Clec4A4 is a regulatory receptor for dendritic cells that impairs inflammation and T-cell immunity

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1 Received 1 Oct 215 Accepted 8 Mr 216 Pulished 12 Apr 216 DOI: 1.138/ncomms11273 OPEN Clec4A4 is regultory receptor for dendritic cells tht impirs inflmmtion nd T-cell immunity Tomofumi Uto 1,, Tomohiro Fuky 1,, Hideki Tkgi 1, Keiichi Arimur 1,2, Tkeshi Nkmur 1,3, Noy Kojim 4, Bernrd Mlissen 5 & Ktsuki Sto 1,6 Dendritic cells (DCs) comprise severl susets tht re criticlly involved in the initition nd regultion of immunity. Clec4A4/DC immunoreceptor 2 (DCIR2) is C-type lectin receptor (CLR) exclusively expressed on CD8 conventionl DCs (cdcs). However, how Clec4A4 controls immune responses through regultion of the function of CD8 cdcs remins uncler. Here we show tht Clec4A4 is regultory receptor for the ctivtion of CD8 cdcs tht impirs inflmmtion nd T-cell immunity. Clec44 / CD8 cdcs show enhnced cytokine production nd T-cell priming following Toll-like receptor (TLR)-medited ctivtion. Furthermore, Clec44 / mice exhiit TLR-medited hyperinflmmtion. On ntigenic immuniztion, Clec44 / mice show not only ugmented T-cell responses ut lso progressive utoimmune pthogenesis. Conversely, Clec44 / mice exhiit resistnce to microil infection, ccompnied y enhnced T-cell responses ginst microes. Thus, our findings highlight roles of Clec4A4 in regultion of the function of CD8 cdcs for control of the mgnitude nd qulity of immune response. 1 Division of Immunology, Deprtment of Infectious Diseses, Fculty of Medicine, University of Miyzki, 52 Kihr, Kiyotke, Miyzki, , Jpn. 2 Deprtment of Orl nd Mxillofcil Surgery, Fculty of Medicine, University of Miyzki, 52 Kihr, Kiyotke, Miyzki, , Jpn. 3 Deprtment of Otolryngology, Hed nd Neck Surgery, Fculty of Medicine, University of Miyzki, 52 Kihr, Kiyotke, Miyzki, , Jpn. 4 Deprtment of Applied Biochemistry, Toki University, Kit-knme, Hirtsuk-shi, Kngw, Jpn. 5 Centre d Immunologie de Mrseille-Luminy, Université de l Méditerrnnée, Cse 96, Institut Ntionl de l Snté et de l Recherche Médicle U631, nd Centre Ntionl de l Recherche Scientifique UMR612, Mrseille 13288, Frnce. 6 Jpn Science nd Technology Agency, Precursory Reserch for Emryonic Science nd Technology (PRESTO), Hon-cho, Kwguchi, Sitm , Jpn. These uthors contriuted eqully to this work. Correspondence nd requests for mterils should e ddressed to K.S. (emil: ktsuki_sto@med.miyzki-u.c.jp). NATURE COMMUNICATIONS 7:11273 DOI: 1.138/ncomms

2 NATURE COMMUNICATIONS DOI: 1.138/ncomms11273 Dendritic cells (DCs) re essentil ntigen (Ag)-presenting cells (APCs) linking innte nd dptive immunity, which comprise heterogeneous susets, functionlly clssified into conventionl DCs (cdcs) nd plsmcytoid DCs (pdcs) 1 3. DCs ct s sentinels to sense the invding pthogens through vriety pttern-recognition receptors (PRRs), including Toll-like receptors (TLRs). Most of the memers of TLRs, except for TLR3, ssocite myeloid fctor88 (MyD88) on inding of the lignds, nd initites the ctivtion of vrious intrcellulr signlling cscdes, including interleukin (IL)-1 receptor-ssocited kinse 1/4 (IRAK1/4), tumour necrosis fctor (TNF) receptor-ssocited fctor 6 (TRAF6), nd inhiitor of nucler fctor kb (NF-kB) kinse- (IKK-) nd their downstrem, which further ctivte NF-kB nd mitogen-ctivted protein kinses (MAPKs), including ERK, JNK nd p38, re involved in the secretion of proinflmmtory cytokines, while the ctivtion of IKK- nd its downstrem interferon (IFN) regultory fctor (IRF)-7 re crucil for the production of type I IFN (IFN-I) (refs 4 6). The recognition of the lignds y TLR3 or TLR4 lso cuses their recruitment of the dpters Toll/IL-1 receptor (IL-1R) domin-contining dpter-inducing interferon (IFN)- (TRIF), which intercts with TANK-inding kinse 1 (TBK1) nd ctivtes, nd downstrem IRF-3 for the production of IFN-I, while TRIF lso ctivtes MAP kinses nd NF-kB leding to the production of proinflmmtory cytokines 4,5. DCs process nd present exogenous Ags to CD4 þ T cells through mjor histocomptiility complex clss (MHC) II-restricted pthwy for induction of CD4 þ effector T (CD4 þ T eff ) cells 7,8. DCs lso prime CD8 þ T cells medited y clssicl MHC I presenttion pthwy of endogenously synthesized proteins nd MHC I-dependent cross-presenttion pthwy of exogenous Ags for the genertion of cytotoxic T lymphocytes (CTLs) 7,8. Mouse CD11c þ cdcs in lymphoid orgns consist of two mjor susets, clssified into CD8 þ cdcs (cdc1) minly residing in the T-cell zone nd CD8 cdcs (cdc2) loclizing in the red pulp nd mrginl zone, including CD4 þ CD8 cdcs nd CD4 CD8 cdcs, which my exert distinct functions in immune responses 1 3. CD8 þ DCs pper to e specilized for the phgocytosis of ded cells nd the cross-presenttion of cell-ssocited nd solule Ags to induce strong CTL responses 7 1. In contrst, CD8 cdcs re more efficient thn CD8 þ DCs in MHC II-dependent presenttion pthwy to initite CD4 þ T-cell responses 7,11. C-type lectin receptors (CLRs) hve een known s PRRs recognizing wide vriety of glycn structures present on pthogens, commensls nd self-glycoproteins using their crohydrte recognition domin (CRD) in their extrcellulr portion, nd the lignd inding of CLRs on the surfce of myeloid cells often induces their internliztion nd/or intrcellulr signlling events 12,13. Severl CLRs ct s ctivtion receptors tht trnsduce intrcellulr signls vi n integrl immunoreceptor tyrosine-sed ctivtion motif (ITAM)-like motif within their cytoplsmic tils, or vi interction with ITAM-ering Fc receptor (FcR)g dptor molecules 13,14. Other CLRs possess n immunoreceptor tyrosine-sed inhiition motif (ITIM) in their cytoplsmic portions, nd intrcellulr signlling from these CLRs suppresses or prdoxiclly enhnces cellulr ctivtion DC susets reportedly express unique repertoires of CLRs tht re involved in Ag internliztion nd the routing to MHC I nd II loding comprtments, therey influencing T-cell responses 7,18. In stedy stte, DEC-25 nd Clec9A re predominntly expressed on CD8 þ DCs 7,18. In contrst, CD8 cdcs specificlly express Clec4A4 (DCIR2) recently found to e recognized y the 33D1 monoclonl ntiody (ma), which hs een widely utilized s enchmrk for the detection of mouse cdcs 7,19,2. Clec4A4 hs the potentil to deliver negtive signls due to the presence of n ITIM in its cytoplsmic portion tht possily ffects the cellulr ctivtion of CD8 cdcs. However, the physiologicl role for Clec4A4 in the regultion of immune responses through control of the function of CD8 cdcs remins uncler ecuse its function is poorly chrcterized reltive to those of other CLRs. In this study, we show y chrcterizing Clec4A4-deficient mice tht Clec4A4 cts s unique regultory CLR in the context of TLR-medited ctivtion of CD8 cdcs. We further demonstrte tht DCIR2 plys