TLR10 Is a B Cell Intrinsic Suppressor of Adaptive Immune Responses Nicholas J

TLR10 Is a B Cell Intrinsic Suppressor of Adaptive Immune Responses Nicholas J. Hess, Song Jiang, Xinyan Li, Yue Guan and Richard I. Tapping This information is current as of October 1, 2021. J Immunol published online 12 December 2016 http://www.jimmunol.org/content/early/2016/12/09/jimmun ol.1601335 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2016/12/09/jimmunol.160133 Material 5.DCSupplemental

Why The JI? Submit online. http://www.jimmunol.org/

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average by guest on October 1, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 12, 2016, doi:10.4049/jimmunol.1601335 The Journal of Immunology

TLR10 Is a B Cell Intrinsic Suppressor of Adaptive Immune Responses

Nicholas J. Hess,*,1 Song Jiang,*,†,1 Xinyan Li,* Yue Guan,* and Richard I. Tapping*,†

Toll-like receptors play a central role in the initiation of adaptive immune responses with several TLR agonists acting as known B cell mitogens. Despite thousands of publications on TLRs, the function of TLR10 remains unknown. We have found that Ab- mediated engagement of TLR10 on primary human B cells suppresses B cell proliferation, cytokine production, and signal transduction. When challenged with either a T independent or T dependent Ag, TLR10 transgenic mice exhibit diminished Ab responses. Adoptive transfer of splenic B cells into B cell–deﬁcient mice revealed that the suppressive effects on Ag-speciﬁc humoral immune responses are entirely B cell intrinsic. Our results demonstrate that TLR10 has a functional role within the B cell lineage that is distinct from that of other TLR family members and may provide a potential therapeutic target for diseases characterized by dysregulated B cell activity. The Journal of Immunology, 2017, 198: 000–000. Downloaded from

s central elements of the innate immune system, TLRs MyD88, which is required for transducing signals that ultimately provide a first line of immune defense against infec- culminate in proinflammatory gene expression (7, 8). A tious agents. Through direct sensing of bacterial, fungal, TLR activation not only induces classic inflammatory mediators or viral components TLRs activate intracellular signaling events but also provides a critical link between the innate and adaptive that drive the cellular expression and release of immune mediators. arms of the immune response (9, 10). The ability of TLRs to in- These activation events not only drive inflammatory processes, but duce adaptive responses is best understood through their actions http://www.jimmunol.org/ also initiate and orchestrate the longer-term protective responses on dendritic cells; however, TLR subsets are also expressed on of the adaptive immune system (1). B cells where they have direct stimulatory activity. For example, Humans possess 10 TLR family members, numbered 1–10, certain TLR agonists are well-known T independent (TI) Ags for which are differentially expressed in leukocytes and the epithelial B cells. In addition, B cell–intrinsic TLR activation has been cells of mucosal surfaces (2–4). Subsets of TLRs that are shown to promote Ab production and class-switching responses to expressed on the plasma membrane stimulate the production of both TI and T dependent (TD) Ags (11–13). Importantly, TLR- classic proinflammatory molecules whereas other TLRs expressed mediated B cell activation has been shown to be a major driver in endosomal compartments are best known for their ability to of disease progression in various mouse models of autoimmune by guest on October 1, 2021 stimulate the production of type I IFNs (5, 6). All TLRs are type 1 disease. In addition to studies in mice, genome-wide association transmembrane receptors comprised of extracellular leucine rich studies and in vitro studies with patient cells have identified a repeat domains and an intracellular toll/IL-1 receptor homology significant role for TLRs in promoting both the progression and (TIR) signaling domain. TLRs signal via ligand-induced recep- severity of autoimmune diseases, particularly systemic lupus tor dimerization in which two juxtaposed TIR domains act as a erythematosus (14–16). scaffold for the recruitment of proximal adaptor molecules. With TLRs have been the subject of intense research over the last the exception of TLR3, which solely utilizes TIR domain–containing decade, providing a fairly clear picture of the ligand recognition, adaptor-inducing IFN-b (TRIF), TLRs use the proximal adaptor signaling, and biologic functions of TLRs 1–9, but not TLR10. To date, TLR10 remains an orphan receptor with no agreed function in part due to the murine TLR10 gene being disrupted by several *Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, retroviral insertions making classical knockout studies impossible. IL 61801; and †College of Medicine, University of Illinois at Urbana-Champaign, Human TLR10, which was initially cloned and sequenced in 2001 Urbana, IL 61801 (17), is most homologous to TLRs 1 and 6, and intact orthologs 1 N.J.H. and S.J. contributed equally to this work. of the TLR10 gene have been found in every other sequenced ORCIDs: 0000-0001-5720-9305 (N.J.H.); 0000-0003-4890-4327 (S.J.). mammal to date, including several rodent species (18, 19). Received for publication August 2, 2016. Accepted for publication November 11, We have previously shown that similar to TLR1, TLR10 co- 2016. operates with TLR2 in the sensing of triacylated lipopeptide ag- This work was supported by National Institute of Allergy and Infectious Diseases of onists. However, TLR10, either alone or in cooperation with TLR2, National Institutes of Health Grant 1R01-AI097639. fails to induce typical TLR-associated signaling events including S.J., X.L., Y.G., and R.I.T. designed the study; N.J.H., S.J., and X.L. performed the research; N.J.H. and S.J. analyzed the data; N.J.H. and R.I.T. wrote the manuscript. activation of NF-kB, IL-8 or IFN-b–driven reporters (20). More recently, we and others have reported that TLR10 is able to sup- Address correspondence and reprint requests to Prof. Richard I. Tapping, University of Illinois at Urbana-Champaign, B103 CLSL, MC110, 601 S. Goodwin Avenue, press both MyD88-dependent and independent signaling in Urbana, IL 61801. E-mail address: [email protected] mononuclear cell preparations, ultimately inhibiting the produc- The online version of this article contains supplemental material. tion of inflammatory mediators including IL-6 and IFN-b (21, 22). Abbreviations used in this article: MNC, mononuclear cell; NP, nitrophenol; TD, We report in this study that TLR10 is functionally expressed on T cell dependent; TI, T cell independent; TIR, toll/IL-1 receptor homology; TRIF, the surface of primary human B cells and is able to suppress re- TIR domain–containing adaptor-inducing IFN-b. sponses mediated by a variety of B cell costimulatory signals. Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 Furthermore, we show that in a TLR10 knockin mouse model,

