of March 31, 2018.This information is current as

Double-Stranded RNA SensingFragment and Cell Surface TLR3 in Roles of the Cleaved N-Terminal TLR3

Saitoh and Kensuke MiyakeKanno, Takuma Shibata, Natsuko Tanimura, Shin-ichiroh Yusuke Murakami, Ryutaro Fukui, Yuji Motoi, Atsuo

http://www.jimmunol.org/content/193/10/5208doi: 10.4049/jimmunol.1400386October 2014;

2014; 193:5208-5217; Prepublished online 10J Immunol 

MaterialSupplementary

6.DCSupplementalhttp://www.jimmunol.org/content/suppl/2014/10/10/jimmunol.140038

Referenceshttp://www.jimmunol.org/content/193/10/5208.full#ref-list-1

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The Journal of Immunology

Roles of the Cleaved N-Terminal TLR3 Fragment and CellSurface TLR3 in Double-Stranded RNA Sensing

Yusuke Murakami,* Ryutaro Fukui,* Yuji Motoi,* Atsuo Kanno,*,† Takuma Shibata,*

Natsuko Tanimura,* Shin-ichiroh Saitoh,* and Kensuke Miyake*,†

TLR3 senses viral dsRNA in endolysosomes. The TLR3 ectodomain is cleaved by proteases such as cathepsins in endolysosomes. It

remains controversial whether the N-terminal fragment of TLR3 ectodomain (TLR3N) is cleaved off or remains associated with the

C-terminal TLR3 fragment (TLR3C). In addition to endosomes, TLR3 is reported to be expressed on the surface of human fibro-

blasts, but not of human monocyte-derived dendritic cells. Less is known about roles of TLR3N and cell surface TLR3 in dsRNA

sensing. In this study, we show the cleavage site of the TLR3 ectodomain and cell surface expression of TLR3 on mouse primary

immune cells. TLR3C, which started at 343S, was associated with TLR3N. Both TLR3N and TLR3C were required for activation of

IFN-b and NF-kB promoters by dsRNA, demonstrating that dsRNA is sensed by the TLR3N+C complex. Newly established mAbs

to mouse TLR3 revealed that cell surface TLR3 was highly expressed on splenic CD8+ dendritic cells and marginal zone B cells.

Cell surface expression of TLR3 on these cells was dependent on the TLR-specific transporter Unc93B1. Although cell surface

TLR3 was only weakly expressed on macrophages, TLR3 mAb specifically enhanced TLR3 responses to dsRNA. These results

demonstrate that dsRNA is sensed by the TLR3N+C complex and that cell surface TLR3 is a promising target for modulating

TLR3 responses. The Journal of Immunology, 2014, 193: 5208–5217.

Toll-like receptors recognize a variety of microbialproducts. Cell surface TLRs, including TLR4/MD-2,TLR1/TLR2, and TLR6/TLR2, recognize bacterial

membrane lipids, whereas TLR3, TLR7, TLR8, and TLR9 recognizemicrobial nucleic acids (NAs) in intracellular organelles (1–3). Theself/pathogen discrimination by NA-sensing TLRs is error prone andneeds to be strengthened by compartmentalization of NA sensing inendolysosomes, not on the cell surface. Although self-derived NA israpidly degraded by nucleases, microbial NAs are resistant to deg-radation because they are encased in bacterial cell walls or viralparticles. Microbial NAs, therefore, more easily reach endolyso-somes and stimulate NA-sensing TLRs than self-derived NAs (4, 5).To compartmentalize NA sensing by TLRs in endolysosomes,

two mechanisms have been proposed. The first mechanism dependson the proteolytic cleavage of TLRs in endolysosomes (6, 7). NAsensing is activated by proteolytic cleavage of TLR ectodomains

after trafficking to endolysosomes. After proteolytic cleavage,requirements of N-terminal TLR fragments in NA sensing have

been controversial. For example, the ectodomain of TLR9, a sen-

sor for ssDNA, is cleaved by asparagine endopeptidase and/or

cathepsins in endolysosomes (8, 9). Although TLR9C alone is

reported to sense DNA in the previous study (7), another study

showed that the N-terminal TLR9 fragment (TLR9N) remains

associated with C-terminal TLR9 fragment (TLR9C) and that

TLR9N association is essential for DNA sensing (10). TLR7,

a ssRNA sensor, is also proteolytically cleaved (11). The TLR7N

fragment is linked to the TLR7C fragment by a disulfide bond,

which is indispensable for TLR7 proteolytic cleavage and re-

sponses (11). Furthermore, the structure of the complex consisting

of TLR8 and a small chemical ligand has revealed that TLR8 is

also cleaved, but TLR8N is associated with TLR8C (12). TLR8N

and TLR8C both have ligand binding domains and dimerization

interfaces (12), demonstrating an indispensable role of TLR8N in

ligand binding and signaling. TLR3, a dsRNA sensor, is cleaved,

but the precise cleavage site has been only suggested (13, 14).

TLR3N is suggested to be associated with TLR3C and required

for dsRNA sensing (14, 15). However, the direct evidence for

TLR3N requirement in dsRNA sensing has not been shown.The second mechanism for compartmentalization of NA sensing

is based on TLR transportation. Trafficking of NA-sensing TLRs out

of endoplasmic reticulum (ER) is regulated by Unc93B1, a multiple

transmembrane protein (16, 17). In Unc93b13d/3d mice harboring

loss-of-function mutation, NA-sensing TLRs fail to respond to NAs,

because all the NA-sensing TLRs remain uncleaved in the ER (16,

17). Enforced TLR9 localization to the cell surface causes systemic

lethal inflammation (18). Nonetheless, cell surface expression of

TLR9 on splenic dendritic cells (DCs) is recently shown (10),

suggesting a functional role of cell surface TLR9 in ssDNA sensing.

