metolism, ngiogenesis nd inflmmtion, ut were onsidered orphn lignds euse no reeptors were identified 3,11,12. Here we show tht the immune-inhiitory reeptor humn leukoyte immunogloulin-like reeptor B2 () nd its mouse orthologue pired immunogloulin-like reeptor (PIRB) re reeptors for severl ANGPTLs. nd PIRB re expressed on humn nd mouse HSCs, respetively, nd the inding of ANGPTLs to these reeptors supported ex vivo expnsion of HSCs. In mouse trnsplnttion ute myeloid leukemi models, defiieny in intrellulr signlling of PIRB resulted in inresed differentition of leukemi ells, reveling tht PIRB supports leukemi development. Our study indites n unexpeted funtionl signifine of lssil immune-inhiitory reeptors in mintenne of stemness of norml dult stem ells nd in support of ner development. We used multiple pprohes, inluding expression loning, to identify the reeptor(s) for ANGPTLs. Humn, when etopilly expressed on BF3 ells, enled the ells to speifilly ind glutthione S-trnsferse (GST) ANGPTL5 s determined y flow ytometry (Fig. 1). is memer of the immune-inhiitory B-type sufmily of LILR reeptors 13 nd ontins four immunogloulin domins nd three immunoreeptor tyrosine-sed inhiitory motifs. Using flow ytometry nlysis, we further demonstrted tht - overexpressing 293T ells demonstrted enhned inding to severl ANGPTLs, espeilly ANGPTL2 nd GST ANGPTL5 (Fig. 1 nd Supplementry Fig. 1, ). ANGPTL2 nd GST ANGPTL5 lso ound to LILRB3- nd LILRB5-overexpressing ells, lthough with lower ffinity thn to -expressing ells (Supplementry Tle 1). In ddition, ANGPTL1 nd ANGPTL7 ound to 293T ells overexpressing LAIR1 ref. 14. (Supplementry Tle 1 nd Supplementry Fig. 2). ANGPTLs did not ind to LILRAs, LILRB1 or LILRB4 (Supplementry Tle 1). Beuse ANGPTL2 nd GST ANGPTL5 ound to - expressing ells etter thn other ANGPTLs, we further ssessed the moleulr intertion etween ANGPTL2/ANGPTL5 nd. ANGPTL5 inding ANGPTL5 inding IB:Anti-Flg IB:Anti-Flg ,33 GFP ontrol GFP + ontrol Control ANGPTL1 ANGPTL2 ANGPTL Tie-2 GFP ANGPTL4 GST ANGPTL5 ANGPTL6 ANGPTL7 Flg Flg+ Flg+ Flg Flg GFP IB:Anti- IB:Anti- ANGPTL inding ANGPTL inding d e + + 1, 7,5 5, K d = 5.5 nm 2, γ-ounts 125 I-ANGPTL5 (nm) γ-ounts 5, 4, 3, 2, 1, ANGPTL5 (nm) Figure 1 Cell-surfe inds to ANGPTLs., Flow ytometry nlysis of GST ANGPTL5 Flg inding to uninfeted BF3 ells or MSCV GFP, MSCV Tie-2 GFP- or MSCV GFP-stly infeted BF3 ells. Men fluoresene intensities re indited., Flow ytometry nlysis of indited Flg-tgged ANGPTLs inding to -trnsfeted 293T ells., ANGPTL2 nd ANGPTL5 ound to the ECD of ut not Tie-2 in onditioned medium () of otrnsfeted 293T ells. d, e, Conentrtiondependent speifi (d) nd ompetitive (e) 125 I GST ANGPTL5 inding to stly expressed BF3 ells (n 5 3). Error rs denote s.e.m. IB, immunolotting. 1 Deprtments of Physiology nd Developmentl Biology, University of Texs Southwestern Medil Center, Dlls, Texs 7539, USA. 2 Key Lortory of Cell Differentition nd Apoptosis of Chinese Ministry of Edution, Shnghi Jio Tong University Shool of Mediine, Shnghi 225, Chin. 3 Deprtment of Medil Biohemistry, University of the Ryukyus, Okinw , Jpn. 4 Deprtment of Immunology, University of Texs Southwestern Medil Center, Dlls, Texs 7539, USA. 5 Deprtment of Onologil Sienes, Mount Sini Shool of Mediine, New York, New York , USA. 6 Deprtment of Pthology, University of Cliforni Sn Diego, L Joll, Cliforni 9293, USA. These uthors ontriuted eqully to this work. MONTH 212 VOL NATURE 1