crucil role in the regultion of innte nd dptive immune responses under inflmmtory conditions in vivo. Collectively, these findings indicte tht Clec4A4 provides fine tuning of the function of mjor cdc susets to generte pproprite immune responses. Results Clec4A4 suppresses the function of cdcs. To ddress the function of Clec4A4 in cdcs, one mrrow (BM)-derived DCs (BMDCs) s well s cdc cell lines (DC2.4) (ref. 21), which did not express of Clec4A4 on the cell surfce (Supplementry Fig. 1,), were trnsfected with icistronic retrovirl vector crrying n internl riosome entry site followed y green fluorescent protein (IRES-GFP; mock-gfp) or C terminus FLAGtgged Clec4A4-IRES-GFP (Clec4A4-GFP) (Supplementry Fig. 1,), nd their production of cytokines ws nlysed fter stimultion with vrious TLR lignds. Following stimultion with Pm3CSK4 (TLR1/TLR2 lignds), poly(i:c) (TLR3 lignd), lipopolyscchride (; TLR4 lignd) or (TLR9 lignd), BMDCs or DC2.4-expressing Clec4A4-GFP showed lower production of IL-6 IL-12p4, IFN- nd TNF- thn BMDCs or DC2.4-expressing mock-gfp (Fig. 1; Supplementry Fig. 1c,d; Supplementry Fig. 2). To ddress how Clec4A4 regultes the TLR-medited cytokine production in cdcs, we exmined the signlling mechnism y which the expression of Clec4A4 cused the reduced production of cytokines in cdcs. BMDCs or DC2.4-expressing Clec4A4-GFP showed lower phosphoryltion of NF-kB p65 suunit, ERK, JNK nd p38 s well s IRF-3 or IRF-7 thn BMDCs or DC2.4-expressing mock-gfp fter stimultion with or (Fig. 1; Supplementry Fig. 1e; Supplementry Fig. 2). We lso investigted the ctivtion sttus of the ITIM in Clec4A4 fter the ligtions of TLRs nd Clec4A4. The stedy-stte phosphoryltion of the ITIM in Clec4A4 s well s its ssocition with SHP-1 nd SHP-2 ws oserved in BMDCs expressing Clec4A4-GFP, while crosslinking of Clec4A4 with nti-flag ma, ut not stimultion with, enhnced its phosphoryltion sttus nd recruitment of these phosphtses (Fig. 1c). We lso oserved tht tyrosine-specific phosphtse ctivity in the immunoprecipitte with Clec4A4 ws further enhnced following crosslinking of Clec4A4 with nti-flag ma (Fig. 1d). Clec4A4 consists of 236 mino cid, in which Y68 to L236 or I5 to V1 correspond to the extrcellulr portion or the ITIM sequence, respectively. To clrify the role of extrcellulr portion or ITIM in the suppressive function of Clec4A4 on the ctivtion of cdcs, we generted BMDCs expressing Clec4A4 mutnt lcking extrcellulr portion (Clec4A4 DY68 L236 -GFP) or ITIM in cytoplsmic tils (Clec4A4 DI5 V1 -GFP). Following stimultion with or, there ws no mrked difference in the TLR-medited cytokine production mong BMDCs expressing mock-gfp, BMDCs expressing Clec4A4 DY68 L236 -GFP, nd BMDCs expressing Clec4A4 DI5 V1 -GFP (Fig. 1; Supplementry Fig. 2), indicting tht the inhiitory effect of 2 NATURE COMMUNICATIONS 7:11273 DOI: 1.138/ncomms

3 NATURE COMMUNICATIONS DOI: 1.138/ncomms11273 ARTICLE IL-6 (pg ml 1 ) 5, 4, 3, 2, 1, Mock-GFP Clec4A4-GFP Clec4A4 N184Q -GFP Clec4A4 ΔY68-L236 -GFP Clec4A4 ΔI5-V1 -GFP (min): Phospho-p65 p65 Phospho-ERK IL-12p4 (pg ml 1 ) 5, 4, 3, 2, 1, Mock-GFP Mock-GFP Clec4A4-GFP Clec4A4 N184Q -GFP Clec4A4 ΔY68-L236 -GFP Clec4A4 ΔI5-V1 -GFP IFN-β (pg ml 1 ) Clec4A4-GFP , 1,5 1, 5 Mock-GFP Clec4A4-GFP Clec4A4 N184Q -GFP Clec4A4 ΔY68-L236 -GFP Clec4A4 ΔI5-V1 -GFP c IP:αFLAG : αflag IB:αp-Tyr IB:αSHP-1 IB:αSHP-2 IB:αFLAG TNF-α (pg ml 1 ) + + 2,5 2, 1,5 1, Mock-GFP Clec4A4-GFP Clec4A4 N184Q -GFP Clec4A4 ΔY68-L236 -GFP Clec4A4 ΔI5-V1 -GFP ERK Phospho-JNK JNK Phospho-p38 p38 Phospho-IRF-3 IRF-3 d Asornce t 41 nm : + + αflag: + + IP: αflag Figure 1 Retrovirl trnsduction of Clec4A4 suppresses the TLR-medited ctivtion of cdcs. () BMDCs expressing mock-gfp, Clec4A4-GFP, Clec4A4 N186Q -GFP, Clec4A4 DY68 L236 -GFP or Clec4A4 DI5 V1 -GFP were stimulted or not stimulted with the indicted, nd the production of cytokines ws mesured y enzyme-linked immunosorent ssy (ELISA). Dt re the men±s.d. from three individul smples in single experiment. Po.1 compred with BMDCs expressing mock-gfp or mong groups (nlysis of vrince (AN), Bonferroni s multiple comprison test). () BMDCs expressing mock-gfp or Clec4A4-GFP were stimulted or not stimulted with for the period indicted, t which time cells were lysed. Totl lyste ws nlysed using A specific for p65, ERK, JNK, p38 nd IRF-3 or for phosphorylted versions of these proteins. (c,d) BMDCs expressing mock- GFP or Clec4A4-GFP were stimulted or not stimulted with in comintion with or without crosslinking of Clec4A4 with nti-flag M2 ma for 3 min. (c) The immunoprecipitte with nti-flag M2 ma ws nlysed using nti-phosphotyrosine (p-tyr) ma, nti-shp-1 ma, nti-shp-2 ma or nti-flag M5 ma. (d) The immunoprecipitte with nti-flag M2 ma or control IgG ws nlysed for protein tyrosine phosphtse ctivity. Dt re the men±s.d. from three individul smples in single experiment. Po.1 compred with the immunoprecipitte with nti-flag M2 ma otined from unstimuted cells (AN, Bonferroni s multiple comprison test). All dt re representtive of t lest three independent experiments. Clec4A4 on the TLR-medited ctivtion of cdcs requires oth extrcellulr portion nd ITIM in cytoplsmic tils. Collectively, these results indicte tht retrovirl trnsfection of Clec4A4 suppresses the TLR-medited cytokine production medited through the impired ctivtion of the downstrem signlling cscdes in cdcs. CRD glycn interctions medite homotypic Clec4A4 inding. To identify the lignds of Clec4A4, we creted CLR fusion protein, which consists of the extrcellulr domin of Clec4A4 nd the Fc frgment of humn immunogloulin G (IgG) (Clec4A4-huIgFc). As the CRD of the fmily of Clec4As potentilly recognizes oligoscchride moieties 17,22, we exmined whether Clec4A4-huIgFc would ind to neoglycolipids (NGL) constructed with oligoscchrides nd diplmitoylphosphtidylglycerol (DPPG) 23 s crrier lipid (Fig. 2,). Clec4A4- huigfc, ut not huigfc nd Clec9A-huIgFc, exhiited more potent specific inding to mnnotriose (Mn3)-DPPG or lcto-n-fucopentose 3 (LNFP3 (Lewis x ))-DPPG thn DPPE lone or lcto-n-tetrose (LNT)-DPPG. On the other hnd, the specific inding of Clec4A4-huIgFc, ut not huigfc nd Clec9A-huIgFc, to intennry N-linked core pentscchride (BNCP)-DPPG, which contins N-cetylglucosmine (GlcNAc) nd Mn3 residues, ws higher thn Mn3-DPPG. We lso oserved tht Clec4A4-huIgFc, ut not huigfc nd Clec9A-huIgFc, specificlly ound to silognglioside-gm2 (silo-gm2) consisting of N-cetylglctosmine (GlNAc) nd lctocererosides ws higher thn tht to lctocererosides. As the CRD of SIGNR1 lso reportedly recognizes severl NATURE COMMUNICATIONS 7:11273 DOI: 1.138/ncomms