Human TLR3 is also shown to be expressed on the surface of

fibroblasts, but not of DCs (19, 20). Moreover, human TLR3

overexpressed in HEK293 cells is shown to be expressed on the cell

surface (13, 21). Unc93B1 overexpression augments cell surface

*Division of Innate Immunity, Department of Microbiology and Immunology, Instituteof Medical Science, University of Tokyo, Tokyo 108-8639, Japan; and †Laboratory ofInnate Immunity, Center for Experimental Medicine and Systems Biology, Institute ofMedical Science, University of Tokyo, Tokyo 108-8639, Japan

Received for publication February 10, 2014. Accepted for publication September 14,2014.

This work was supported in part by a contract research fund from the Ministry ofEducation, Culture, Sports, Science and Technology for the Program of Japan Initiativefor Global Research Network on Infectious Diseases; a Grant-in-Aid for ExploratoryResearch; a Grant-in-Aid for Scientific Research on Innovative Areas; a Grant-in-Aidfor Scientific Research (A); and the Translational Research Network Program.

Address correspondence and reprint requests to Prof. Kensuke Miyake, Division ofInnate Immunity, Department of Microbiology and Immunology, Institute of MedicalScience, University of Tokyo, 4-6-1 Shirokanedai, Minatoku, 108-8639, Tokyo,Japan. E-mail address: kmiyake@ims.u-tokyo.ac.jp

The online version of this article contains supplemental material.

Abbreviations used in this article: BM, bone marrow; BM-cDC, BM-derived con-ventional DC; BM-MC, BM-derived macrophage; CBB, Coomassie Brilliant Blue;DC, dendritic cell; ER, endoplasmic reticulum; HA, hemagglutinin; LRM, leucine-rich motif; LRR, leucine-rich repeat; MZ, marginal zone; NA, nucleic acid; pA,polyclonal Ab; PVDF, polyvinylidene difluoride; StAv, streptavidin.

Copyright� 2014 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/14/$16.00

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expression of TLR3 (21). Despite this progress, cell surface ex-pressions of mouse TLR3 on primary immune cells remain unclar-ified. Furthermore, functional roles of cell surface TLR3 in responsesto dsRNA have not been clear yet.TLR3 is most highly expressed in CD8+ cross-presenting DCs

(22). TLR3 induces rapid and massive production of type I IFN andproinflammatory cytokines. TLR3-deficient human patients showthat TLR3 is crucial for the defense against HSV-1 encephalitis(23). TLR3 also has a role in sensing UV radiation-dependent celldamage (24). These results demonstrate an essential role of TLR3in activating immune responses during virus infection and tissuedamage. It is important to understand how TLR3 responses arecontrolled. To study mechanisms regulating TLR3 responses, thecurrent study determined the cleavage site of the TLR3 ectodomainand revealed requirements for TLR3N in dsRNA sensing by TLR3.Furthermore, cell surface TLR3 on CD8+ DCs and marginal zone(MZ) B cells was detected by novel mAbs against mouse TLR3.TLR3 mAb enhanced dsRNA-dependent TLR3 responses withoutany activation with TLR3 mAb alone, suggesting that cell surfaceTLR3 can be a target for modulating TLR3 responses.

Materials and MethodsGeneration of anti-mouse TLR3 mAbs

To establish anti-mouse TLR3 mAbs, BALB/c background Tlr32/2 micewere immunized with Ba/F3 cells expressing Flag-63His-conjugated mouseTLR3 (Ba/F3-mTLR3fH) in CFA/IFA used as adjuvants. Next, to boost theimmunization, the mice were immunized three times with Ba/F3-mTLR3fHin PBS. Three days after the final immunization, splenic cells were fusedwith SP2/O myeloma cells. Hybridomas producing anti-TLR3 mAb wereselected by flow cytometry staining of Ba/F3 cells expressing mTLR3fH.The novel mAbs, named CaT3 and PaT3, were used in this study.

Mice, reagents, and cells

C57BL/6N mice were purchased from Japan SLC, and Tlr32/2 mice onC57BL/6N background were provided by S. Akira (Osaka University,Osaka, Japan). The Unc93b13d/3d mice were provided by B. Beutler(University of Texas, Dallas, TX) and the Scripps Research Institute. Allanimal experiments were conducted with the approval of the Animal Re-search Committee of the Institute of Medical Science, University of Tokyo.

Biotinylated anti-Flag Ab (clone M2) and anti-Flag M2 agarose affinitygel were purchased from Sigma-Aldrich. Rabbit GFP polyclonal Ab (pAb)and mAb (clone FM264) were purchased from Invitrogen and MBL, re-spectively. The pAb to the N-terminal fragment of TLR3 (TLR3N) waspurchased from Millipore.