2 Co-trnsfetion of ANGPTL2 or ANGPTL5 with extrellulr domin (ECD) fused to humn IgG F () into 293T ells followed y immunopreipittion nd western lot showed tht oth ANGPTL2 nd ANGPTL5 interted with the ECD of, ut not tht of Tie-2 (Fig. 1 nd Supplementry Fig. 1). The diret intertions etween ANGPTLs nd were onfirmed y in vitro oimmunopreipittion, using purified ANGPTL2 Flg or GST ANGPTL5 nd (Supplementry Fig. 1d) nd y surfe plsmon resonne (SPR; Supplementry Fig. 3). A liquid-phse inding ssy with 125 I-lelled GST ANGPTL5 demonstrted tht the intertion etween ANGPTL5 nd ell-surfe ws speifi nd sturle, with hlf mximl sturtion of the intertion s nm (Fig. 1d, e). Although untgged ANGPTLs ind to, the type or the position of tgging ould ffet the inding (Supplementry Tle 2). Beuse severl ANGPTLs support expnsion of HSCs 4 12, we sought to determine whether ANGPTLs ound to or LAIR1 on primry humn ord lood ells. Flow ytometry nlysis showed tht ANGPTLs 1, 2, 5 nd 7 ll ound to 1 humn ord lood ells, nd tht ANGPTL2 nd GST ANGPTL5 hd higher ffinities (Fig. 2, Supplementry Fig. 4 nd Supplementry Tle 1). The inding of ANGPTL1 nd ANGPTL7 to LAIR1 1 humn ord lood ells ws reltively wek (Supplementry Fig. 5), nd we therefore foused on studying the inding of ANGPTL2 nd ANGPTL5 to in susequent experiments. We determined whether ws expressed on humn HSCs. Flow ytometry nd quntittive (q)rt PCR nlyses showed tht ws expressed on the surfe of 4 95% of humn ord lood CD34 1 CD38 CD9 1 ells (95% in the experiment shown in Fig. 2 nd Supplementry Fig. 6); this popultion is enrihed for HSCs. GST ANGPTL5 tretment indued inresed phosphoryltion of lium/ lmodulin-dependent protein kinse (CAMK)-2 nd -4 in humn ord lood mononuler ells (Supplementry Fig. 7). It is of note tht CAMK4 is required for mintenne of the poteny of HSCs 15. Suppression of expression with short hirpin RNAs effetively redued ANGPTL inding (Supplementry Fig. 8). Importntly, the silening of resulted in deresed repopultion of humn ord lood HSCs s mesured y reonstitution nlysis in non-oese dieti/severe omined immunodefiient (NOD/SCID) mie (1% repopultion from ultured knokdown ells ompred to 17% repopultion from ultured norml ells in medium STF; Fig. 2). Together, these dt indite tht ANGPTL5 supports expnsion of humn ord lood HSCs 1 in proess t lest prtilly medited y the surfe reeptor. PIRB is the mouse memrne orthologue of humn LILRBs 16,17. ANGPTL2, ANGPTL3 nd GST ANGPTL5 ound to PIRB s determined y flow ytometry (Fig. 3 nd Supplementry Fig. 9) nd oimmunopreipittion (Fig. 3 nd Supplementry Fig. 1). As with humn ord lood HSCs, mouse HSCs were lso enrihed for PIRB expression (Fig. 3 nd Supplementry Fig. 11). To study the funtion of PIRB in mouse HSCs, we used PIRBdefiient () mie 18, in whih four exons enoding the trnsmemrne domin nd prt of the intrellulr domin were deleted. ells freshly isolted from 3-week-old mie hd signifintly deresed CAMK4 phosphoryltion, nd inding of ANGPTL to PIRB indued phosphoryltion of PIRB, reruitment of SHP-1 nd SHP-2 (lso known s PTPN6 nd