4 NATURE COMMUNICATIONS DOI: 1.138/ncomms11273 Asornce (OD45 nm) c αhuigfc-pe Mn3 Mnα1 Mnα1 6 Mn 3 BNCP GlcNcβ1 GlcNcβ1 2Mnα1 2Mnα1 LNT Glβ1 Lewis x 4GlcNcβ1 3Glβ1 4Glc Glβ1 4GlcNAcβ1 3 3Glβ1 4Glc Fucα huigfc Clec4A4-huIgFc SIGNR1-huIgFc Clec9A-huIgFc DPPG Mn3-DPPG huigfc... BNCP-DPPG Clec4A4 -huigfc GFP LNT-DPPG 6 Mn 3 Lctocererosides Gl Glc Cer Asilo-GM2 GlNAc Gl Glc Cer SIGNR1 -huigfc Clec9A -huigfc DC2.4 Mock-GFP Clec4A4-GFP oligoscchride residues of glycn 23, SIGNR1-huIgFc showed higher specific inding to Mn3-DPPG thn DPPG lone, wheres the inding of SIGNR1-huIgFc with BNCP-DPPG or Lewis x -DPPG ws comprle to tht with Mn3-DPPG or LNT-DPPG. Furthermore, there ws no different in the inding of SIGNR1-huIgFc to lctocererosides nd silo-gm2. Tken together, these results indicte tht the CRD of Clec4A4 specificlly recognizes D-mnnose (Mn), L-fucose (Fuc), GlcNAc nd GlNAc moieties on glycn. We further exmined whether Clec4A4-huIgFc directly ound to DC2.4-expressing Clec4A4-GFP ecuse the extrcellulr Lewis x -DEEG Lctocererosides Asilo-GM2 Figure 2 Self-interction of Clec4A4 through the inding of CRD nd glycns. () Schemtic digrm of structures of the oligoscchrides in NGL. Gl, D-glctose; Glc, D-glucose. () The inding of huigfc, Clec4A4- huigfc, SIGNR1-huIgFc or Clec9A-huIgFc to glycns ws mesured y ELISA. Dt re the men±s.d. from three individul smples in single experiment. Po.1 compred with DEEG or mong groups (nlysis of vrince (AN), Bonferroni s multiple comprison test). (c) The inding of huigfc, Clec4A4-huIgFc, SIGNR1-huIgFc or Clec9A-huIgFc to DC2.4, DC2.4-expressing mock-gfp, nd DC2.4-expressing Clec4A4-GFP ws nlysed y flow cytometry with nti-huigfc-pe. Dt re presented y dot plot, nd numers represent the proportion of the inding of huigfc, Clec4A4-huIgFc, SIGNR1-huIgFc or Clec9A-huIgFc in ech qudrnt. All dt re representtive of t lest three independent experiments. domin of Clec4A4 possesses severl predicted N-glycosyltion sites (Fig. 2c). While Clec4A4-huIgFc, ut not huigfc nd Clec9A-huIgFc, ound to DC2.4 nd DC2.4-expressing mock- GFP, the specific inding of Clec4A4-huIgFc to DC2.4-expressing Clec4A4-GFP ws significntly higher thn tht to DC2.4 nd DC2.4-expressing mock-gfp, indicting tht Clec4A4 intercts with itself on cdcs. To lesser degree, similr results were oserved in the inding of SIGNR1-huIgFc to DC2.4, DC2.4-expressing mock-gfp nd DC2.4-expressing Clec4A4-GFP. Since the self-interction of Clec4A4 would e medited y the inding of the CRD with oligoscchride resides on glycns, nd the N186 of Clec4A4 is only N-glycosyltion site in the CRD (ref. 17), we therefore generted BMDCs expressing glycosyltion mutnt of Clec4A4 (Clec4A4 N186Q -GFP) lcking the N-glycosyltion site in the CRD. The introduction of Clec4A4 N186Q -GFP displyed lower inhiitory effect on the cpcity of BMDCs to secrete cytokines fter stimultion with or s compred with tht of Clec4A4-GFP (Fig. 1; Supplementry Fig. 2), suggesting tht the N-glycosyltion of CRD is required for self-interction of Clec4A4 for the suppressive effect on the TLR-medited ctivtion of cdcs. Clec4A4 inhiits the TLR-medited ctivtion of CD8 cdcs. To investigte the physiologicl functions of Clec4A4 in the control of the immune responses through regultion of the function of CD8 cdcs, we creted Clec44 / mice (Supplementry Fig. 3 e), which were vile nd helthy. As expected, CD8 cdcs from Clec44 / mice lcked cell surfce expression of Clec4A4 (Fig. 3,). Histologicl nlysis of Spl otined from Clec44 / mice confirmed tht the expression of Clec4A4 ws not detected, while CD11c þ DCs were normlly loclized (Fig. 3c). CD8 cdcs otined from wild-type () mice nd Clec44 / mice hd similr expressions of MHC I (H-2K )nd MHC II (I-A/I-E) s well s CD11c nd costimultory molecules (Fig. 3d,e) under stedy-stte conditions. We lso oserved similr ility of CD8 cdcs etween mice nd Clec44 / mice to ctivte CD4 þ nd CD8 þ T cells using ovlumin ()-specific T-cell receptor (TCR) trnsgenic OT-II CD4 þ TcellsndOT-ICD8 þ T cells (refs 6,24; Fig. 3f,g). To ddress the influence of deficiency of Clec4A4 on the functions of CD8 cdcs, we nlysed the TLR-medited chnges of CD8 cdcs from mice nd Clec44 / mice. Wheres the injection of enhnced the expression of MHC I, MHC II, nd costimultory molecules on CD8 cdcs, nd their ility to ctivte OT-II CD4 þ T cells nd OT-I CD8 þ T cells in mice, compred with those from untreted mice, the expression levels of these molecules nd their ility to ctivte T cells were further enhnced in Clec44 / mice (Fig. 3e g; Supplementry Fig. 4). Collectively, the deficiency of Clec4A4 promotes the TLRmedited upregultion of MHC nd costimultory molecules in CD8 cdcs, leding to their enhncement of T-cell ctivtion. Becuse the induced expression of Clec4A4 suppresses TLRmedited cytokine production in cdcs, we compred the cytokine production of CD8 cdcs etween mice nd Clec44 / mice (Fig. 4). Clec44 / CD8 cdcs showed higher secretion of IL-6, IL-12p4, IFN- nd TNF- fter stimultion with Pm3CSK4, poly(i:c), nd thn CD8 cdcs. We lso oserved tht Clec44 / CD8 cdcs showed greter phosphoryltion of NF-kB p65 suunit, ERK, JNK nd p38 s well s IRF-3 or IRF-7 fter stimultion with or (Supplementry Fig. 5). These results indicte tht the deficiency of Clec4A4 enhnces the ility of CD8 cdcs to produce proinflmmtory 4 NATURE COMMUNICATIONS 7:11273 DOI: 1.138/ncomms

5 NATURE COMMUNICATIONS DOI: 1.138/ncomms11273 ARTICLE cytokines nd type I IFN, which is ssocited with the incresed ctivtion of NF-kB, MAPKs nd IRFs in response to TLR lignds. Clec4A4 suppresses the TLR-medited inflmmtion. We exmined the roles of Clec4A4 in the TLR-medited inflmmtion in vivo. The ppliction of Pm3CSK4, poly(i:c), or cused serum secretion of IL-6, IL-12p4, IFN- nd TNF- in mice (Fig. 5 nd Supplementry Fig. 6). On the other hnd, Clec44 / mice exhiited prominent elevtion in the serum production of these cytokines following injection with vrious TLR lignds, compred with mice (Fig. 5; Supplementry Fig. 6). We lso demonstrted tht Clec44 / mice showed more susceptility to lethlity induced y microil peritonitis thn mice, nd this lethlity ws correlted with the more mrked secretion of serum proinflmmtory cytokines 24 h following the dministrtion of het-killed Escherichi coli (Fig. 5,c). We next compred the proportion of leukocytes in the spleen etween mice nd Clec44 / mice under stedy-stte nd TLR lignd-induced inflmmtory conditions (Supplementry Fig. 7). In the stedy-stte conditions, Clec44 / mice exhiited regulr myeloid nd lymphoid immune cell comprtments in the spleen. The dministrtion of to mice incresed the weight of spleen nd the solute numer of leukocytes, with elevted frequencies of CD11c B22 þ B cells nd CD11c þ B22 þ pdcs nd reduced proportion of other leukocytes. On the other hnd, Clec44 / mice showed greter weight of spleen nd more splenocytes thn mice fter injection with. Furthermore, Clec44 / mice exhiited higher frequency of CD11c B22 þ B cells, nd lower frequencies of CD3 þ T cells, CD4 