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601335 2 TLR10 SUPPRESSES B CELL ACTIVATION

TLR10 is able to suppress both TI and TD Ab production, secondary Ab is used to further cross-link the primary Ab and to prevent showing that human TLR10 is a functional receptor with a novel IgG FcR-mediated inhibition. After preincubation, cells are stimulated anti-inflammatory function in B cells. with a subset of the following: aIgM (20 mg/ml), aCD40 (0.1 mg/ml), the TLR7/8 agonist R848 (100 ng/ml), or the TLR9 agonist CpG (2 mg/ml). For the PCR superarray, total RNA was isolated after 24 h of stimulation Materials and Methods and sampled using a T cell and B cell Activation PCR Array (SA Bio- Reagents sciences). Also after 24 h, cell-free supernatants were assayed with a MIP- 1b Ab pair (Life Technologies) using a 1:20 dilution. Proliferation was All cells were grown in RPMI 1640 supplemented with 10% FBS, 2 mM assayed using an ELISA-based BrdU detection kit (Roche). In short, a 13 glutamine and 13 non-essential amino acids. Anti-IgM and anti-mouse solution of BrdU was added to the cells. After 24 h, the supernatant was IgG Abs were purchased from the Jackson Laboratories. Anti-CD40 was removed and the cells fixed to the plate. Cells were then incubated with a purchased from R&D Systems. R848 and Class C CpG were purchased HRP–conjugated-anti-BrdU Ab for 2 h prior to washing and the addition of from InvivoGen. Phospho-specific Abs p38 (clone D3F9), JNK (clone tetramethylbenzidine for color detection at 450 nm. 81E11), Syk Y525/526 (C87C1), Akt S473 (D9E), and b-actin (clone 13E5) were purchased from Cell Signaling Technologies. The isotype Human B cell signaling control Ab (clone MOPC-21) was purchased from BioLegend. m Two aTLR10 Abs, 3C10C5 and 5C2C5, were generated with the as- Tonsillar B cells were preincubated with an isotype control (20 g/ml) or a m sistance of the University of Illinois Immunological Resource Center. A TLR10 Ab (20 g/ml) for 30 min prior to stimulation. After 15 min, cell lysates were generated in radioimmunoprecipitation assay buffer (150 mM soluble TLR10 fragment consisting of the extracellular domain of TLR10 NaCl, 50 mM Tris-HCl pH 8.0, 1% NP-40 and 1 mM EDTA) supple- (aa 20–474) was purified following the protocol we previously used for other TLRs (23). Female BALB/c mice were injected every 3 wk with the mented with Halt protease and phosphatase inhibitor (Thermo Scientific). TLR10 Ag and checked for serum Ab activity after the third immunization. Equivalent amounts of protein lysate were separated by SDS-PAGE, Splenic cells were fused with SP2/0 tumor cells using polyethylene glycol transferred to polyvinylidene difluoride membranes and blotted against and selected under hypoxanthine-aminopterin-thymidin media. All hy- specific phospho-antibodies (Cell Signaling Technologies). Downloaded from bridoma clones were counter screened against plate-bound soluble TLR1 Generation of TLR10 transgenic mice to remove cross-reactive clones. Lastly, a subset of clones were chosen to determine their ability to stain human embryonic kidney (HEK) cells A constitutive human TLR10 knockin mouse expressing the full-length transfected with TLR10 or a CMV empty vector. From this screen, human TLR10 gene was generated by the UIUC Transgenic Facility. two clones, 3C10C5 and 5C2C5, were selected for future studies The BAC clone #1148D18 containing the full coding region of TLR10 with (Supplemental Fig. 1). The aTLR10 clone 3C10C5 was chosen for licensing its native promoter was purchased from Empire Genomics. BAC DNA was and is currently commercially available from several different biotechnology isolated with Qiagen’s Large Construct Kit with genomic DNA removed http://www.jimmunol.org/ companies. by endonuclease digestion. Approximately 80 mg of plasmid DNA was digested with 100 U of BsrB1 and HF-Not1 to release a 16,584 bp frag- Flow cytometry ment containing the full TLR10 transcript along with ∼4 kb of the up- ∼ Approximately 2–4 3 106 cells per test were blocked in flow buffer (13 PBS, stream and 3 kb of the downstream sequence. Excised DNA was 10% rabbit serum) for 30 min. Cells were centrifuged and resuspended in recovered with Qiagen’s Large Fragment Extraction Kit before continuing 100 ml of flow buffer containing primary Ab at 10 mg/ml and incubated for with the transgenic process. A total of 54 transgenic pups from the C57BL/6 30 min. After washing, the cells were suspended in 100 mlofflowbuffer genetic background were received and screened for TLR10 insertion by PCR. Southern blot analysis revealed that seven of the transgenic pups containing aMouse IgG1-Biotin Conjugate (Jackson Laboratories) for carried the transgene and that six founders possessed at least 10 copies of the 30 min. Cells were washed again and suspended in 100 mlofflowbuffer transgene. Three lines were selected for further characterization of TLR10