PTPN11, respetively) nd CAMK4 tivtion (Supplementry Figs 12 nd 13). These results suggest tht Control ANGPTL1 ANGPTL2 ANGPTL3 ANGPTL inding ANGPTL inding , , ANGPTL4 GST ANGPTL5 ANGPTL6 ANGPTL , CD38.5% CD34 Figure 2 medites the effet of ANGPTL in supporting the repopultion of humn ord lood HSCs., Flow ytometry nlysis of indited Flg-tgged ANGPTLs inding to 1 humn ord lood mononuler ells (FACSAri). Men fluoresene intensities re indited., Representtive flow ytometry plots for the o-stining of CD34, CD38, CD9 nd in humn ord lood mononuler ells (FACSCliur). 2 NATURE VOL MONTH 212 Nture nture1195.3d 1/5/12 12:59:34 95% of CD34 + CD38 CD9 + CD9 Repopultion (%) Fresh Ctrl Fresh STF STF STF, Humn ord lood CD34 1 ells infeted with shrna-enoding virus () or ontrol (Ctrl) srmle shrna virus were trnsplnted into sulethlly irrdited NOD/SCID mie efore or fter ulture for 1 dys. SCF1TPO1Flt3L (STF) or STF1ANGPTL5 (STF) ws used in the ulture. Shown is the humn donor repopultion fter 2 months (n ). P,.5. Error rs denote s.e.m.

3 ANGPTL inding Control ANGPTL2 ANGPTL IB:Anti-Flg IB:Anti-Flg + +PIRB F PIRB F PIRB F Figure 3 ANGPTLs ind PIRB nd support the repopultion of mouse? HSCs., Flow ytometry nlysis of Flg ANGPTL2 or GST ANGPTL5 Flg inding to PIRB-trnsfeted 293T ells., ANGPTL2 inds to the ECD of PIRB ut not Tie-2 in the onditioned medium () of otrnsfeted 293T ells., PIRB is expressed on mouse one mrrow () Lin 2 S-1 1 Kit 1 PIRB Repopultion (%) Gte on Lin d PIRB -Kit Control LSK PIRB 1.4% 92.4% HSCs HSCs HSCs+ANGPTL2 HSCs+ANGPTL2 S-1 S-1 3 weeks 6 weeks 16 weeks (LSK) ells. Isotype ontrol is indited in the left pnel. d, Competitive reonstitution of 8-dy ultured progenies of input equivlent to 25 Lin 2 S- 1 1 Kit 1 CD34 2 Flk-2 2 one mrrow HSCs from wild-type () or donors (n 5 5). SCF, TPO nd FGF-1, with or without ANGPTL2, were used in ulture. HSCs, ultured HSCs. P,.5. Error rs denote s.e.m. ertin ANGPTLs my e the lignds of PIRB tht tivte CAMK4 in vivo. Beuse SHP-2 nd CAMK4 re required for the repopultion of HSCs 15,19, nd the hemil inhiition of CAMK2, homologue of CAMK4, indues differentition nd suppresses prolifertion of myeloid leukemi ells 2, we sought to determine whether PIRB ws importnt for HSC tivity. Although the dult mie hve ertin immune nd neuronl defets, they re grossly norml in hemtopoiesis 16,18. Interestingly, ompetitive repopultion showed tht fetl liver HSCs hd pproximtely 5% deresed repopultion tivity (Supplementry Fig. 14). Moreover, lthough ANGPTL2 nd ANGPTL5 hd little effet on ex vivo expnsion of dult HSCs, they supported ex vivo expnsion of dult wildtype HSCs (Fig. 3d nd Supplementry Fig. 14), s we previously demonstrted 2. Colletively, our results indite tht ANGPTLs ind humn nd mouse PIRB to support HSC repopultion. On the sis of our in silio nlysis of pool of 9,4 smples desried previously 21, the level of messenger RNA is t lest fourfold higher in the humn ute monolsti nd monoyti leukemi ells (M5 sutype of ute myeloid leukemi (AML)) thn in other AML ells (Supplementry Fig. 15). As humn ute monolsti nd monoyti leukemi ells re often ssoited with rerrngement of mixed-linege leukemi (MLL; histone methyltrnsferse deemed positive glol regultor of gene trnsription), we used retrovirl MLL AF9 