þ T cells nd CD8 þ T cells thn mice following injection with. Tken together, these results indicte tht the deficiency of Clec4A4 ugments TLR-medited inflmmtion in vivo. Clec44 ttenutes the responses of CD4 þ T cells. We exmined how Clec4A4 controls CD4 þ T-cell responses through the regultion of the function of CD8 cdcs. In vitro nlysis reveled tht Clec44 / CD8 cdcs displyed n enhnced cpcity to generte -specific IFN-g-producing CD4 þ T cells (T helper 1; T H 1 cells) nd IL-17-producing CD4 þ T cells (T H 17 cells) when compred with CD8 cdcs (Fig. 6 d; Supplementry Fig. 8,). To determine the roles of Clec4A4 in the cpcity of CD8 cdcs to prime Ag-specific CD4 þ T cells in vivo, OT-II CD4 þ T cells, which were lelled with croxyfluorescein dicette-succinimidyl ester (CFSE), were doptively trnsferred into mice, nd their division ws monitored 3 dys fter dministrtion of solule protein under stedy-stte or inflmmtory conditions (Fig. 6e,f; Supplementry Fig. 8c). While similr Ag-specific division of OT-II CD4 þ T cells etween mice nd Clec44 / mice ws oserved fter systemic injection of protein, Clec44 / mice exhiited the enhncement of this response compred with mice following the dministrtion of protein plus vrious TLR lignds. We lso compred the responses of Ag-specific CD4 þ T cells etween mice nd Clec44 / mice. At 14 dys fter immuniztion with protein nd complete Freund s djuvnt (CFA), CD4 þ T cells from Clec44 / mice showed more prominent prolifertion nd secretion of IFN-g s well s the genertion of T H 1 cells thn those from mice (Fig. 6g i). Similrly, Clec44 / mice hd the enhnced Ag-specific prolifertion nd production of IFN-g nd IL-17 s well s the ugmented genertion of T H 1 cells nd T H 17 cells compred with mice following immuniztion with plus (Supplementry Fig. 8d f). Tken together, these results indicte tht the deficiency of Clec4A4 potentites the responses of Ag-specific CD4 þ T eff cells in vivo. Clec4A4 reduces the responses of CD8 þ T cells. We ddressed the influence of the deficiency of Clec4A4 on the cpcity of CD8 cdcs to ctivte of CD8 þ T cells through the cross-presenttion of solule Ag in vivo. On immuniztion with protein plus vrious TLR lignds, Clec44 / mice displyed significnt enhncement of Ag-specific prolifertion of doptively trnsferred OT-I CD8 þ T cells compred with mice (Fig. 7,; Supplementry Fig. 9). To exmine the influence of the deficiency of Clec4A4 on the induction of CTLs through the cross-presenttion of solule Ag, we quntified the genertion of Ag-specific CD8 þ T cells sed on inding with the MHC I- tetrmer nd intrcellulr expression of IFN-g 6 dys fter immuniztion with protein comined with vrious TLR lignds nd nti-cd4 ma (Fig. 7c f; Supplementry Fig. 9,c). Clec44 / mice hd more mrked ugmenttion in the induction of MHC I- tetrmer þ CD44 high CD8 þ T cells nd CD8 þ IFN-g þ T cells thn mice. Collectively, these results indicte tht the deficiency of Clec4A4 promotes the ility of CD8 cdcs to ctivte CD8 þ T cells through cross-presenttion in vivo. Clec4A4 meliortes experimentl utoimmune encephlitis. We ssessed the impct of the deficency of Clec4A4 on the initition nd progression of experimentl utoimmune encephlitis (EAE). Immuniztion of mice with myelin oligodendrocyte glycoprotein peptide (MOGp) together with CFA elicited EAE (Fig. 8,), which ws ccompnied y the genertion of Ag-specific CD4 þ T-cell prolifertion, nd the induction of T H 1 cells nd T H 17 cells following ex vivo recll ntigenic stimultion, s well s the secretion of nti- MOGp-specific IgG (Fig. 8c g). In contrst, Clec44 / mice showed more progressive development of EAE thn mice (Fig. 8,). Furthermore, Clec44 / mice displyed higher Ag-specific prolifertion of CD4 þ T cells thn mice (Fig. 8c). Indeed, CD4 þ T cells from Clec44 / mice not only exhiited more potent production of IFN-g nd IL-17 ut lso hd higher frequency of T H 1 cells nd T H 17 cells thn mice (Fig. 8d f). On the other hnd, Clec44 / mice showed higher production of nti-mogp-specific IgG thn mice (Fig. 8g). Collectively, the deficiency of Clec4A4 ggrvtes the development of T-cell-medited utoimmune disese through the excessive ctivtion of CD8 cdcs. Clec4A4 suppresses host protection ginst cteril infection. To determine the role of Clec4A4 in the host protection ginst cteril infections through the regultion of the function of CD8 cdcs, we exmined the host protective immune responses ginst Listeri monocytogenes expressing (LM-) 25. While mice succumed to cteril infection within 11 dys, Clec44 / mice showed n enhnced survivl rte (Po.1), which ws ssocited with significnt reduction in splenic cteril urden, nd prominent elevtion in serum productions of IL-6, IL-12p4 nd IFN-g (Supplementry Fig. 1 c). Furthermore, Clec44 / mice displyed the enhnced nti-cteril responses of CD4 þ T cells following cteril infection when compred with mice (Supplementry Fig. 1d f). NATURE COMMUNICATIONS 7:11273 DOI: 1.138/ncomms

6 NATURE COMMUNICATIONS DOI: 1.138/ncomms11273 Collectively, these results indicte tht the deficiency of Clec4A4 promotes nti-cteril protective immune responses through the ugmenttion of the function of CD8 cdcs unless the inflmmtion cuses the microil septic shock. Discussion As descried in this mnuscript, our iochemicl nd genetic results clerly demonstrte tht Clec4A4 cts s unique regultory CLR for the TLR-medited ctivtion of CD8 cdcs tht is criticl for control of the mgnitude nd qulity of innte nd dptive immune responses. To our knowledge, our findings re the first descriing tht Clec4A4 known s DC-specific surfce mrker molecule suppresses the cellulr ctivtion nd mturtion of CD8 cdcs, while most PRRs expressed on DCs reportedly ct s ctivtion receptors 1 3. Furthermore, the Clec4A4-medited regultion of CD8 cdcs ws shown to impct on immune responses in vivo. The fmily of Clec4As is the only group of clssicl CLRs with n ITIM in their cytoplsmic til 17,26. In shrp contrst to Clec4A4, Clec4A2 (DCIR1) is expressed on vrious immune cells, such s B cells, monocytes/mcrophges nd DCs 17,22,27.It hs een shown tht the triggering of humn Clec4A with specific ma not only elicits its internliztion to endosoml/lysosoml comprtments leding to Ag presenttion, ut lso results in inhiition of the TLR9-medited secretion of type I IFN on humn pdcs nd the TLR8-medited production of proinflmmtory cytokines on humn cdcs, lthough which signlling pthwy is elicited fter the ligtion of Clec4A, leding to the inhiition of TLR signlling, remins unresolved 17,28,29. Here we show tht retrovirl trnsfection of Clec4A4 into cdcs impirs TLR-medited cytokine production, possily through suppression of the ctivtion of MyD88- or TRIF-dependent ctivtion of NF-kB, MAPKs nd IRFs. Consistently, deficiency of Clec4A4 specificlly leds to the hyperresponsiveness