J774 cells were cultured in high-glucose DMEM supplemented with 10%FBS, penicillin-streptomycin-glutamine (Life Technologies), and 50 mM2-ME. Ba/F3 cells were cultured in RPMI 1640 medium supplementedwith IL-3, 10% FBS, penicillin-streptomycin-glutamine, and 50 mM 2-ME.Bone marrow (BM)–derived macrophage (BM-MC) was prepared. In brief,BM cells were plated at 1 3 107 cells per 10 ml with 10% FCS-DMEMsupplemented with 100 ng/ml mouse rM-CSF (PeproTech, Rocky Hill, NJ)in 10-cm cell culture dishes.

Plasmid constructs

The C terminus of mouse TLR3, 7, 8, and 9 was tagged with Flag-63His.They were amplified by PCR and cloned into retroviral pMX, pMXpuro,or pMXneo vectors (provided by Dr. T. Kitamura, Tokyo, Japan). Wealso constructed the short hairpin RNA-targeting Unc93B1 in retroviralvector pSSCH. For the constructs encoding TLR3 N fragments (Figs. 3and 4), TLR3 fragments corresponding to aa 1–342 (TLR3N342), 1–346(TLR3N346), or 1–356 (TLR3N356) were cloned into pMX vector. Forthe TLR3C constructs, TLR3 fragments corresponding to aa 1–35, thesignal peptide, and following 10 aa, fused to aa 343–905 (TLR3C343),347–905 (TLR3C347), or 357–905 (TLR3C357), were cloned into thepMX vector with tandem HA-tag. The In-Fusion HD cloning kit(Clontech Laboratories) and Rapid DNA Ligation kit (Roche AppliedScience) were used for cloning.

Retroviral transduction

pMX and pMXpuro vectors were transfected into Plat-E packaging cellswith FuGene6 (Roche Applied Science). After 2 d of incubation, superna-

tants were obtained as virus suspensions. Ba/F3 and BakB cells were infectedby virus suspensions mixed with DOTAP (Roche Applied Science).

N-terminal amino acid sequencing

TLR3-GFP was stably expressed in the RAW264.7 cell line. Next, 5 3 109

transfectants were collected and lysed in the buffer, 150 mM NaCl, 50 mMTris-HCl (pH 7.4), 1.0% Triton X-100, and 13 complete EDTA-free mixture(Roche Applied Science). Anti-GFP agarose beads were added to the lysateand incubated overnight at 4˚C. Beads were collected and washed twice bywashing buffer, 150 mM NaCl, 30 mM Tris-HCl (pH 7.4), and 0.1% TritonX-100; and bound protein was eluted in elusion buffer, 150 mM NaCl, 30mM glycine-HCl (pH 2.5), and 0.1% Triton X-100 and neutralization buffer,1 M Tris-HCl (pH 8.0). Eluted sample was concentrated and dialyzed inbuffer (10 mM NaCl, 0.1% Triton X-100) by Amicon Ultra (Millipore).Concentrated sample was dissolved in the sample buffer, 2% SDS, 10%glycerol, 62.5 mM Tris-HCl (pH 6.8), 0.025% bromophenol blue, and 5%2-ME, and subjected to SDS-PAGE. Separated protein was transferred topolyvinylidene difluoride (PVDF) membranes. Transferred protein was vi-sualized by Coomassie brilliant blue, and the membrane containing TLR3Nand 3C was cut out. The membrane was subjected to N-terminal amino acidsequencing by Edman degradation (APRO Science).

Immunoprecipitation

Cells were collected and washed twice with 13 PBS. Washed cells werelysed with 1% Triton X-100 lysis buffer [20 mM Tris-HCl (pH 7.4), 150mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 10% glycerol, 1 mM DTT, and 13complete EDTA-free mixture (Roche Applied Science)]. After incubationfor 30 min on ice, lysates were centrifuged and debris was removed. Thecell lysates were rotated for 2 h at 4˚C with beads coupled with anti-Flag oranti-TLR3 mAbs. The beads were then washed with 0.1% Triton X-100washing buffer [20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM CaCl2,1 mM MgCl2, 10% glycerol, 1 mM DTT] three times. Immunoprecipitateswere subjected to SDS-PAGE after boiling in reduced sample buffer [125mM Tris-HCl (pH 6.8), 20% glycerol, 4% SDS, 10% 2-ME, and 0.005%bromophenol blue] or nonreduced sample buffer [125 mM Tris-HCl(pH 6.8), 20% glycerol, 4% SDS, and 0.005% bromophenol blue]. Afterelectrophoresis, samples were transferred to PVDF membranes and sub-jected to immunoblotting.

Luciferase assay

HEK293 cells (1 3 106 cells/well) were transiently transfected in 6-wellplates using Lipofectamine 2000 reagent (Invitrogen) with pMX, pMX-TLR3,or -TLR3 mutant vector (5 mg), together with a luciferase-linked IFN-bpromoter reporter gene, p55C1B-Luc (100 ng). Twenty-four hours aftertransfection, cells were harvested and seeded into 96-well plates (53 104 perwell). After 24 h, cells were stimulated with medium alone or poly(I:C) for6 h. Stimulated cells were lysed using lysis buffer (Promega), and luciferaseactivities were measured. Data are expressed as mean values with SD.

ELISA

J774 cells and BM-MCs plated at 5 3 104 per well on 96-well plates werestimulated with indicated TLR ligands for 24 h. The supernatant wassubjected to ELISA for RANTES production (R&D Systems).