trnsplnttion mouse model 22,23 to further exmine the role of PIRB in regultion of AML development. Wild-type or donor Lin 2 ells infeted y retrovirl MLL AF9 internl riosome entry site (IRES) yellow fluoresent protein (YFP) were used to indue AML s previously desried 22,23. We exmined PIRB expression in YFP 1 M-1 1 Kit 1 ells tht my e enrihed for AML-inititing tivity 22,23, nd found tht out 8% of YFP 1 M-1 1 Kit 1 ells were PIRB 1 (Fig. 4). We next investigted whether PIRB ws required for the indution of AML y MLL AF9. Mie trnsplnted with MLL AF9-trnsdued wildtype ells developed AML nd died within pproximtely 5 weeks, wheres those trnsplnted with MLL AF9-trnsdued ells were resistnt to the indution of MLL AF9 nd developed AML muh more slowly (Fig. 4 nd Supplementry Fig. 16). The signifintly delyed development of the leukemi ws orrelted with out 5% redution in numers of white lood ells in irultion nd muh less severe infiltrtion of myeloid leukemi ells into the liver nd spleen (Fig. 4, d). Consistently, PIRB defiieny used n pproximtely 5% redution of YFP 1 M-1 1 Kit 1 ells in oth one mrrow nd peripherl lood (Fig. 4d). There were more CD3 1 or B22 1 ells in mie tht reeived MLL AF9-trnsdued donor ells thn in those given wild-type ells (Fig. 4d). These results demonstrte tht PIRB-medited signlling is ssoited with fster AML development nd greter numers of YFP 1 M-1 1 Kit 1 AML ells in vivo. We further ssessed whether PIRB potentilly regultes differentition nd self-renewl of AML ells. Colony-forming unit (.f.u.) > ssys showed tht extrinsi ANGPTL stimultion led to inresed.f.u. numers in wild-type ut not AML ells, gin inditing tht PIRB diretly medites the effets of ANGPTLs (Supplementry Fig. 16d). In ddition, wild-type AML ells formed mostly ompt olonies, wheres ells tended to form more diffuse ones (Fig. 4e). The formtion of diffuse olonies indites high differentition potentil 24. The inhiition of differentition of AML ells y PIRB is in ordne with previous reports tht PIRB inhiits differentition of myeloid-derived suppressive ells 25 nd osteolsts 26,s well s our dt showing tht endogenous ANGPTLs inhiit differentition nd inrese replting effiieny of hemtopoieti progenitors (Supplementry Fig. 17). Moreover, primry olony-forming units were unle to form seondry olonies upon replting (Fig. 4f), inditing tht PIRB supports self renewl of AML.f.u. ells. Finlly, we nlysed the moleulr signlling triggered y the inding of ANGPTLs to PIRB in AML ells. AML ells hd deresed phosphoryltion of phosphtse SHP-2 (Fig. 4h), whih is known to e ssoited with LILRB reeptors nd is n onogene tht supports leukemi development 13,16,18,27. ANGPTLs lso stimulted MONTH 212 VOL NATURE 3

4 Enrihment sore Enrihment sore Control 3.88% 1 d PIRB M % YFP Survivl (%) Dys YFP Reltive weight M Liver Spleen PB Reltive weight 8 Reltive ell numer e Kit.8 1 Gr-1 M CD PB f 3, 2,5.f.u. numers 2, 1,5 1, Kit g LSC signture_somerville NES: 1.24 FDR q-vl:.88 4 B22 Myeloid differentition_a NES: 1.37 FDR q-vl:.15 First Figure 4 PIRB suppresses differentition nd enhnes development of MLL AF9 AML., PIRB expression on YFP 1 M-1 1 Kit 1 AML ells s determined y flow ytometry., Survivl urve of mie reeiving MLL AF9- infeted or hemtopoieti progenitors (n 