of CD8 cdcs to vrious TLR lignds for the secretion of cytokines medited y the ugmented ctivtion of NF-kB, MAPKs nd IRFs. Thus, our findings strongly suggest tht Clec4A4 genertes n inhiitory signl for the TLR-medited downstrem cscdes to rogte the ctivtion of CD8 cdcs. Previous studies hve shown tht the phosphorylted ITIM in humn Clec4A is le to interct with SHP-1 nd SHP-2 using pervndte tretment 3 or peptide contining the phosphorylted form of ITIM 31, lthough their physiologicl relevnce remins uncler. We demonstrted tht Clec4A4 mutnt lcking ITIM in cytoplsmic tils hd no inhiition on the TLR-medited cytokine production in cdcs, suggesting tht the inhiitory effect of Clec4A4 on the TLR-medited ctivtion of cdcs directly involves the ITIM-medited intrcellulr signlling. Furthermore, Clec4A4 constitutively ssocited with SHPs under stedy-stte phosphoryltion of its ITIM, nd the ligtion of Clec4A4 not only potentited its phosphoryltion sttus, ut lso enhnced the recruitment of these phosphtses nd their functions. As the deficiency of SHPs reportedly mplified the TLR-medited downstrem signlling cscdes for the enhnced production of IFN-I nd proinflmmtory cytokines, SHP-1 hs een shown to lock the MyD88- dependent cytokine secretion y suppressing the ctivtion of NF-kB, MAPK, IRAK1, IKK- nd IKK- medited through directly inding nd dephosphorylting them, wheres SHP-2 hs een shown to inhiit the TLR3/4-medired cytokine production in TRIF-dependent signlling due to the direct interction with TBK1 (refs 4,32 34). Therefore, the moleculr inhiitory mchinery in the Clec4A4-medited suppression of TLR lignd-induced cytokine production could involve the ssocition of SHP-1 or SHP-2 with the phosphorylted ITIM in Clec4A4, leding to the impirment of MyD88- or TRIF-dependent ctivtion of the downstrem signlling pthwy. Anlysis of Clec4A4 mutnt lcking extrcellulr portion suggests tht the excessive expression of memrne-nchored ITIM might e insufficient for the suppressive effect on the TLR-medited ctivtion of cdcs when extrcellulr portion of Clec4A4 is lcking. Furthermore, glycosyltion mutnt of Clec4A4 lcking the N-glycosyltion site in the CRD, which possily prevented the formtion of self-interction, reduced the inhiitory effect. Thus, the self-interction of Clec4A4 in cis nd/or trns through the inding of its CRD with oligoscchride resides on glycns could e required for the suppressive effect on the TLR-medited ctivtion of cdcs. Similr to the proposed concept for the msking of Siglecs 35, the potentil interction of Clec4A4 with glycns present on Clec4A4 itself nd cis lignds existed on neighouring glycoproteins could occupy its CRD ecuse we clerly demonstrted tht the stedy-stte phosphoryltion of ITIM in Clec4A4 in cdcs. Indeed, we showed tht the solule form of Clec4A4 specificlly ound to Mn, Fuc, GlcNAc nd GlNAc moieties on glycns nd Clec4A4-expressing cdc trnsfectnts, while it lso ound to their control trnsfectnts to lesser extent. Thus, it is intriguing to hypothesize tht Clec4A4 constitutively ssocites with itself in ddition to other djcent glycoproteins (for exmple, SIGNR1) medited through the inding of CRD with oligoscchride resides on glycns, nd the inhiitory signlling vi ITIM in Clec4A4 could potentilly occur under stedy-stte conditions, resulting in lowering of the responsiveness of CD8 cdcs to TLR-medited ctivtion. Different from our oservtion on the suppressive role of Clec4A4 in the TLR-medited ctivtion of CD8 cdcs, the deficiency of Clec4A2 reportedly did not ffect the response of BMDCs to stimultion 36, despite the fct tht these two Clec4As shre similr extrcellulr domin nd cytoplsmic portions. It remins uncler how distinct Clec4A4s led to different cellulr responses, the kinetics, ffinity nd specificity of glycn inding, or the vlency of enggement of Figure 3 Deficiency of Clec4A4 modultes TLR-medited ctivtion of CD8 cdcs. (,) The expression of Clec4A4 nd cell surfce molecules on splenocytes () nd CD11c þ DCs () otinedfrommicendclec44 / mice ws nlysed y flow cytometry. Dt re presented y dot plot, nd numers represent the proportion in ech qudrnt. (c) Immunofluorescent microscopic nlysis ws performed on frozen horizontl sections. Sections were stined for CD19 (green), CD3e (lue) nd Clec4A4 (red) or CD11c (red). (d) The expression of Clec4A4 nd cell-surfce molecules on CD8 cdcs otined from mice nd Clec44 / mice ws nlysed y flow cytometry. Dt re presented y histogrm, nd numers represent men fluorescence intensity (MFI). (e)mice(n ¼ 6) nd Clec44 / mice (n ¼ 6) were injected with or without, nd CD8 cdcs were otined 24 h fter injection. The expression of cell surfce molecules on CD8 cdcs ws nlysed y flow cytometry. Dt re MFI±s.d. from six individul smples in single experiment. (f,g)mice(n ¼ 6) nd Clec44 / mice (n ¼ 6) were not injected (left pnel) or injected (right pnel) with, nd CD8 cdcs were otined 24 h fter injection. CD45.1 þ OT-II CD4 þ Tcells(f) or CD45.1 þ OT-I CD8 þ Tcells(g) wereculturedwithcd8 cdcs (1 4 )otinedfrom mice nd Clec44 / mice in the presence or sence of peptide (1 mm; f) or peptide (1 nm; g), nd the prolifertion ws mesured y [ 3 H]thymidine incorportion. Dt re the men±s.d. from six individul smples in single experiment. Po.1 compred with mice (nlysis of vrince (AN), Bonferroni s multiple comprison test). All dt re representtive of t lest three independent experiments. 6 NATURE COMMUNICATIONS 7:11273 DOI: 1.138/ncomms

7 NATURE COMMUNICATIONS DOI: 1.138/ncomms11273 ARTICLE ech Clec4A, s well s how cell-type-specific expression potentilly ccounts for the distinct signlling through the ITIM-medited regultion of cell function. Wheres vrious immune cells, including DCs nd nonhemtopoietic cells, hve een reported to express vrious TLRs to respond to ech lignd 37, the contriution of CD8 cdcs to the TLR-medited responses nd their regultory mechnism in vivo remins uncler. In line with the ugmented TLRmedited cytokine production y Clec44 / CD8 cdcs, nlysis of in vivo responsive to TLR lignds nd cteril peritonitis reveled tht Clec44 / mice exhiited hyperinflmmtory response ssocited with the excessive > Clec4A4.3 >.1.4 >.1.3 >.1.2 >.1.2 >.1.4 >.1 Clec4A CD11c CD4 CD8α CD11 B22 Siglec-H CD4 CD8 B22 c CD19/Clec4A4/CD3ε CD19/Clec4A4/CD3ε CD19/CD11c/CD3ε CD19/CD11c/CD3ε d CD11c Clec4A4 CD4 CD8 CD86 H-2K I-A/I-E e CD4 (MFI) 1, CD8 (MFI) CD86 (MFI) 1, H-2K (MFI) 8, 6, 4, 2, I-A/I-E (MFI) 6, 4,5 3, 1,5 f [ 3 H] thymidine uptke ( 1 3 ) 4 p [ 3 H] thymidine uptke ( 1 3 ) p g [ 3 H] thymidine uptke ( 1 3 ) 3 p [ 3 H] thymidine uptke ( 1 3 ) p NATURE COMMUNICATIONS 7:11273 DOI: 1.138/ncomms