Cell staining and flow cytometry

BM cells and splenocytes were stained with fluorescein-conjugated Absspecific for the following markers in flow cytometry analysis: CD4 (GK1.5),CD8a (53-6.7), CD11b (M1/70), CD11c (HL3), PDCA-1 (927), B220(RA3-6B2), CD21 (7G6), CD23 (B3B4), Ly6C (HK1.4), Ly6G (1A8), andGr-1 (RB6-8C5). Biotinylated anti-TLR3 (CaT3 and PaT3) mAbs wereestablished in this study. For preparation of single-cell splenocyte sus-pensions, spleens were minced by scissors and incubated for 30 min at37˚C in RPMI 1640 with 0.09 U ml–1 Liberase TL (Roche) and 0.1 mgml–1 DNase I (Roche). Finally, suspended splenocytes were teasedthrough nylon mesh after pipetting and subjected to cell staining.

The cell staining was performed in staining buffer, 13 PBS with 2.5%FBS and 0.1% NaN3. Single-cell suspensions were incubated on ice for15 min with biotinylated mAbs diluted in staining buffer. Then cells werewashed and incubated with PE-streptavidin (StAv; BioLegend) andfluorescein-conjugated mAbs for 15 min. Stained cells were analyzed bythe FACSCalibur or FACSAria flow cytometers (BD Biosciences).

Cell surface biotinylation and precipitation with StAv

Wild-type, Tlr32/2, or Unc93b13d/3d BM-MCs were washed twice withHBSS and subjected to cell surface biotinylation with EZ-Link Sulfo-

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NHS-LC-LC Biotin (Thermo Scientific Protein Biology Products) for 1 hat 4˚C. After washing with HBSS, BM-MCs were lysed in 1% TritonX-100 lysis buffer (20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM CaCl2,1 mM MgCl2, 10% glycerol, 1 mM DTT, and 13 Complete EDTA-freemixture [Roche Applied Science]). Cell lysate was incubated on ice for 30min and centrifuged to remove debris. Supernatant was collected and rotatedat 4˚C for 2 h with StAv-conjugated Dynabeads (Veritas). After incubation,beads were washed three times with 0.1% Triton X-100 washing buffer (20mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 10%glycerol, and 1 mM DTT). Immunoprecipitates were subjected to SDS-PAGE after boiling in sample buffer (125 mM Tris-HCl [pH 6.8], 20%glycerol, 4% SDS, and 0.005% bromophenol blue). After electrophoresis,samples were transferred to PVDF membrane and subjected to immuno-blotting with TLR3N pAb.

Immobilized Abs

PaT3 or CaT3 was coated at 10 mg/ml on the 96-well plate and incubatedat 37˚C for 1 h. Then the plates were washed twice with 13 PBS.

Quenching of Alexa488-labeled mAbs

J774 cells were cultured with Alexa488-labeled Abs at 4˚C or 37˚C for24 h. Then these cells were washed twice with 13 PBS and stained withAnti-Alexa488 Ab (Life Technologies) at 4˚C for 20 min. After washingthe cells, mAb uptake was measured by the FACSCalibur flow cytometers(BD Biosciences).

Immunoprecipitation of TLR3 mAb-binding TLR3

J774 cells were cultured with CaT3, PaT3, or IgG1 control Ab at 37˚C for24 h. After washing with 13 PBS, these cells were lysed in 1% TritonX-100 lysis buffer [20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM CaCl2,1 mM MgCl2, 10% glycerol, 1 mM DTT, and 13 Complete EDTA-freemixture (Roche Applied Science)]. Cell lysate was incubated on ice for 30min and centrifuged to remove debris. Supernatant was collected and rotatedat 4˚C for 8 h with protein G Sepharose 4 Fast Flow (Amersham Bio-sciences). After incubation, beads were washed three times with 0.1% TritonX-100 washing buffer (20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mMCaCl2, 1 mM MgCl2, 10% glycerol, and 1 mM DTT). Immunoprecipitateswere subjected to SDS-PAGE after boiling in sample buffer (125 mM Tris-HCl [pH 6.8], 20% glycerol, 4% SDS, and 0.005% bromophenol blue). Afterelectrophoresis, samples were transferred to PVDF membrane and subjectedto immunoblotting with TLR3N pAb.

Statistical analysis

Data from triplicate samples were statistically analyzed by Student t test.The p value ,0.01 was considered to be significant.

ResultsEstablishment of novel anti-mouse TLR3 mAbs

To study endogenous TLR3, novel two mAbs against mouse TLR3were established. The specificity of the mAbs was confirmed bymembrane-permeabilized staining (Fig. 1A). The mAbs, CaT3 andPaT3, reacted to TLR3, but not TLR7, TLR8, or TLR9. NeitherPaT3 nor CaT3 showed cross-reactivity with human TLR3. Fur-thermore, the mAbs immunoprecipitated TLR3, but not TLR7, froma pro-B cell line Ba/F3 expressing TLR3 or TLR7 (Fig. 1B). En-dogenous TLR3 was next precipitated from J774 macrophage cellline by TLR3 mAbs and immunoprobed with the pAb againstTLR3N. Two signals were detected in J774 cells (Fig. 1C). We alsostudied Unc93B1-silenced J774, which showed ∼90% reduction inUnc93B1 mRNA (Supplemental Fig. 1). The ∼120-kDa signal wasimmunoprecipitated from Unc93B1-silenced J774 cells and is likelyto be full-length, uncleaved TLR3 (TLR3F) in the ER. The ∼70-kDasignal disappeared in Unc93B1-silenced J774 cells (Fig. 1C), indi-cating that this signal is TLR3N. These results demonstrate that CaT3and PaT3 are able to bind to both uncleaved and cleaved TLR3.