5 15); P,.5., Comprison of the sizes of spleen, liver nd numers of peripherl lood (PB) ells of the mie trnsplnted with wild-type () MLL AF9 ells nd MLL AF9 ells t 28 dys fter trnsplnttion (n 5 6). d, Representtive flow ytometry plots showing tht AML mie hve deresed M-1 1 Kit 1 ells nd inresed differentited ells reltive to mie trnsplnted with ells SHP-2 phosphoryltion (Supplementry Fig. 13). Similr to untrnsformed ells, AML ells hd deresed CAMK4 tivtion (dt not shown). Furthermore, wild-type M-1 1 Kit 1 ells hd muh greter expression of leukemi initition/mintenne genes 22,23, ut mrkedly deresed expression of myeloid-differentition genes s determined y DNA mirorry nlyses (Fig. 4g). qrt PCR onfirmed the inresed expression of severl HOXA genes, Meis1, Ey1, My nd Mef2 in wild-type M-1 1 Kit 1 ells thn their ounterprts (Supplementry Fig. 18); these genes re ritil for initition or mintenne of MLL-rerrnged AML (refs 22, 23). Similr to the MLL AF9 model, the defiieny of PIRB in the AML1 ETO9 leukemi model led to deresed numers of leukemi progenitors nd inresed numers of differentited ells (Supplementry Fig. 19). Colletively, these results indite tht the inding of ANGPTLs to PIRB promotes leukemi development, proly through inhiiting differentition of AML ells. nd PIRB re known to ind to other lignds, inluding vrious MHC lss I moleules 28 nd myelin inhiitors 17.Itwille importnt to investigte the in vivo ontext in whih these different lignds ind LILRB nd indue signlling. As ANGPTLs n e undntly expressed y mny types of ells, inluding those from endorine orgns 11 nd potentil one mrrow nihe (endothelium nd dipoytes 9,11 ), nd n e indued y hypoxi 11, these sereted ftors my hve importnt diret nd indiret effets on the tivities of HSCs nd leukemi stem ells in vivo. Although the LILRB/PIRB reeptors were reported to suppress tivtion of differentited 4 NATURE VOL MONTH 212 Nture nture1195.3d 1/5/12 12:59:38 t 28 dys fter trnsplnttion. e, Comprison of olony-forming tivity of nd MLL AF9 1 one mrrow ells. Shown is typil morphology of nd olony-forming units. f, MLL AF9 one mrrow ells hve mrkedly deresed olony-forming ility in seond replting (n 5 3). g, gene set enrihment nlysis plots evluting hnges in leukemi initition/mintenne nd myeloid-differentition gene signtures upon PIRB signlling depletion in or MLL AF9 M-1 1 Kit 1 AML ells. P,.5. Error rs denote s.e.m. FDR q-vl, flse-disovery rte q-vlue; NES, normlized enrihment sore. immune ells nd inhiit neurite outgrowth of neurl ells 16,17, they support HSC repopultion nd inhiit differentition of AML ells. This result suggests the importne of these inhiitory reeptors in mintenne of stemness of norml stem ells nd support of leukemi development. In ontrst to the stimultory reeptors suh s interferon reeptors or Toll-like reeptors tht tivte nd indue differentition of HSCs upon inflmmtion 29, nd PIRB my funtion s sensors of inflmmtion through inding to the inflmmtory ANGPTLs 12 nd proteting HSCs from exessive tivtion nd exhustion. Adult stem ells nd ner ells proly require oth stimultory reeptors nd inhiitory reeptors to mintin the lne of their ell ftes. METHODS SUMMARY Plsmid ytomeglovirus (V) Kozk humn Angiopoietin-1 nd ANGPTLs 1, 2, 3, 4, 6 nd 7 with Flg tgs t the