containing a streptavidin-APC conjugate (Jackson Laboratories). After by guest on October 1, 2021 washing, cells were fixed in 300 ml of 4% paraformaldehyde for 15 min after expression in various tissues by RT-PCR and immunoprecipitation. Line 5–1 which they were washed and resuspended in 100 ml of flow buffer. All was chosen for further experimental characterization. Genotyping of the 9 labeling steps were performed on ice. Cells were analyzed on a BD FACS mice was performed using forward- 5 -CAGTGGAACACTTTCAGATCC- 9 9 9 Canto II Flow Cytometer operated by the Roy J. Carver Biotechnology 3 and reverse-5 -CCAAGGGTGTGTTGTTAGC-3 primers for TLR10. All mice used for experiments were between the ages of 4–12 wk. For consis- Center at the University of Illinois. tency, only males were used in our experiments. Reverse transcriptase PCR Splenic B cell isolation RNA was isolated using a RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions. The cDNA was prepared with a 1:1 mixture of oligo- Murine splenocytes were isolated by manual disruption of the spleen dT and random primers (Invitrogen), 1 mg of RNA and the Superscript III in sterile PBS followed by passage through a 40 mM filter. MNCs were enzyme (Invitrogen). Reverse transcriptase negative samples were generated further purified by centrifuging through a Ficoll-Paque gradient. B cells by withholding Superscript III from the reaction mixture. PCR was were isolated using STEMCELL Technologies’ EasySep Mouse B Cell performed using AmpliTaq Gold 360 Master Mix (Applied Biosystems). Pri- Isolation Kit (Cat #19854A). mers were used for TLR10 (forward -59-CAGTGGAACACTTTCAGATCC-39, reverse - 59-CCAAGGGTGTGTTGTTAGC-39); HPRT1 (forward - 59-TGGG- Murine B cell stimulation CTTACCTCACTGCTTT-39,reverse-59-CTAATCACGACGCTGGGACT-39). Murine splenic B cells were isolated as described above and prepared at a concentration of 2–3 3 106 B cells/100 ml of media in BD Falcon tubes. Human B cell isolation Prior to stimulation, cells were incubated in 10 nM CFSE (Life Technol- Peripheral blood B cells were isolated by centrifuging whole blood over a ogies) in PBS for 20 min at room temperature in the dark. Cells were then Ficoll-Paque solution (GE Healthcare) at 11003 g for 10 min with 0 brake washed and resuspended in media followed by stimulation with aIgM to obtain mononuclear cells (MNCs). B cells were isolated from the MNCs (20 mg/ml), aCD40 (1 mg/ml) or CpG (4 mg/ml). After 72 h cell pellets using a MACS B Cell Isolation Kit II (Milteny Biotech) according to the were prepared for flow and run on a BD FACS Canto II flow cytometer to manufacturer’s instructions. Tonsillar tissue, obtained from surgical dis- measure CFSE fluorescence in the FITC channel. Additionally, cell-free cards at Carle Foundation hospital, was homogenized and passed through a supernatants were collected and IL-6 was measured by paired Ab ELISA 40 mM filter. MNCs were obtained by centrifuging the filtered homogenate (Life Technologies). over a Ficoll-Paque solution as described above. MNCs were mixed at a 1:50 ratio with previously isolated human RBCs and a Human B Cell Immunization and nitrophenol-specific Ab ELISA Enrichment RossetteSep (STEMCELL Technologies) Ab mixture prior Age and sex matched mice were immunized with either nitrophenol (NP) to centrifuging over a Ficoll-Paque solution with the resulting buffy coat . (27)-CGG or NP (7)-LPS (BioSearch Technologies) mixed in a 1:1 ratio consisting of 95% B cells. with Imject Alum (ThermoScientific). Each mouse received a 200 ml in- Human B cell stimulation assays traperitoneal injection of NP (27)-CGG or NP (7)-LPS equal to 100 and 20 mg of Ag respectively. Prior to immunization along with 7 and 14 d Isolated human B cells were preincubated with either an isotype control after immunization, blood was collected from the submandibular vein and (20 mg/ml) or aTLR10 (3C10C5 at 20 mg/ml) Ab with the addition of a the resulting serum stored. NP-specific ELISA plates were prepared by secondary aMouse IgG1 F(ab)92 fragment (20 mg/ml) for 30 min. The coating NP (5)-BSA (BioSearch Technologies) onto ThermoScientific The Journal of Immunology 3

Nunc plates at concentration of 10 mg/ml in Coating Buffer B (4.3 g high levels of TLR10 RNA message observed in lymphoid tissues NaHCO3, 5.3 g Na2CO3,1LH2O) and incubated at 4˚C overnight. Plates believed to originate from B cells. Our findings prompted us to 3 were then blocked in Assay Buffer (1 PBS, 2% BSA, 0.05% Tween 20) investigate the function of this highly uncharacterized TLR in this for 1 h. Samples used a 1:10,000 dilution for IgM and IgG1 and a 1:100 dilution for IgG2b, IgG2c, IgG3, and IgA. An eight-point standard curve immune cell. was created by taking pooled undiluted serum from previous NP injections and creating serial dilutions starting at 1:50 or 1:5000 for each respective TLR10 Ab-mediated suppression of primary human B cells isotype. Samples and standards were allowed to incubate for 1 h. Biotin conjugated goat anti-mouse detection Abs (Southern Biotech) specific to Upon appropriate costimulation, clonal B cells undergo prolifer- each isotype were added at 50 ng/ml and allowed to incubate for 1 h. ation and differentiation to Ab-producing plasma cells. To deter- Streptavidin-HRP (Jackson ImmunoResearch) was added at 0.1 mg/ml and mine if TLR10 can suppress human B cell proliferation, we incubated for 30 min before developing in an o-phenylenediamine solu- assessed the effect of an aTLR10 mAb on B cell proliferation tion. Wells were washed in ELISA Wash Buffer (13 PBS, 0.05% Tween 20) between each step and measured at 490 nm. induced by costimulation through pairwise engagement of the BCR, TLR7, and the CD40 receptor. Compared to an isotype B cell transfer control Ab, the aTLR10 Ab suppressed the proliferation of Splenic B cells were isolated as described previously from between five and B cells, isolated from six different donors, irrespective of the nine male mice per group. Splenocytes were pooled for subsequent B cell stimulus (Fig. 2A). TLR10 engagement also suppressed B cell isolation and then resuspended in sterile PBS at a concentration of 1E8 proliferation induced by Staphylococcus aureus Cowan strain, a 7 B cells/ml. A total of 200 ml, equaling 2E B cells was injected via i.v. well-established B cell mitogen. Together, these results suggest injection into the caudal tail vein of male mMT mice and allowed to colonize for 24 h before immunization. To further boost the immune re- that TLR10 serves to dampen B cell activation mediated by either TI or TD mechanisms. action, LPS was added to the NP (27)-CGG immunizations equivalent to Downloaded from 5 mg of LPS per mouse. B cell stimulation results in changes in the expression of a wide Statistics range of genes that function to drive functional B cell responses. To more broadly examine the effect of TLR10 engagement on B cell All statistics were performed using the GraphPad Prism software. Signi- activation, we costimulated B cells through the BCR and TLR9 for ficance was determined by a t test using the Holm-Sidak method. 24 h in the presence of either aTLR10 or an isotype control Ab. After stimulation, RNA was collected and the expression of an Results array of lymphocyte specific activation genes was measured by http://www.jimmunol.org/ TLR10 expression in primary lymphocytes real-time PCR. Of the 84 genes tested, 39 exhibited statistically To assess human TLR10 expression, lymphocytes from whole significant changes as either an increase or decrease in expression blood of healthy individuals were labeled with an anti-TLR10 Ab following B cell stimulation. Compared to an isotype control Ab, developed in our laboratory. To detect weakly expressed TLR10, a aTLR10 affected 33 of the 39 genes by consistently dampening biotin-streptavidin labeling procedure was employed prior to flow the increase or decrease in individual gene expression normally cytometric analysis (see Materials and Methods). Compared to an induced by B cell stimulation (Fig. 2A, Supplemental Table I). isotype control Ab, a shift in TLR10 fluorescence was detected in The three genes that exhibited the greatest degree of suppression