Determination of the cleavage site of the TLR3 ectodomain

To study TLR3 proteolytic cleavage, the N-terminal sequence of thecleaved TLR3 fragments was determined. TLR3-GFPwas expressedin RAW264.7 macrophage cell line and purified with anti-GFPmAb.

Eluted fractions were analyzed by SDS-PAGE and CoomassieBrilliant Blue (CBB) staining (Fig. 2A). Immunoprobing with anti-GFP Ab identified TLR3F-GFP and TLR3C-GFP. The ∼70-kDasignal was predicted as TLR3N from its apparent molecular mass.The N-terminal amino acid sequences of TLR3C and TLR3N weredetermined. TLR3C and TLR3N started at 343S and 26T, respec-tively (Fig. 2B). The TLR3 ectodomain consists of tandem repeatsof 22 leucine-rich repeats (LRRs). TLR3C contains LRR12-LRR22(Fig. 2C). The result that TLR3N started at 26T experimentallydemonstrated the signal peptide cleavage site at 25S.

PaT3 specifically binds to TLR3N

Based on the determined amino acid sequence, TLR3Nor TLR3Cwasexpressed in Ba/F3 cells and subjected to membrane-permeabilizedstaining. PaT3 bound to TLR3N, but not TLR3C (Fig. 2D),whereas CaT3 bound to neither fragment. CaT3, however, reactedwith the Ba/F3 cells coexpressing TLR3N and TLR3C (Fig. 2D).PaT3 and CaT3 did not interfere with the binding of the othermAb, demonstrating that these two mAbs react with completelydifferent epitopes on the TLR3 ectodomain (Supplemental Fig. 2).

Independently expressed TLR3N and TLR3C were associatedwith each other

According to the previous reports (14, 15), dsRNA sensing byTLR3C alone is still controversial. One report shows that TLR3Cresponds to dsRNA (15), whereas another report shows thatTLR3C fails to respond to dsRNA (14). After TLR3 cleavage,TLR3N is suggested to be still associated with TLR3C (14), butthe requirement of TLR3N for TLR3 responses has not beenclarified. We previously showed that the TLR9N+C complex, butnot TLR9C alone, responds to ssDNA (10). Expression of TLR9Nenables TLR9 responses in TLR9C-expressing cell lines. Fur-thermore, TLR9 responses in Tlr92 /2 BM-cDCs are comple-mented by TLR9N and TLR9C, but not by TLR9C alone.These previous results indicate that coexpression of TLR3N andTLR3C leads to the assembly of TLR3N+TLR3C complex. Nota-bly, CaT3 reacted with the cells expressing both TLR3N andTLR3C, but not those expressing TLR3N or TLR3C alone(Fig. 2D). These results suggest that CaT3 binds to TLR3N+Ccomplex. To confirm that separately expressed TLR3N and TLR3Care associated with each other, TLR3N and TLR3C-hemagglutinin(HA) were expressed in Ba/F3 cells and immunoprecipitated byPaT3 or anti-HA, and coprecipitation of the other TLR3 fragmentwas detected. TLR3N alone showed faster migration than TLR3Nendogenously cleaved from TLR3 (Fig. 2E, lanes 4 and 5), prob-ably due to distinct posttranslational modification such as glyco-sylation. TLR3C was coprecipitated with TLR3N by PaT3 (Fig. 2E,lane 4), whereas coprecipitation of TLR3N with TLR3C was dif-ficult to detect (Fig. 2E, lane 9). PaT3 is likely to stabilize the as-sociation between TLR3N and TLR3C (see below).

Requirement of TLR3N for dsRNA responses

To address a role of TLR3N in dsRNA sensing by TLR3, TLR3Nand TLR3C were separately expressed in HEK293 cells togetherwith the luciferase reporter plasmid p55C1B for IFN-b promoteractivity. Comparable expression of TLR3 or its fragments wasevaluated by membrane-permeabilized staining with PaT3 or anti-HA mAb (Supplemental Fig. 3). dsRNA-dependent IFN-b pro-moter activation was not found in HEK293 cells expressingTLR3N truncated at 342, 346, or 356 (N342, N346, or N356) orthose expressing TLR3C starting at 343, 347, or 357 (C343, C347,or C357). TLR3 responses were observed by coexpression ofN342 with C343, N346 with C347, or N356 with C343, but not bycoexpression of N356 with C357 or N342 with C357 (Fig. 3A).

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TLR3-dependent NF-kB activation was next studied by using Ba/F3cells expressing the reporter construct NF-kB-GFP. TLR3, TLR3N,and TLR3C were expressed in this cell line, and expression of eachfragment was confirmed by membrane-permeabilized staining withPaT3 for TLR3N and anti-HA for TLR3C (Supplemental Fig. 4).These cells were stimulated with poly(I:C), and GFP induction wasevaluated by flow cytometry (Fig. 3B). Poly(I:C)-dependent GFPinduction was not seen in cells expressing any single fragment.TLR3-dependent GFP induction was detected by combined ex-pression of N342+C343 or N346+C347, but not of N356+C357(Fig. 3B). These results are consistent with those obtained by IFN-binduction assay and demonstrate that dsRNA is sensed by theTLR3N+C complex, not by TLR3C alone.