roxy terminus were used for trnsfetion. ANGPTL2 Flg ws purified using M2 resin. Purified GST ANGPTL5 ws purhsed from Anov. Bterilly expressed Flg ANGPTL2 (with Flg t the mino terminus) nd ANGPTL2 Flg (with Flg t the C terminus) were onstruted in pet-26(1) vetor, nd GST ANGPTLs Flg in pgex vetor, nd expressed nd purified from teri. Murine stem ell virus (MSCV) IRES green fluoresent protein (GFP) or ontrol retrovirus-infeted BAF3 ells, V-driven LILRA-, LILRB-, PIRB-, or LAIR1-trnsfeted 293T ells, or humn mononuler ord lood ells were used in inding ssys. See the Methods for detiled experimentl methods for flow ytometry, oimmunopreipittion, SPR, liquid-phse inding, ulture, trnsplnttion,.f.u. nd gene set enrihment nlysis (GSEA). Mie were mintined t the University of Texs Southwestern Medil

5 Center niml fility. All niml experiments were performed with the pprovl of University of Texs Southwestern Committee on Animl Cre. Full Methods nd ny ssoited referenes re ville in the online version of the pper t Reeived 15 July 211; epted 29 Mrh 212. = Pulished online XX Zhng, C. C., K, M., Iizuk, S., Huynh, H. & Lodish, H. F. Angiopoietin-like 5 nd IGFBP2stimulteex vivo expnsionofhumn ordlood hemtopoietistemells s ssyed y NOD/SCID trnsplnttion. Blood 111, (28). 2. Zhng, C. C. et l. Angiopoietin-like proteins stimulte ex vivo expnsion of hemtopoieti stem ells. Nture Med. 12, (26). 3. Zhng, C. C. & Lodish, H. F. Cytokines regulting hemtopoieti stem ell funtion. Curr. Opin. Hemtol. 15, (28). 4. Huynh, H. et l. IGFBP2 sereted y tumorigeni ell line supports ex vivo expnsion of mouse hemtopoieti stem ells. Stem Cells 26, (28). 5. Chou, S. & Lodish, H. F. Fetl liver hepti progenitors re supportive stroml ells for hemtopoieti stem ells. Pro. Ntl Ad. Si. USA 17, (21). 6. Lin, M. & Zon, L. I. Geneti nlyses in zerfish revel tht ngiopoietin-like proteins 1 nd 2 re required for HSC development during emryogenesis. Am. So. Hemtol. 5th Ann. Meeting Astrt (28). 7. Khoury, M. et l. Mesenhyml stem ells sereting ngiopoietin-like-5 support effiient expnsion of humn hemtopoieti stem ells without ompromising their repopulting potentil. Stem Cells Dev. 2, (211). 8. Drke, A. C. et l. Humn CD34 1 CD133 1 hemtopoieti stem ells ultured with growth ftors inluding Angptl5 effiiently engrft dult NOD SCID Il2r 2/2 (NSG) mie. PLoS ONE 6, e18382 (211). 9. Zheng, J., Huynh, H., Umikw, M., Silvny, R. & Zhng, C. C. Angiopoietin-like protein 3 supports the tivity of hemtopoieti stem ells in the one mrrow nihe. Blood 117, (211). 1. Zheng, J. et l. Ex vivo expnded hemtopoieti stem ells overome the MHC rrier in llogenei trnsplnttion. Cell Stem Cell 9, (211). 11. Hto, T., Tt, M. & Oike, Y. The role of ngiopoietin-like proteins in ngiogenesis nd metolism. Trends Crdiovs. Med. 18, 6 14 (28). 12. Tt, M. et l. Angiopoietin-like protein 2 promotes hroni dipose tissue inflmmtion nd oesity-relted systemi insulin resistne. Cell Met. 1, (29). 13. Brrow, A. D. & Trowsdle, J. The extended humn leukoyte reeptor omplex: diverse wys of modulting immune responses. Immunol. Rev. 224, (28). 14. Meyrd, L. LAIR nd ollgens in immune regultion. Immunol. Lett. 128, (21). 15. Kitsos, C. M. et l. Clmodulin-dependent protein kinse IV regultes hemtopoieti stem ell mintenne. J. Biol. Chem. 28, (25). 