a subpopulation of the gated lymphocytes (Fig. 1A). Costaining were CCL3, CCL4, and AICDA. The chemokines CCL3 and by guest on October 1, 2021 revealed that all lymphocytes expressing TLR10 also expressed CCL4, also known as MIP-1a and MIP-1b, respectively, are each the B cell marker CD19, but not the T cell marker CD3 (Fig. 1B). potent chemoattractants secreted by lymphocytes that function to The presence of cell surface TLR10 on B cells is consistent with drive lymphocyte migration in the germinal center. AICDA is

FIGURE 1. TLR10 is expressed on the plasma membrane of primary human B cells. (A)Flow cytometry of lymphocytes gated from whole blood. Cells were triple labeled for TLR10 expression (see Materials and Methods) using either an isotype control or TLR10 Ab. (B) Lymphocytes were stained as in (A) for TLR10 with the addition of either the T cell marker CD3 or the B cell marker CD19 to differentiate the lymphocyte population into its constituent components. 4 TLR10 SUPPRESSES B CELL ACTIVATION

FIGURE 2. Primary human B cell activation is suppressed by an aTLR10 Ab. Human primary B cells were isolated from peripheral blood of healthy donors by negative selection and preincubated with either an isotype control or TLR10 mAb for 30 min prior to stimulation. (A) B cells were stimulated with the indicated agonists for 96 h with BrdU added after 72 h of stimulation. BrdU incorpo- ration was assayed by ELISA on six different donors. (B) B cells were stimulated with aIgM and CpG for 24 h after which RNA was isolated and assayed for activation using a B cell PCR super array. Bars represent the fold expression of stimulated cells compared with control cells, which were left unstimulated in the presence of the isotype control Downloaded from Ab for 30 min. (C) B cells were stimulated with the indicated agonists. Cell-free supernatants were collected after 24 h and assayed for MIP-1b by ELISA. *p , 0.1, **p , 0.05, ***p , 0.01. http://www.jimmunol.org/ necessary for both somatic hypermutation and class-switch re- TLR-mediated activation events but also broadly regulates B cell combination during clonal expansion in the germinal center. activation induced by a variety of stimuli. To conﬁrm that the suppression observed in the RNA array translates to functional effects, secretion of MIP-1 b (CCL4) was Suppression of BCR signaling through TLR10 measured from B cells stimulated in the presence of either isotype Given the broad suppressive activity of TLR10, we next assessed control or the aTLR10 Ab. TLR10 Ab engagement suppressed the the effect of the aTLR10 Ab on a number of early signaling events

production of MIP-1 b induced by pairwise costimulation of either that drive B cell activation. To this end, primary human B cells by guest on October 1, 2021 the BCR and TLR7 or the BCR and CD40, which serve to mimic were stimulated through the BCR either alone or with TLR7 or TI and TD activation, respectively (Fig. 2C). TLR10 engagement CD40 as a costimulus. Compared to isotype control, we observed also suppressed MIP-1 b production induced by S. aureus Cowan that the aTLR10 Ab was able to suppress the phosphorylation of strain. These results suggest that the TLR10 not only suppresses the MAPK JNK but not P38 or ERK. Additionally, the aTLR10

FIGURE 3. BCR signaling is suppressed by a TLR10 mAb on primary human B cells. Primary human B cells were isolated from tonsillar tissue by homogenization, density gradient centrifugation, and a negative selection Ab mixture. B cells were preincubated with an isotype control or TLR10 mAb for 30 min prior to stimulation with the indicated agonist for 15 min after which the cells were lysed and frozen at 220˚C. (A) Lysates were probed for phosphorylated targets of TLR signaling. Data is representative of three independent experiments (two for pP38). (B) Blots were normalized against b-Actin and densities shown relative to the unstimulated isotype control cells. Error bars represent the SEM. *p , 0.1, **p , 0.05. The Journal of Immunology 5