Cell surface expression of TLR3 on CD8+ DCs and MZ B cells

Human TLR3 is expressed on the surface of fibroblasts, but notof DCs, and TLR3 mAb inhibits TLR3 responses in fibroblasts

(19, 20), suggesting a functional role of cell surface TLR3. In con-trast, cell surface expression of mouse TLR3 on primary immunecells has never been reported. In a previous study, membrane-permeabilized staining with anti-TLR3 shows that CD8+ cDCsubset expresses TLR3 more highly than other cDC subsets (22).We performed cell surface and membrane-permeabilized stain-ing of splenocytes with PaT3. Membrane-permeabilized stainingrevealed that TLR3 was expressed in cDCs, but not pDCs(Fig. 4A). The specificity of staining was verified by stainingTlr32/2 DCs. Consistent with the previous report (22), highestTLR3 expression was observed in CD8+ cDCs. Cell surface TLR3was detected on CD8+ cDCs and very weakly on the other cDCsubsets (Fig. 4A). Splenic B cell subsets were next studied. TLR3was detected in both follicular and MZ B cells by membrane-permeabilized staining, whereas only MZ B cells expressedTLR3 on the cell surface (Fig. 4B). Cell surface TLR3 was alsodetected on BM-MCs (Fig. 4C). Although TLR3 was detectable

FIGURE 1. Establishment of anti-mouse TLR3. (A) Ba/F3

cells expressing TLR3, 7, 8, and 9 were subject to membrane-

permeabilized staining with two anti-TLR3 mAbs or anti-Flag

mAb, followed by goat anti-mouse IgG-FITC. All the TLRs

were tagged with the Flag epitope at the C terminus, enabling

detection by anti-Flag. Gray histograms show staining with

the second reagent alone. (B) Ba/F3 cells or those expressing

TLR3-Flag or TLR7-Flag were subjected to immunoprecipi-

tation with anti-TLR3 or anti-Flag mAb and immunoprobing

with anti-Flag mAb. (C) J774 cells or Unc93B1-silenced J774

cells were subjected to immunoprecipitation with anti-TLR3

and immunoprobing with anti-TLR3 pAb.

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by membrane-permeabilized staining of BM-derived cDCs(BM-cDCs), cell surface TLR3 was hard to detect.

Unc93B1 is required for cell surface expression of cleavedTLR3

To gain insight into a mechanism regulating cell surface expressionof TLR3, Unc93b13d/3d mice were studied by using PaT3. InUnc93b13d/3d mice harboring loss-of-function mutation, NA-sensing

TLRs fail to respond to NAs, because all the NA-sensing TLRs failedto exit the ER (16, 17). Cell surface TLR3 was not detected onsplenic CD8a+ cDCs (Fig. 4D), demonstrating an essential role ofUnc93B1 in cell surface expression of TLR3.Next, we asked the origin of cell surface TLR3. If cell surface

TLR3 comes from the ER, uncleaved TLR3 is likely to be enrichedon the cell surface. BM-MCs were subjected to cell surface bio-tinylation, immunoprecipitation with StAv or PaT3, and immuno-

FIGURE 2. TLR3C starts at 343S, and Pat3 mAb binds to TLR3N. (A) TLR3-GFP expressed in the RAW264 macrophage line was subjected to pu-

rification by anti-GFP mAb. Eluted fractions (Fr. 1–5) were analyzed by SDS-PAGE and CBB staining. The right panel shows immunoprobing with GFP

mAb of the immunoprecipitated with GFP mAb from RAW cells expressing TLR3-GFP (lane 1) or plain RAW264 (lane 2). (B) The amino acid sequences

of mouse and human TLR3. The determined N-terminal amino acid sequences of TLR3C and TLR3N are underlined. The signal peptide is indicated by

a line. (C) Schematic representation of proteolytically cleaved TLR3. (D) Open histograms show membrane-permeabilized staining with anti-TLR3 mAbs

or anti-HA mAb of Ba/F3 cells expressing TLR3, TLR3N, or TLR3C-HA. TLR3C lacks the region from 36 to 342. Gray histograms show staining with the

second reagent alone. (E) Ba/F3 cells expressing TLR3N, TLR3C-HA, or TLR3F-HAwere subjected to immunoprecipitation with anti-TLR3N or anti-HA

mAb and immunoprobing with anti-HA (top) or anti-TLR3 (bottom) Abs.

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blotting with anti-TLR3 Ab. In Unc93b13d/3d BM-MCs, TLR3 wasnot detected in the precipitate with StAv (Fig. 4E, lane 2), dem-onstrating cell surface–specific biotinylation. Only cleaved TLR3was detected by immunoprecipitation with StAv, and no increase inuncleaved TLR3 was observed when compared with immunopre-cipitation with PaT3 (Fig. 4E, compare upper and middle in lane 1),indicating that cleaved TLR3 is a predominant form on the cellsurface. These results demonstrate that cleaved TLR3 is predomi-nantly expressed on the cell surface.

PaT3 enhances TLR3 responses

The importance of cell surface TLR3 in dsRNA sensing wasaddressed by studying effects of PaT3 and CaT3 on TLR3 re-

sponses. Cell surface TLR3 was detected on J774 cells. J774 cellswere stimulated with poly(I:C) in the presence of TLR3 mAb, andupregulation of costimulatory molecules and RANTES produc-tion were analyzed. Interestingly, PaT3, but not CaT3, augmentedpoly(I:C)-dependent upregulation of CD40 and CD69 (Fig. 5A).RANTES production in response to poly(I:C) was also upregu-lated by PaT3 (Fig. 5B). RANTES production induced by lipid Awas not altered by TLR3 mAbs. Next, the effect of TLR3 mAbon poly(I:C) responses of BM-MCs was studied. Although cellsurface TLR3 was only weakly detected on BM-MCs (Fig. 4C),poly(I:C)-induced RANTES production was augmented by PaT3(Fig. 5C). PaT3 did not enhance lipid A–dependent RANTES pro-duction by BM-MCs. Augmentation of IFN-b mRNA induction by

FIGURE 3. dsRNA sensing by TLR3N+C.