8 NATURE COMMUNICATIONS DOI: 1.138/ncomms11273 IL-6 (pg ml 1 ) IL-6 (pg ml 1 ) c IL-6 (pg ml 1 ) d IL-6 (pg ml 1 ) 2, 1,5 1, 5 1,5 1, ,5 1, , 3, 2, 1, IL-12p4 (pg ml 1 ) IL-12p4 (pg ml 1 ) IL-12p4 (pg ml 1 ) IL-12p4 (pg ml 1 ) , 3, 2, 1, production of cytokines. These findings suggest tht Clec4A4 increses the threshold of responsiveness of CD8 cdcs to TLR lignds s well s other pthogen-ssocited moleculr ptterns to prevent excessive detrimentl to inflmmtion. IFN-β (pg ml 1 ) IFN-β (pg ml 1 ) IFN-β (pg ml 1 ) IFN-β (pg ml 1 ) TNF-α (pg ml 1 ) TNF-α (pg ml 1 ) TNF-α (pg ml 1 ) TNF-α (pg ml 1 ) 3, 2, 1, , 3, 2, 1, 4, 3, 2, 1, Figure 4 Deficiency of Clec4A4 enhnces the ility of CD8 cdcs to produce cytokines in response to TLR lignds. CD8 cdcs otined from mice nd Clec44 / mice were stimulted or not stimulted with Pm3CSK4 (), poly(i:c) (), (c) nd (d), nd the production of cytokines ws mesured y ELISA. Dt re the men±s.d. from three individul smples in single experiment. Po.1 compred with mice (nlysis of vrince (AN), Bonferroni s multiple comprison test). All dt re representtive of t lest three independent experiments. Although there hve een severl reports descriing the role for CD8 cdcs in the induction of the responses of CD4 þ T cells in vivo y the Ag trgeting to this DC suset vi 33D1 ma 7,2, how CD8 cdcs instruct nd regulte the responses of CD4 þ T cells in vivo remins uncler. Our in vitro nlysis showed tht the deficiency of Clec4A4 promoted the ility of CD8 cdcs to generte Ag-specific T H 1/T H 17 cells. Furthermore, the deficiency of Clec4A4 not only enhnced Ag-specific priming of CD4 þ T cells ut lso ugmented CD4 þ T eff -cell responses in vivo. These oservtions strongly suggest tht the deficiency of Clec4A4 leds to enhncement of the cpcity of CD8 cdcs to upregulte the expression of MHC nd costimultory molecules, nd to produce cytokines fter the ligtion of TLRs, resulting in the promoted development of CD4 þ T eff cells from nive CD4 þ T cells in vivo under inflmmtory conditions. Thus, Clec4A4 could regulte APC function of CD8 cdcs for tight control of the direction of the responses of CD4 þ T eff cells in vivo. CD8 þ cdcs hve reportedly shown to e superior to CD8 cdcs to prime CD8 þ T cells for the genertion of CTL response through cross-presenttion, possily owing to the differences in expression of proteins ssocited with Ag processing for MHC I presenttion 1,7,8. Notly, the deficiency of Clec4A4 cused further increse in the priming of Ag-specific CD8 þ T cells nd induction of CTLs in vivo when solule Ag ws immunized, demonstrting the potentil cross-presenttion cpcity of CD8 cdcs for the efficient genertion of CTLs. Therefore, Clec4A4 could strictly suppress the TLR-medited mplifiction of the expression of severl proteins involved in cross-presenttion to ctivte CD8 þ T cells in CD8 cdcs under pthophysiologicl conditions. It hs een shown tht Clec42 (Dcir1) / mice exhiited n excertion of the pthogenesis in collgen-induced rthritis (CIA), possily due to the excessive expnsion of CD11c þ DCs 36. Furthermore, the expression of CD8, CD86 nd MHC II on CD11c þ DCs in CIA-induced Clec42 / mice ws t norml level for their stedy-stte conditions, indicting tht DCIR1 could not e involved in the mturtion nd ctivtion of CD11c þ DCs 36. On the other hnd, our results demonstrted tht Clec44 / mice displyed progression of the pthogenesis of EAE, which is ccompnied y the enhnced genertion of MOG-specific T H 1 cells nd T H 17 cells s well s mssive production of nti-mogp-specific IgG. Therefore, in shrp contrst to the suppressive role of Clec4A2 in the expnsion of CD11c þ DCs for the development of utoimmune diseses, Clec4A4 could specificlly downregulte the mturtion nd ctivtion of CD8 cdcs rther thn their expnsion, leding to impirment of the pthogenic responses of CD4 þ T eff cells, which is responsile for meliortion of the pthogenesis of utoimmune diseses. While cdcs hve reportedly een involved in host defenses ginst cteril infections medited through the recognition of pthogen-ssocited moleculr ptterns y severl ctivting PRRs 5,38, the role of CLRs in the ortive ctivtion of cdcs leding to the inhiition of nti-microil immunity remins to e understood. On cteril infection, the deficiency of Clec4A4 not only led to the ugmenttion of nti-cteril CD4 þ T eff cell-responses, ut lso displyed the prominent elimintion of the invded cteri, resulting in the reduction of the mortlity. These oservtions led us to hypothesize tht the interction of the CRD of Clec4A4 with the glycn present on microes lso cuse the impired ctivtion of CD8 cdcs, which hmpers nti-microil host defense. In conclusion, we descried tht Clec4A4 constitutes CLR endowed with criticl negtive regultory function in CD8 cdcs, in which it prevents the induction of excerted 8 NATURE COMMUNICATIONS 7:11273 DOI: 1.138/ncomms

9 NATURE COMMUNICATIONS DOI: 1.138/ncomms11273 ARTICLE Serum IL-6 (ng ml 1 ) Survivl (%) Time fter injection (h) Serum IL-12p4 (ng ml 1 ) Time fter injection of E. coli (h) Time fter injection (h) c Serum IL-6 (pg ml 1 ) Time fter injection (h) 6, 4,5 3, 1,5 Serum IL-12p4 (pg ml 1 ) 1,5 1, Time fter injection (h) Figure 5 Deficiency of Clec4A4 leds to TLR-medited hyperinflmmtory response in vivo. () mice (n ¼ 6) nd Clec44 / mice (n ¼ 6) were injected with, nd serum production of cytokines ws mesured t the indicted time fter injection y ELISA. Dt re the men±s.d. from six individul smples in single experiment. Po.1 compred with mice (nlysis of vrince (AN), Bonferroni s multiple comprison test). (,c) mice (n ¼ 1) nd Clec44 / mice (n ¼ 1) were injected with het-killed E. coli.() Survivl rte ws monitored t the indicted times for 6 h fter injection of het-killed E. coli. Po.1 compred with mice (Kpln Meier log-rnk test). (c) Serum production of cytokines ws mesured 24 h fter injection with het-killed E. coli y ELISA. Dt re the men±s.d. from 1 individul smples in single experiment. Po.1 compred with mice (nlysis of vrince (AN), Bonferroni s multiple comprison test). All dt re representtive of t lest three independent experiments. Serum IFN-β (ng ml 1 ) Serum TNF-α (ng ml 1 ) Serum TNF-α (pg ml 1 ) 4, 3, 2, 1, inflmmtion nd pthologicl T-cell responses, nd therefore mintins immune homeostsis nd prevents immunopthogenesis. Conversely, Clec4A4 lso contriutes to the suversion of nti-microil host protective immune responses medited through the ortive ctivtion of CD8 cdcs, which is linked to the microil evsion from immune surveillnce. Thus, our findings proposed the concept tht regultory CLR inding to endogenous lignds on cdcs nd pthogens plys n crucil role in immune homeostsis nd host defense. Trgeting this unique CLR with ntiody (A) my constitute new therpies for utoimmune nd inflmmtory disorders s well s infectious diseses nd cncer. Methods Mice. The following 8- to 12-week-old femle mice were used in this study: C57BL/6 mice (Jpn Cle), B6.CD45.1 þ OT-I TCR trnsgenic mice hrouring -specific CD8 þ T cells 6,24, nd B6.CD 45.1 þ OT-II TCR trnsgenic mice hrouring -specific CD4 þ T cells 6,24, nd B6.Clec44 / mice s descried elow. All mice were red nd mintined in specific pthogen-free conditions in the niml fcility t University of Miyzki in ccordnce with institutionl guidelines of the Animl Experiment Review Bord. Genertion of Clec44 / mice. The trgeting vector for Clec44 / mice ws constructed in the pbluescript vector y using 4.