Ab was able to suppress Akt signaling when stimulated with Suppression of ex vivo transgenic B cell activation aBCR and R848, suggesting TLR10 may also be able to target the Given that TLR10 is expressed by human B cells we examined PI3 kinase pathway (Fig. 3). isolated splenic B cells from TLR10 transgenic mice and found that the receptor was expressed at both the mRNA and protein level Generation of TLR10 transgenic mice (Fig. 4B, 4C). To assess the effects of TLR10 expression, we To better study TLR10, we sought to develop a suitable animal compared the proliferative responses of splenic B cells as a result model. Because TLR10 is a pseudogene in mice, and orthologs of ex vivo stimulation. Compared to non-transgenic control mice, of the TLR10 gene are present in all other rodents and splenocytes from TLR10 transgenic mice exhibited less prolifer- mammals examined thus far, it seemed reasonable to believe ation when stimulated with aIgM/aCD40 according to the CFSE that human TLR10 would function in mice. Therefore, the full- profile of viable cells as determined by their forward and side length human TLR10 gene, including several kb of the 59 and scatter characteristics. Although aIgM/CpG stimulation showed 39 untranslated region, was stably integrated in the mouse similar trends, the data did not reach statistical significance genome (see Materials and Methods). A total of three TLR10 (p = 0.116). Because we did not measure absolute numbers of transgenic founder lines were established that both expressed dividing cells, the data do not address potential differences in detectable levels of TLR10 and successfully passed the activation-induced cell death (Fig. 5A, 5B). Transgenic B cells, transgene to offspring. Of the three founder lines, line 5–1 irrespective of either of the two stimulatory conditions we tested, was selected for further functional assessment based on in- exhibited significantly decreased CD69 expression and IL-6 se- termediate levels of TLR10 expression and sizable litters. cretion compared with non-transgenic control mice (Fig. 5C, 5D).

Line 5–1 exhibits reverse transcriptase–dependent expression These data demonstrate that human TLR10 is capable of sup- Downloaded from of TLR10 in a variety of different tissues (Fig. 4A). TLR10 pressing murine B cell activation ex vivo and that TLR10- transgenic mice are viable, breed well, and do not exhibit any mediated suppression of B cells is conserved across different overt physical abnormities. mammalian species. http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 4. Human TLR10 transgenic mice stably express TLR10 on B cells. (A) Mouse organs were harvested and homogenized in Trizol with a Dounce homogenizer prior to RNA isolation from each indicated tissue. RNA was reverse transcribed in the presence and absence of reverse transcriptase [RT(-)] and checked for TLR10 expression by PCR. (B) Isolated murine B cell RNA was isolated from three non-transgenic and three transgenic mice before preparing cDNA and assaying for TLR10 as in (A). (C) Murine splenocytes from three non-transgenic and three transgenic mice were triple labeled for TLR10 and the B cell markers B220 and IgM. Histogram shows the TLR10 ﬂuorescence compared with an isotype control Ab from a representative non-transgenic and transgenic mouse. Bar graph shows the D mean ﬂuorescence intensity from the three transgenic and non-transgenic mice. 6 TLR10 SUPPRESSES B CELL ACTIVATION Downloaded from

FIGURE 5. Primary TLR10 transgenic murine B cell activation is suppressed compared with non-transgenic control mice. Murine spleens from three transgenic and three non-transgenic mice were collected and pooled, and a single-cell suspension was obtained. Murine B cells were further purified bya http://www.jimmunol.org/ negative selection Ab mixture. (A–C) B cells were preincubated with CFSE prior to stimulation with the indicated agonists for 72 h. After stimulation, live cells were gated by forward and side scatter characteristics. (A) Representative example of a fluorescent CFSE B cell cycling profile. The percentage of divided B cells (B) and CD69 expression (C) was analyzed from three different replicates based upon the marker present in (A). (D) Pooled B cells were stimulated for 24 h with the indicated agonists and cell-free supernatant collected to measure mIL-6 production. Data is representative of three independent experiments. *p , 0.1, **p , 0.05, ***p , 0.01.

The ex vivo experiments on splenic B cells were conducted ence in the steady-state serum concentrations between the TLR10 without the addition of an aTLR10 Ab suggesting that the ob- transgenic mice and the non-transgenic control mice for any of the served suppression is the direct result of heterologous expression Ig isotypes (Supplemental Fig. 2). Together, these results dem- by guest on October 1, 2021 of human TLR10. This prompted us to assess whether TLR10 onstrate that human TLR10 transgene expression in mice does not transgenic mice had deﬁciencies in either B cell development or grossly effect B cell development and Ab production. Ab production. To assess the former, we examined the ratio and abundance of the major B cell subpopulations in the bone marrow, Transgenic B cells are suppressed in vivo blood, spleen, and lymph nodes but no differences were observed To assess the effect of TLR10 expression on B cell responses between TLR10 transgenic and non-transgenic littermate control in vivo, we immunized transgenic or non-transgenic control mice mice (Supplemental Table II). Additionally, there was no differ- with the TI Ag, and B cell mitogen, LPS, which was conjugated to

FIGURE 6. TLR10 transgenic mice exhibit suppressed type 1 TI Ab responses. Six transgenic and non-transgenic age-matched male mice were immunized with 10 mg of NP-LPS by i.p. injection. Serum was collected 1 d preimmunization and 7 and 14 d postimmunization. Serum was assayed for the presence of NP-speciﬁc Abs by ELISA. Arbitrary units were calculated by a seven-point serially diluted standard curve of previously immunized mouse serum and then normalized to day 0. *p , 0.1, **p , 0.05, ***p , 0.01. The Journal of Immunology 7