(A) HEK293 cells were transfected with

the reporter plasmid for IFN-b promoter

and indicated expression vectors. The

TLR3 fragments used are TLR3N trun-

cated at 342, 346, or 356 (N342, N346, and

N356) and TLR3C starting at 343, 347, or

357 (C343, C347, and C357). Transfected

cells were stimulated with poly(I:C), as in-

dicated. Luciferase activity was determined.

The results are represented by average value

with SD. (B) Ba/F3 cells expressing indi-

cated TLR3 fragments and NF-kB-GFP

were stimulated with poly(I:C) or a TLR2

ligand Pam3CSK4. NF-kB–dependent GFP

induction was determined by flow cytometry.

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PaT3 was also observed (Fig. 5C). In contrast to these strong effects,PaT3 alone did not induce any activation in J774 and BM-MCs.To gain insight into a mechanism underlying the PaT3-dependent

enhancement of TLR3 responses, PaT3 internalization was firststudied. J774 cells were incubated with Alexa488-labeled PaT3or CaT3 at 4 or 37˚C and analyzed by flow cytometry afterquenching the fluorescence of the mAb on the cell surface. Allthe mAbs, including control IgG1 mAb, were internalized at37˚C (Fig. 6A, lower). Considering that quenching did not alterfluorescence intensity, the mAbs were unlikely to remain on thecell surface. To confirm that internalized anti-TLR3 mAbs stillbound to TLR3, internalized mAbs were precipitated by proteinG and immunoblotting with anti-TLR3 Ab was performed. TLR3was precipitated by CaT3 or PaT3, but not by IgG1 control mAb(Fig. 6B). These results suggest that internalized anti-TLR3mAbs still bind to TLR3.Requirement of PaT3 internalization in the enhancing effect

of PaT3 was next asked. J774 cells were stimulated with immobi-

lized PaT3. Immobilized PaT3 would ligate TLR3 on the cell sur-face without internalization. No amplification was observed byimmobilized PaT3 (Fig. 6C), suggesting that PaT3 internalizationis required for the enhancing effect of PaT3.Considering that PaT3, but not CaT3, showed the enhancing

effect, it is important to find the PaT3-specific effect on TLR3.TLR3N was required for TLR3 responses, and association betweenTLR3N and TLR3C was more clearly demonstrated by PaT3 thananti-HA Ab capturing TLR3C (Fig. 2E). We hypothesized thatPaT3 may strengthen the association between TLR3N andTLR3C. To address this possibility, TLR3C-HA was immuno-precipitated by anti-HA Ab in the presence of PaT3 or CaT3 mAbfrom the Ba/F3 cells expressing TLR3C-HA and TLR3N.Coprecipitation of TLR3N with TLR3C was clearly enhancedin the presence of PaT3, but not CaT3 (Fig. 6D). Poly(I:C) stim-ulation did not alter TLR3N coprecipitation. These results indicatethat PaT3 enhances TLR3 signaling in endosomes by stabilizingthe association between TLR3N and TLR3C, indicating an im-

FIGURE 4. Cell surface expression of TLR3.

(A–C) Splenic cells from wild-type or Tlr32/2 mice

were subjected to cell surface and membrane-per-

meabilized staining with anti-TLR3 mAb PaT3

together with cell type markers. Open histograms

show TLR3 expression on DC subsets (A), B cell

subsets (B), BM-MCs (C), and BM-cDCs (C). (D)

Open histograms show cell surface TLR3 on

splenic CD8+ cDCs from indicated mice. (E) BM-

MCs from wild-type, Unc93b13d/3d, or Tlr32/2

mice were subjected to cell surface biotinylation,

immunoprecipitation with StAv (upper) or PaT3

(middle), and immunoblotting with anti-TLR3 pAb

(upper and middle). Whole-cell lysates are immu-

noprobed with anti-EEA1 pAb as a loading control.

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portant role of association between TLR3N and TLR3C in TLR3responses.

DiscussionTLR ectodomains consist of LRRs, the tandem repeats of a leucine-rich motif (LRM). LRM is typically 24-aa length and adopts a loopstructure, beginning with a short b-strand followed by a b-turn(25). The extra residues in LRM form a loop that protrudes fromthe LRR solenoid. A tentative loop in TLR9 ectodomain is locatedbetween LRR14 and LRR15 and shown to be cleaved (10). Asimilar loop is located in the LRR11 of the TLR3 ectodomain,which is predicted to be cleaved (14). Consistent with this pre-diction, our present study experimentally determined the cleavagesite at 343S, which resides in the protruding loop of LRR11.Association of TLR3N and TLR3C was previously suggested by

the result that cleaved TLR3 fragments are detectable only underdenatured, but not nondenatured condition (14). However, pro-teolytic cleavage of the TLR3 ectodomain in endolysosomes leads