-k genomic frgment (left rm) upstrem of Clec44 exon 1 nd 2.-k genomic frgment (right rm) downstrem of exon 2 cloned from modified cteril rtificil chromosome clone, RP23-265M17 (Children s Hospitl Oklnd Reserch Institute), contining the complete Clec44 gene (gene symol Clec44). The left rm ws generted y PCR using the following oligonucleotides: left rm forwrd (5 -CGCCTCGAGGTGATG AATCAAAGATTTAACAGAATGTA-3 ) nd left rm reverse (5 -CGCGTCGACT TCAAGAAAAGACCTGCCTCCCTCAGAAAGCACAA-3 ). The 4.-k PCR frgment ws digested with XhoI nd SlI, nd ligted into ech site of pbluescript. The right rm ws generted y PCR using the following oligonucleotides: right rm forwrd (5 -CGCGTCGACGTAAGTATCCTGCACACATCAATGGGCCT TGTCTG-3 ) nd right rm reverse (5 -CGCACTAGTGTTCCTCAGAA AATTGGACATATTACTAC-3 ), digested with SlI nd SpeI, nd ligted into ech site of the trgeting vector. Ech of the 5 nd 3 primers ws lso tgged (indicted in itlics) with XhoI nd SlI sites for the left rm or SlI nd SpeI sites for the right rm, respectively. A SlI restriction site ws engineered in plce of the strt codon in exon 1. The pires2-egfp-loxp-cre/neo r -loxp uto-deleter cssette 39 ws cloned into the SlI site inserted into the trgeting vector. Finlly, the trgeting construct ws utted to PMC1-DT negtive-selection cssette nd linerized. The linerized trgeting construct ws introduced y electroportion into C57BL/ 6-derived Bruce4 recominnt emryonic stem cell nd neomycin-resistnt clones were first screened for homologous recomintion y PCR utilizing pir of the following oligonucleotides corresponding to sequence outside of the 5 left rm nd to the EGFP site: Primer 1: 5 -GAGTACCTTCTAGGTCTATGTGA CTTGACT-3, nd Primer 2: 5 -ATATAGACGTTGTGGCTGTTGTAGTTGT A-3. EcoRV-digested genomic DNA of positive clones ws then screened y Southern lotting with 3 externl single-copy proe corresponding to.57-k frgment (Supplementry Fig. 3f), which ws mplified y PCR using the oligonucleotides 5 -TTGGTGAAAATTAAAATCACATTCA-3 nd 5 - TGGCATTATAATTAGCTGACACTGA-3. When tested on EcoRV-digested DNA, it hyridized either to 8.3-k frgment or to 7.6-k recominnt frgment. Emryonic stem cell clones ering the correctly trgeted locus were injected into BALB/c lstocysts, nd chimeric mle offspring, in which the utodeleter cssette ws self-excised during the mle germline trnsmission, were mted with femle C57BL/6 mice to otin heterozygotes, which were then crossed to otin homozygotes. Trnsmission of the trgeted llele ws confirmed y PCR with Primer 1 nd Primer 2 s descried ove. CRE-medited deletion of the floxed Neo r cssette cn e visulized y the presence of 6.3-k frgment using EcoRV-digested DNA hyridized with the 3 externl single-copy proe s descried ove. The mutnt mice were cross-mted for more thn nine genertions with C57BL/6 mice, nd 8- to 12-week-old femle Clec44 þ / þ littermtes were used s mice. Cell isoltion. To prepre single-cell suspensions from spleen, PLNs nd MLNs, tissue smples were digested with collgense type III (Worthington Biochemicl) t 37 C for 2 min, nd were ground etween glss slides. Splenocytes were treted with red lood cell lysis uffer (Sigm-Aldrich) efore suspension. BM cells were flushed from the femurs nd tiis. Single-cell suspensions were otined y forcing through 1-mm cell striner (BD Biosciences). CD4 þ T cells nd CD8 þ T cells were purified from the spleen with mouse CD4 T-lymphocyte Enrichment Set-DM nd mouse CD8 T-lymphocyte Enrichment Set-DM (oth from BD Biosciences). CD11c þ DCs were purified y AutoMACS with mouse CD11c NATURE COMMUNICATIONS 7:11273 DOI: 1.138/ncomms

10 NATURE COMMUNICATIONS DOI: 1.138/ncomms11273 (N418) Microeds (Miltenyi Biotec). For the preprtion of CD11c high CD8 cdcs, splenocytes were depleted of T cells, B cells nd grnulocytes with iotin-conjugted A cocktil nd nti-iotin Microeds from CD4 þ Dendritic Cell Isoltion kit (Miltenyi Biotec) ccording to the mnufcturer s directions with some modifictions, nd then sujected to purifiction using mouse CD11c (N418) Microeds. In some experiments, CD11c þ DCs were sorted into CD11c high CD8 cdcs with high purity (ech 499%) using FACSAriII cell sorter (BD Biosciences) with fluorescein-conjugted mas. BMDCs were generted y culturing BM cells with grnulocyte mcrophge colony-stimulting fctor (GM-CSF, 2 ng ml 1, Wko Pure Chemicl Industries) for 8 dys 4,41. Retrovirus-medited gene trnsfer. Totl RNA from CD8 cdcs (1 6 ) ws extrcted with TRIzol (Life Technologies), nd cdna ws synthesized with oligo(dt) 2 s primer using the SuperScript III First-Strnd Synthesis System for RT PCR kit (Life Technologies). The full-length cdna of C terminus FLAG-tgged Clec44 (NCBI: AY397673) ws mplified y PCR using GoTq Green Mster Mix (Promeg) with pir of specific primers (5 -CGCGGA TCCGCCGCCATGGCTTCAGAAATC-3 nd 5 -CGCCTCGAGTCACTTGTC ATCGTCGTCCTTGTAGTCTAAGTATATTTTCTT-3 ). Ech of the 5 nd 3 primers ws lso tgged (indicted in itlics) with BmH1 nd XhoI sites, while kosc sequence or FLAG sequence ws indicted with n underline or n underline in itlics. The PCR product ws sucloned into pcr4-topo using TA TOPO Cloning Kit for Sequencing (Life Technologies) nd the nucleotide sequence ws confirmed with n ABI31xl utomted sequencer (Applied Biosystems) nd the fluoresceinted dye termintor cycle sequencing method. Alterntively, the DNA sequence of Clec4A4 DI5 V1, Clec4A4 DY68 L236 or Clec4A4 N186Q, which were tgged with BmH1 nd XhoI sites in 5 nd 3 terminls, ws custom-mde using GeneArt (Life Technologies). After restriction enzyme digestion, the DNA sequence ws cloned into the sites of the pmx-ires-gfp vector 41,42, nd ws trnsfected into retrovirl pckging cell line, Phoenix 41,42, with LipofectAMINE Plus Regent (Life Technologies). The culture superntnt of Phoenix fter 24 h of culture ws collected nd centrifuged t 8,g for 16 h t 4 C to concentrte the virus. The retrovirl vector ws trnsfected into BM for the genertion of BM-DCs s descried ove or DC2.4 cell lines together with DOTAP Liposoml Trnsfection Regent (Roche) y centrifugtion t 2,g for 1 h t 32 C, nd trnsfectnts were susequently collected to exmine GFP expression y using FACSAriII cell sorter. Genertion of humn IgG Fc fusion protein. The DNA sequences contining the extrcellulr domin lcking the signl sequence nd intrcellulr domin of Clec44 (NCBI: AAR31149 mino cids: ) were custom-mde using GeneArt, nd the 5 nd 3 ends