16. Tki, T., Nkmur, A. & Endo, S. Role of PIR-B in utoimmune glomerulonephritis. J. Biomed. Biotehnol. 211, (211). 17. Atwl, J. K. et l. PirB is funtionl reeptor for myelin inhiitors of xonl regenertion. Siene 322, (28). 18. Syken, J., Grndpre, T., Knold, P. O. & Shtz, C. J. PirB restrits oulr-dominne plstiity in visul ortex. Siene 313, (26). 19. Chn, R. J. et l. Shp-2 heterozygous hemtopoieti stem ells hve defiient repopulting ility due to diminished self-renewl. Exp. Hemtol. 34, (26). 2. Si, J. & Collins, S. J. Ativted C2 1 /lmodulin-dependent protein kinse II is ritil regultor of myeloid leukemi ell prolifertion. Cner Res. 68, (28). 21. Lukk, M. et l. A glol mp of humn gene expression. Nture Biotehnol. 28, (21). 22. Krivtsov, A. V. et l. Trnsformtion from ommitted progenitor to leukemi stem ell initited y MLL AF9. Nture 442, (26). 23. Somerville, T. C. & Clery, M. L. Identifition nd hrteriztion of leukemi stem ells in murine MLL AF9 ute myeloid leukemi. Cner Cell 1, (26). 24. Lvu, C., Szilvssy, S. J., Slny, R. & Clery, M. L. Immortliztion nd leukemi trnsformtion of myelomonoyti preursor y retrovirlly trnsdued HRX- ENL. EMBO J. 16, (1997). 25. M, G. et l. Pired immunogloin-like reeptor-b regultes the suppressive funtion nd fte of myeloid-derived suppressor ells. Immunity 34, (211). 26. Mori, Y. et l. Inhiitory immunogloulin-like reeptors LILRB nd PIR-B negtively regulte osteolst development. J. Immunol. 181, (28). 27. Chn, R. J. & Feng, G. S. PTPN11 is thefirst identified proto-onogenetht enodes tyrosine phosphtse. Blood 19, (27). 28. Shiroishi, M. et l. Humn inhiitory reeptors Ig-like trnsript 2 (ILT2) nd ILT4 ompete with CD8 for MHC lss I inding nd ind preferentilly to HLA-G. Pro. Ntl Ad. Si. USA 1, (23). 29. Bldridge, M. T., King, K. Y. & Goodell, M. A. Inflmmtory signls regulte hemtopoieti stem ells. Trends Immunol. 32, (211). Supplementry Informtion is linked to the online version of the pper t Aknowledgements We thnk S. Armstrong for the MSCV MLL AF9 IRES YFP onstrut, H. Hos for the V ANGPTL6 Flg plsmid, T. Tki for providing the PIRB knokout mie to S.-H.C., X.-J. Xie for inding nlysis, nd UTSW Genomis nd Mirorry Core fility for DNA rry experiments. S.-H.C. thnks support from NIH. C.C.Z. ws supported y NIH grnt K1 CA 1299, Amerin Soiety of Hemtology Junior Fulty Awrd, Mrh of Dimes Bsil O Connor Sholr Awrd, DOD PR93256, CPRIT RP142, nd the Grielle s Angel Foundtion. Author Contriutions J.Z., M.U., C.C. nd C.C.Z. were responsile for the study design, identifition of reeptors, inding, signlling nd funtionl ssys, dt nlysis nd writing of the mnusript. J.L., X.C., C.Z., H.H., X.K., R.S. nd X.W. were responsile for inding nd signlling ssys nd dt nlysis. J.Y. nd S.-H.C. rried out the lignd-inding ssys, H.-Y.W. rried out AML hrteriztion, nd A.P.C. nd E.S.W. rried out the SPR ssy nd dt nlysis. Author Informtion DNA mirorry dt re ville for downlod from the GEO under ession numer GSE Reprints nd permissions informtion is ville t The uthors delre no ompeting finnil interests. Reders re welome to omment on the online version of this rtile t Correspondene nd requests for mterils should e ddressed to C.C.Z. (Ale.Zhng@UTsouthwestern.edu). MONTH 212 VOL NATURE 5