FIGURE 7. TLR10 transgenic mice type 1 TI Ab response suppression is B cell intrinsic. B cells from ﬁve male transgenic and ﬁve male non-transgenic mice were isolated and pooled. Approximately 2E7 B cells were i.v. injected into eight age-matched mMT mice. One day post transfer, mice were immunized with 20 mg of NP-LPS by i.p. injection. Serum was collected and assayed as described in Fig. 6. *p , 0.1, **p , 0.05, ***p , 0.01. Downloaded from

the hapten NP. One and two weeks after immunization, serum which mMT mice, naturally deficient in B cells, received pooled http://www.jimmunol.org/ levels of NP-specific Abs in TLR10 transgenic mice were sig- splenic B cells from either TLR10 transgenic or non-transgenic nificantly decreased in both the IgM and IgG3 subclasses compared control mice prior to immunization with NP-LPS. Initial adoptive with serum levels in immunized non-transgenic control mice transfer experiments showed no difference in the populations of (Fig. 6). NP-specific Ab levels for other Ig subclasses also trended transgenic or non-transgenic B220+ B cells residing in the lower in TLR10 transgenic mice but did not reach significance in spleens of mMT mice 24 h after injection (N.J. Hess, unpublished our studies. These results demonstrate that TLR10 transgenic mice observations). are deficient in TI Ab responses to LPS, however, because TLR10 Similar to the above immunization findings, mice reconstituted is expressed throughout various cells and tissues of the mice the with B cells from TLR10 transgenic mice had significantly lower by guest on October 1, 2021 in vivo effects observed may be extrinsic to B cells. NP-specific IgM Ab responses to NP-LPS than those reconstituted with B cells from non-transgenic control mice. Additionally, NP- Transgenic TLR10 suppression is B cell intrinsic specific Ab levels for all the other Ig isotypes trended lower in To determine whether the in vivo effects of TLR10 on Ab responses mice receiving B cells from TLR10 transgenic mice (Fig. 7). are B cell intrinsic we performed adoptive transfer experiments in Together, these findings show that the suppression of Ab responses

FIGURE 8. TLR10 transgenic mice TD Ab response suppression is B cell intrinsic. B cells were collected and transferred as described in Fig. 7. One day post transfer mice were immunized with 100 mg of NP- CGG and 5 mg of LPS by i.p. injection. Serum was collected and assayed as described in Fig. 6. *p , 0.1, **p , 0.05, ***p , 0.01. 8 TLR10 SUPPRESSES B CELL ACTIVATION by TLR10 to the TI Ag LPS is B cell intrinsic. Our in vitro studies mouse model, it is able to suppress proinflammatory signaling and showed that TLR10 suppresses B cell responses and signaling in cytokine production. We also show that TLR10 engagement with response to CD40 costimulation, suggesting that TLR10 may also an mAb is able to suppress IL-6 production in primary mononuclear regulate immune responses to TD Ags. To assess this, we im- cells (22). In a different study, another group showed that TLR2 munized mMT mice, reconstituted with splenic B cells from either responses to either PAM3CSK4 or whole Borrelia bugdorferi are TLR10 transgenic or non-transgenic mice, with the TD protein Ag suppressed after TLR10 engagement with an mAb. They also use NP-CGG (chicken-g globulin) along with an immune adjuvant. In a transgenic mouse model to confirm that TLR10 is able to sup- addition to IgM, NP-specific serum Ab levels of IgG1, IgG2b and press TLR2-mediated inflammatory signaling (21). Our study IgA were all significantly reduced in mice receiving TLR10 expands upon this work by showing that TLR10-mediated sup- transgenic B cells with other Ab isotypes also trending lower pression is not limited to mononuclear cells and can also suppress (Fig. 8). This shows that, even when presented with costimulatory B cell adaptive immune responses. signals from T cells, TLR10 is able to suppress B cell Ab re- Two other studies have presented an opposing hypothesis that sponses. Taken together, we conclude that TLR10 is a B cell in- TLR10 is a proinflammatory receptor. They use either THP-1 or HT- trinsic suppressor of both TI and TD B cell activation. 29 cell lines to knockdown TLR10 followed by challenge with either H1N1 flu virus or live Listeria monocytogenes.Ineachcase,they reported increased expression of proinflammatory cytokines only Discussion upon infection with a live pathogen (34, 35). This suggests a pos- Since its reported discovery in 2001 (17), the biological function of sible alternative function for TLR10, perhaps in the context of live TLR10 has remained unknown. Recently, several groups have intracellular pathogens, which will require further investigation. ascribed different biological functions for TLR10, which has a Nonetheless, our study has vast implications for many B cell Downloaded from function that is now controversial. We have previously shown that malignancies that are driven by dysregulated TLR activation. It when overexpressed in a monocytic cell line or in a transgenic is now well established that unwanted or overactivation of TLR 7, 8 mouse, TLR10 is able to suppress TLR signaling irrespective and 9 in B cells are drivers of autoimmune diseases. Studies have of either MyD88 or TRIF recruitment (22). In this manuscript, we shown that mice deficient in TLRs 7–9 have attenuated autoim- support the hypothesis that TLR10 is a novel suppressor of mune diseases characterized by autoantibody production as in inflammatory responses and can suppress the adaptive immune rheumatoid arthritis and lupus (16, 36). Additionally, genetic http://www.jimmunol.org/ responses of B cells in vivo. polymorphisms in several TLR signaling components such as Since the discovery of TLRs, much of the focus has been on how IRF5, IRAK1 and TNFAIP3 have been shown to predispose in- they positively regulate inflammation, although more recently dividuals to lupus (37). This manuscript highlights the ability of research has focused on the negative regulation of TLR signaling. TLR10 engagement to suppress B cell responses after TLR acti- Two proteins, SIGIRR and ST2, have been shown to negatively vation, potentially offering a therapeutic opportunity for individ- regulate TLR signaling by sequestering the proximal adaptor uals with B cell autoimmune diseases. MyD88 (24–26). This regulation is most likely not how TLR10 TLR10 remains a vastly understudied TLR that for many years was functions, as TLR10 has the ability to suppress both TLR- believed to have no biological function. In this study, we have shown that by guest on October 1, 2021 dependent and TLR-independent signaling in B cells (22). TLR10 is expressed within human B cells and, through Ab-mediated Additionally, the observations that TLR10 is not under convergent crosslinking, can suppress the activation of human B cells. Further- evolution, has numerous nonsynonymous mutations in its TIR domain more, we have shown that the TLR10 machinery is conserved across and is able to form a stable homodimer may allow TLR10 to recruit mammals by creating a knockin mouse model that expresses the full- novel TIR-domain containing proteins (18, 19, 27). TLR10 contains length human TLR10 gene under its native human promoter, which has several mutations within the BB loop of its TIR domain, which has the ability to suppress murine B cell activation. Lastly, we have shown been shown to be important in TLR signaling. Furthermore, those that the TLR10-mediated suppression is a B cell intrinsic property and is mutations are conserved across different mammalian TLR10 proteins, able to suppress both TI and TD Ab responses. This initial evidence that suggesting it has important implications in TLR10 signaling (18). Much TLR10 is a B cell suppressor may assist in the future development of of TLR biology focuses on the five well-studied TIR-domain containing novel immunotherapies for B cell autoimmune diseases. proteins: MyD88, MAL (TIRAP), TRIF, TRAM and SARM, although there is a possibility that there are additional TIR-domain containing Acknowledgments proteins either understudied or undiscovered (5). Two such examples The authors would like to the thank the Transgenic Mouse Facility at the are the proteins B-cell adaptor for PI3K (BCAP) (28) and B-cell Roy J. Carver Biotechnology Center at the University of Illinois Urbana- scaffold with ankyrin repeats 1 (BANK1) (29), which are novel TIR Champaign for assistance in designing and creating the TLR10 transgenic domain–containing proteins that havebeenlinkedtoTLRsignaling. mouse line used in this study. More importantly, a BCAP knockout mouse was shown to have ele- vated levels of inflammation in response to a Salmonella typhimurium Disclosures infection (28). This highlights the possibility that TLR10 could be The authors have no financial conflicts of interest. recruiting a novel TIR-domain protein to mediate the suppression. There is also a growing body of evidence that there is a large synergy between the BCR, TLRs, and TNF receptors in B cells. References Several key mediators of each pathway have been shown to be 1. Kawai, T., and S. Akira. 2010. The role of pattern-recognition receptors in innate important in the regulation of the complementary pathway such as immunity: update on toll-like receptors. Nat. Immunol. 11: 373–384. 2. Hornung, V., S. Rothenfusser, S. Britsch, A. Krug, B. Jahrsdo¨rfer, T. Giese, the proteins PKD (30), BTK (31), TRAF5 (32), and Syk (33). It is S. Endres, and G. Hartmann. 2002. Quantitative expression of toll-like receptor possible that by suppressing one of these pathways, TLR10 is able 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and to alter B cell signaling, which allows it to suppress several sensitivity to CpG oligodeoxynucleotides. J. Immunol. 168: 4531–4537. pathways simultaneously. 3. Dorner, M., S. Brandt, M. Tinguely, F. Zucol, J.-P. Bourquin, L. Zauner, C. Berger, M. Bernasconi, R. F. Speck, and D. Nadal. 2009. Plasma cell toll-like In support of our study, another manuscript from our laboratory receptor (TLR) expression differs from that of B cells, and plasma cell TLR has shown that when TLR10 is overexpressed in cell lines or in a triggering enhances immunoglobulin production. Immunology 128: 573–579. The Journal of Immunology 9