to the presence of all the fragments, including uncleaved TLR3(TLR3F), TLR3N, and TLR3C. It is difficult to exclude a possi-bility that TLR3N and TLR3C are indirectly associated with eachother by forming a complex with uncleaved TLR3F. In the currentstudy, the determination of the cleavage site enabled separateexpression of TLR3N and TLR3C. Thereby, the association ofTLR3N and TLR3C was directly shown. Interestingly, copreci-pitation of TLR3N was hard to detect in immunoprecipitation ofTLR3C by the Ab to C-terminal–tagged HA. Interaction betweenTLR3N and TLR3C may be unstable in the cell lysate. PaT3 wasable to enhance TLR3N coprecipitation with TLR3C, indicatingthat PaT3 stabilizes the interaction between TLR3N and TLR3C.The persistent association of TLR3C and TLR3N was furthersupported by another TLR3 mAb Cat3, which is likely to reactwith the TLR3N+C complex, but not with either fragment. In theTLR ectodomains, tandem repeats of LRM form a solenoid struc-ture with parallel b-sheets. TLR ectodomains are stabilized by theirinterior hydrophobic cores and hydrogen bonds between parallel

FIGURE 5. TLR3 mAb enhances poly(I:C)

responses. (A and B) J774 cells were stimulated by

poly(I:C) or lipid A at the indicated concentrations

together with PaT3 (▴), CaT3 (n), and control IgG1(X), or without Ab (♦) for 24 h. Cell surface CD40

and CD69 were stained. The results are represented

by MFI value. RANTES production was measured

by ELISA. The results were represented by the

average values with SD from triplicate wells. (C)

BM-MCs were stimulated with poly(I:C) or lipid A

together with PaT3 (▴), CaT3 (n), and control IgG1(X), or without Ab (♦) for 24 h in ELISA or in-

dicated times in quantitative PCR. RANTES pro-

duction was measured by ELISA. (D) For IFN-b

mRNA induction, mRNAwas extracted at 0, 3, and

6 h after poly(I:C) stimulation. IFN-b mRNA was

quantitated by quantitative PCR, and the values

are normalized by hypoxanthine phosphoribosyl-

transferase mRNA.

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b-sheets of LRRs (25). Proteolytic cleavage of the protruding loopis unlikely to alter the interaction between adjacent LRMs.The interaction between TLR3N and TLR3C was found to be

functionally important. TLR3N+C complex, not TLR3C alone,was able to sense dsRNA and activate both IFN-b and NF-kBpromoters. Just 14-aa N-terminal deletion in TLR3C abolishedTLR3N+C responses. Considering that the N-terminal portion ofTLR3C locates in the LRRs, not the protruding loop, 14-aa de-letion may prevent TLR3C association with TLR3N by alteringthe LRR structure. Similarly, the 14-aa extension of TLR3N im-paired TLR3 responses. The extended portion may also impairTLR3N association with TLR3C by competing with the N-terminalportion of TLR3C. These results suggest that the cleavage site ofTLR3 needs to be within the protruding loop, not in the LRRs.Cell surface TLR3 expression on CD8+ DCs and MZ B cells was

shown in the current study. Consistent with the results usingin vitro cell lines (13, 21), cell surface expression of TLR3 onprimary immune cells was dependent on Unc93B1. TLR3 wasweakly detected on the J774 macrophage cell line and BM-MCs.TLR3 mAb PaT3 specifically enhanced TLR3 responses. This isnot simply due to mAb-mediated TLR3 ligation, because PaT3alone did not induce any activation. Moreover, only the mAb toTLR3N PaT3, but not CaT3, had the augmenting effect. Cellsurface TLR3N may have a role in modulating TLR3 responses.Considering that PaT3 specifically augmented TLR3 responses,but not TLR4/MD-2 responses, PaT3 is likely to have a directeffect on TLR3 rather than inducing weak priming signal. PaT3and CaT3 were both internalized, but still bound to TLR3. PaT3,but not CaT3, strengthened the association between TLR3N andTLR3C. PaT3-dependent augmentation of the association betweenTLR3N and TLR3C is likely to enhance TLR3 responses inendolysosomes, suggesting an important role of association be-tween TLR3N and TLR3C in TLR3 responses. Taken togetherwith the previous report showing that human TLR3 mAb is shownto inhibit TLR3 responses in fibroblasts (19), cell surface TLR3may have a role in TLR3 responses, although downstream sig-naling is activated after internalization into endosomes.

Immunoprecipitation of endogenous TLR3 shows that a ma-jority of TLR3 is cleaved in BM-MCs, probably due to rapiddegradation of uncleaved TLR3 (13). Consistent with this, cellsurface biotinylation shows that cell surface TLR3 is mostlycleaved on these cells. Previous study also shows cleavage of cellsurface TLR3 (13). These results indicate that TLR3 traffics fromendolysosomes to the cell surface. Considering that PaT3 mAbenhanced TLR3 responses, cell surface TLR3 is likely to be ac-tivated by dsRNA probably after internalization, suggesting theshuttling of the TLR3N+C complex between the endolysosomalsystems and the plasma membrane. Cell surface TLR3 may bea target for enhancing or inhibiting TLR3 responses. Given thatTLR3 promotes cross-priming in viral infection (26), targeting cellsurface TLR3 by TLR3 mAb may deserve further study in termsof vaccine development.

AcknowledgmentsWe thank Profs. Shizuo Akira (Osaka University) and Bruce Beutler

(University of Texas) for providing mice.

DisclosuresThe authors have no financial conflicts of interest.

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