of the synthesized DNA sequences were lso tgged with EcoRI nd BglII sites, respectively. After restriction enzyme digestion, the synthesized DNA sequence ws cloned into the sites of pfuse-higg2-fc2 (Invivogen), nd trnsfected into FreeStyle 293-F cells (Life Technologies) using 293fectin Trnsfection Regent (Life Technologies) ccording to the mnufcturers instructions. huigfc or Clec4A4-huIgFc fusion protein ws purified from the culture superntnt using HiTrp Protein G HP (GE Helthcre Life Sciences), nd the purified fusion protein ws seprted y 1% SDS PAGE under non-reducing or reducing conditions, respectively, nd stined with Silver Stin kit (Bio-Rd Lortories) to confirm the protein products. Flow cytometry. Cells were stined with fluorescein-conjugted mas (1:1) to mouse CD3e (ct#55366, 145-2C11), CD4 (ct#55346, RM4-5), CD8 (ct#55332, ), CD11c (ct#55261, HL3), CD4 (ct#553723, 3/23), CD44 (ct#55925, IM7), CD45.1 (ct#55871, A2), CD49 (ct#553857, DX5), CD8 (ct#553768, 16-1A1), CD86 (ct#553691, GL1), I-A/I-E (ct#557, M5/ ), B22 (ct#55389, RA3-6B2), H-2K (ct#553569, AF6-88.5), V2 TCR (ct#553289, B2.1), IFN-g (ct#554411, XMG1.2), IL-17A (ct#55952, TC11-18H1), isotype-mtched control ma (ct#553/55994, 187.1; BD Biosciences), dendritic cell mrker (ct# , 33D1; ebioscience), CD11 (ct#1126, M1/7; Biolegend), Siglec-H (ct# , JF5-1C2.4.1; Miltenyi Biotec), nd H-2K tetrmer (ct#ts-51-1c; MBL). For the intrcellulr expression of cytokines, cells were incuted for 4 h with phorol 12-myristte 13-cette (5 ng ml 1 ; Sigm-Aldrich) plus ionomycin (5 ng ml 1 ; Sigm-Aldrich) or peptide (SIINFEKL; 1 mm) plus GolgiPlug (BD Biosciences) during the finl 2 h. Susequently, the cells were resuspended in Fixtion-Permeiliztion solution (BD Cytofix/Cytoperm kit; BD Biosciences) nd intrcellulr cytokine stining ws crried out ccording to the mnufcturer s directions. For stining with recominnt fusion proteins, cells were stined with huigfc (1 mg), Clec4A4-huIgFc (1 mg), SIGNR1-huIgFc (ct#1836-sr, 1 mg), or Clec9A-huIgFc (ct#649-cl, 1 mg; oth from R&D Systems) followed y nti-humn IgG-PE (ct# ; ebiosciences). Fluorescence stining ws nlysed with FACSCliur flow cytometer nd CELLQuest Softwre (oth from BD Biosciences). Culture of CD11c þ DCs. CD11c high CD8 cdcs were cultured with or without Pm3CSK4 (1 mgml 1 ), poly(i:c) (5 mgml 1 ), (1 mgml 1 ), or (.1 mm) for 18 h in 48-well culture pltes (BD Bioscience). Similrly, BMDCs expressing mock-gfp, Clec4A4-GFP, Clec4A4 N186Q -GFP, Clec4A4 DY68 L236 -GFP, or Clec4A4 DI5 V1 -GFP were cultured with or without (1 mgml 1 ) or (.1 mm) for 18 h in 48-well culture pltes. Alterntively, DC2.4, DC2.4-expressing mock-gfp or DC2.4-expressing Clec4A4-GFP (5 1 5 ) were cultured with or without Pm3CSK4 (1 mgml 1 ), poly(i:c) (5 mgml 1 ), (.1 mgml 1 ), or (.1 mm) for 18 h in 48-well culture pltes. The culture superntnts were collected nd stored t 8 C until ssyed for cytokines. Detection of cytokines. Culture superntnts nd ser were ssyed for IFN- (PBL), TNF-, IL-6, IL-12p4 (ebioscience), IL-17A nd IFN-g (Biolegend) using enzyme-linked immunosorent ssy (ELISA) kits ccording to the mnufcturers instructions. Immunolotting. DC2.4, DC2.4-expressing mock-gfp nd DC2.4-expressing Clec4A4-GFP (2 1 6 ) were stimulted or not stimulted with (.1 mgml 1 ) for the period indicted. Alterntively, BMDCs expressing mock-gfp or Clec4A4-GFP s well s CD8 cdcs (2 1 6 ) were stimulted or not stimulted with (1 mgml 1 ) or (1 mgml 1 ) for the period indicted. Totl lyste ws nlysed y SDS PAGE, nd lots were proed with A (1:1,) for NF-kB p65 (ct#8242, D4E12), ERK (ct#912), JNK (ct#9252), p38 (ct#9212), IRF-3 (ct#432, D83B9) (Cell Signling Technology) nd IRF-7 (ct#sc-983, H-246; Snt Cruz Biotechnology) or the phosphospecific A (1:1,) for NF-kB p65 (ct#333, 93H1), ERK (ct#911), JNK (ct#9251), p38 (ct#9211), IRF-3 (ct#4947, 4D4G) nd IRF-7 (ct#14767; Cell Signling Technology) followed y horserdish peroxidse (HRP)-conjugted got nti-rit IgG (ct#774, 1:1,; Cell Signling Technology). In some experiments, BMDCs expressing mock-gfp or Clec4A4-GFP s well s DC2.4, DC2.4-expressing mock-gfp nd DC2.4-expressing Clec4A4-GFP (2 1 6 ) were pretreted with or without the cross-linked nti-flag M2 ma (ct#f184, 1 mgml 1 ; Sigm-Aldrich) with secondry got nti-rit IgG (ct# , 1 mgml 1 ; Jckson ImmunoReserch) for 3 min. Then, cells were stimulted or not stimulted with (1 mgml 1 ) or (1 mgml 1 ) for 3 min. Susequently, the immunoprecipitte with nti-flag M2 ma (F184, 1:1), nti-shp-1 ma (ct#sc-381, G-2, 1:1) or nti-shp-2 ma (ct#sc-28, C-18, 1:1) Figure 6 Deficiency of Clec4A4 increses Ag-specific CD4 þ T-cell responses in vivo. ( d) CD45.1 þ OT-II CD4 þ T cells were cultured with CD8 cdcs otined from mice nd Clec44 / mice in the presence or sence of Pm3CSK4 (1 mgml 1 ), poly(i:c) (5 mgml 1 ), (1 mgml 1 )or (.1 mm) in comintion with peptide under T H 1(,)- or T H 17 (c,d)-polrized culture conditions for 3 dys, nd intrcellulr production of IFN-g (,) or IL-17 (c,d) in the cultured CD4 þ Tcells ws nlysed y flow cytometry. (,c) Dt re presented y dot plot, nd numers represent the proportion of IFN-g þ cells () nd IL-17 þ cells (c) mong gted CD4 þ T cells in ech qudrnt. (,d) Dt re the men percentge of positive cells±s.d. from three individul smples in single experiment. (e,f) CFSE-lelled CD45.1 þ OT-II CD4 þ T cells were trnsferred into mice (n ¼ 6) nd Clec44 / mice (n ¼ 6), nd then the mice were immunized with protein in comintion with or without the indicted TLR lignds. Ag-specific division of CD45.1 þ OT-II CD4 þ T cells ws nlysed 3 dys fter the immuniztion y flow cytometry. (e) Dt re presented y histogrm, nd numers represent the proportion of CFSE dilution mong gted CD45.1 þ OT-II CD4 þ T cells in ech histogrm. (f) Dt re the men percentge of positive cells±s.d. from six individul smples in single experiment. (g i) mice (n ¼ 6) nd Clec44 / mice (n ¼ 6) were immunized with CFA plus protein. At 14 dys fter the immuniztion, spleen CD4 þ Tcells were isolted then cultured with CD11c þ DCs in the presence or sence of protein for the mesurement of prolifertive responses y [ 3 H]thymidine incorportion (g, left pnel) nd production of IFN-g (g, right pnel) y ELISA. Dt re the men±s.d. from six individul smples in single experiment. (h,i) Intrcellulr production of IFN-g in the cultured CD4 þ T cells ws nlysed y flow cytometry. (h) Dt re presented y dot plot, nd numers represent the proportion of IFN-g þ cells mong gted CD4 þ Tcells in ech qudrnt. (i) Dt re the men percentge of positive cells±s.d. from six individul smples in single experiment. Po.1 compred with mice (nlysis of vrince (AN), Bonferroni s multiple comprison test). All dt re representtive of t lest three independent experiments. 1 NATURE COMMUNICATIONS 7:11273 DOI: 1.138/ncomms