4. Bourke, E., D. Bosisio, J. Golay, N. Polentarutti, and A. Mantovani. 2003. The 22. Jiang, S., X. Li, N. J. Hess, Y. Guan, and R. I. Tapping. 2016. TLR10 is a toll-like receptor repertoire of human B lymphocytes: inducible and selective negative regulator of both MyD88-Dependent and -independent TLR signaling. expression of TLR9 and TLR10 in normal and transformed cells. Blood 102: J. Immunol. 196: 3834–3841. 956–963. 23. Ranoa, D. R. E., S. L. Kelley, and R. I. Tapping. 2013. Human 5. Kawasaki, T., and T. Kawai. 2014. Toll-like receptor signaling pathways. Front. lipopolysaccharide-binding protein (LBP) and CD14 independently deliver tri- Immunol. 5: 461. acylated lipoproteins to Toll-like receptor 1 (TLR1) and TLR2 and enhance 6. O’Neill, L. A. J. 2008. When signaling pathways collide: positive and negative formation of the ternary signaling complex. J. Biol. Chem. 288: 9729–9741. regulation of toll-like receptor signal transduction. Immunity 29: 12–20. 24. Kondo, T., T. Kawai, and S. Akira. 2012. Dissecting negative regulation of Toll- 7. Gay, N. J., M. F. Symmons, M. Gangloff, and C. E. Bryant. 2014. Assembly and like receptor signaling. Trends Immunol. 33: 449–458. localization of Toll-like receptor signalling complexes. Nat. Rev. Immunol. 14: 25. Wald, D., J. Qin, Z. Zhao, Y. Qian, M. Naramura, L. Tian, J. Towne, J. E. Sims, 546–558. G. R. Stark, and X. Li. 2003. SIGIRR, a negative regulator of toll-like receptor- 8. Lin, S.-C., Y.-C. Lo, and H. Wu. 2010. Helical assembly in the MyD88-IRAK4- interleukin 1 receptor signaling. Nat. Immunol. 4: 920–927. IRAK2 complex in TLR/IL-1R signalling. Nature 465: 885–890. 26. Brint, E. K., D. Xu, H. Liu, A. Dunne, A. N. J. McKenzie, L. A. J. O’Neill, and 9. Janeway Jr., C. A. 1989. Approaching the asymptote? Evolution and revolution F. Y. Liew. 2004. ST2 is an inhibitor of interleukin 1 receptor and Toll-like re- in immunology. Cold Spring Harb. Symp. Quant. Biol. 54: 1–13. ceptor 4 signaling and maintains endotoxin tolerance. Nat. Immunol. 5: 373–379. 10. Medzhitov, R. 2009. Approaching the asymptote: 20 years later. Immunity 30: 27. Nyman, T., P. Stenmark, S. Flodin, I. Johansson, M. Hammarstro¨m, and 766–775. P. Nordlund. 2008. The crystal structure of the human toll-like receptor 10 cy- 11. Rawlings, D. J., M. A. Schwartz, S. W. Jackson, and A. Meyer-Bahlburg. 2012. toplasmic domain reveals a putative signaling dimer. J. Biol. Chem. 283: 11861– Integration of B cell responses through toll-like receptors and antigen receptors. 11865. Nat. Rev. Immunol. 12: 282–294. 28. Troutman, T. D., W. Hu, S. Fulenchek, T. Yamazaki, T. Kurosaki, J. F. Bazan, 12. Pasare, C., and R. Medzhitov. 2005. Control of B-cell responses by toll-like and C. Pasare. 2012. Role for B-cell adapter for PI3K (BCAP) as a signaling receptors. Nature 438: 364–368. adapter linking Toll-like receptors (TLRs) to serine/threonine kinases PI3K/Akt. 13. Browne, E. P. 2012. Regulation of B-cell responses by toll-like receptors. Im- Proc. Natl. Acad. Sci. USA 109: 273–278. 29. Wu, Y.-Y., R. Kumar, R. Iida, H. Bagavant, and M. E. Alarco´n-Riquelme. 2016. munology 136: 370–379. 14. Koh, Y. T., J. C. Scatizzi, J. D. Gahan, B. R. Lawson, R. Baccala, K. M. Pollard, BANK1 regulates IgG production in a lupus model by controlling TLR7- dependent STAT1 activation. PLoS One 11: e0156302.

