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 September 23, 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 September 23, 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 trans- duction. 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- http://www.jimmunol.org/ These activation events not only drive inflammatory processes, but duce adaptive responses is best understood through their actions 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 September 23, 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.

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