B. A. Beutler, A. N. Theofilopoulos, and D. H. Kono. 2013. Role of nucleic acid- Downloaded from 30. Haxhinasto, S. A., and G. A. Bishop. 2003. A novel interaction between protein sensing TLRs in diverse autoantibody specificities and anti-nuclear antibody- kinase D and TNF receptor-associated factor molecules regulates B cell producing B cells. J. Immunol. 190: 4982–4990. receptor-CD40 synergy. J. Immunol. 171: 4655–4662. 15. Pelka, K., T. Shibata, K. Miyake, and E. Latz. 2016. Nucleic acid-sensing TLRs 31. Kenny, E. F., S. R. Quinn, S. L. Doyle, P. M. Vink, H. van Eenennaam, and L. A. Immunol. Rev. and autoimmunity: novel insights from structural and cell biology. J. O’Neill. 2013. Bruton’s tyrosine kinase mediates the synergistic signalling 269: 60–75. between TLR9 and the B cell receptor by regulating calcium and calmodulin. 16. Shlomchik, M. J. 2009. Activating systemic autoimmunity: B’s, T’s, and tolls. PLoS One 8: e74103. Curr. Opin. Immunol. 21: 626–633. 32. Buchta, C. M., and G. A. Bishop. 2014. TRAF5 negatively regulates TLR sig- 17. Chuang, T., and R. J. Ulevitch. 2001. Identification of hTLR10: a novel human naling in B lymphocytes. J. Immunol. 192: 145–150. http://www.jimmunol.org/ Toll-like receptor preferentially expressed in immune cells. Biochim. Biophys. 33. Ying, H., Z. Li, L. Yang, and J. Zhang. 2011. Syk mediates BCR- and CD40- Acta 1518: 157–161. signaling integration during B cell activation. Immunobiology 216: 566–570. 18. Roach, J. C., G. Glusman, L. Rowen, A. Kaur, M. K. Purcell, K. D. Smith, 34. Regan, T., K. Nally, R. Carmody, A. Houston, F. Shanahan, J. Macsharry, and L. E. Hood, and A. Aderem. 2005. The evolution of vertebrate Toll-like re- E. Brint. 2013. Identification of TLR10 as a key mediator of the inflammatory ceptors. Proc. Natl. Acad. Sci. USA 102: 9577–9582. response to Listeria monocytogenes in intestinal epithelial cells and macro- 19. Mikami, T., H. Miyashita, S. Takatsuka, Y. Kuroki, and N. Matsushima. 2012. phages. J. Immunol. 191: 6084–6092. Molecular evolution of vertebrate Toll-like receptors: evolutionary rate differ- 35. Lee, S. M. Y., K.-H. Kok, M. Jaume, T. K. W. Cheung, T.-F. Yip, J. C. C. Lai, ence between their leucine-rich repeats and their TIR domains. Gene 503: 235– Y. Guan, R. G. Webster, D.-Y. Jin, and J. S. M. Peiris. 2014. Toll-like receptor 10 243. is involved in induction of innate immune responses to influenza virus infection. 20. Guan, Y., D. R. E. Ranoa, S. Jiang, S. K. Mutha, X. Li, J. Baudry, and Proc. Natl. Acad. Sci. USA 111: 3793–3798. R. I. Tapping. 2010. Human TLRs 10 and 1 share common mechanisms of innate 36. Herlands, R. A., S. R. Christensen, R. A. Sweet, U. Hershberg, and

immune sensing but not signaling. J. Immunol. 184: 5094–5103. M. J. Shlomchik. 2008. T cell-independent and toll-like receptor-dependent by guest on October 1, 2021 21. Oosting, M., S.-C. Cheng, J. M. Bolscher, R. Vestering-Stenger, T. S. Plantinga, antigen-driven activation of autoreactive B cells. Immunity 29: 249–260. I. C. Verschueren, P. Arts, A. Garritsen, H. van Eenennaam, P. Sturm, et al. 2014. 37. Delgado-Vega, A., E. Sańchez, S. Lo¨fgren, C. Castillejo-Lo´pez, and Human TLR10 is an anti-inflammatory pattern-recognition receptor. Proc. Natl. M. E. Alarcoń-Riquelme. 2010. Recent findings on genetics of systemic auto- Acad. Sci. USA 111: E4478–E4484. immune diseases. Curr. Opin. Immunol. 22: 698–705.