Soluble CD83 Inhibits T Cell Activation by Binding to the TLR4/MD-2 Complex on CD14+ Monocytes This information is current as Joe M. Horvatinovich, Elizabeth W. Grogan, Marcus Norris, of September 29, 2021. Alexander Steinkasserer, Henrique Lemos, Andrew L. Mellor, Irina Y. Tcherepanova, Charles A. Nicolette and Mark A. DeBenedette J Immunol 2017; 198:2286-2301; Prepublished online 13 February 2017; Downloaded from doi: 10.4049/jimmunol.1600802 http://www.jimmunol.org/content/198/6/2286 Supplementary http://www.jimmunol.org/content/suppl/2017/02/11/jimmunol.160080 http://www.jimmunol.org/ Material 2.DCSupplemental References This article cites 80 articles, 29 of which you can access for free at: http://www.jimmunol.org/content/198/6/2286.full#ref-list-1 Why The JI? Submit online. by guest on September 29, 2021 • 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 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 Author Choice Freely available online through The Journal of Immunology Author Choice option 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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Soluble CD83 Inhibits T Cell Activation by Binding to the TLR4/MD-2 Complex on CD14+ Monocytes Joe M. Horvatinovich,*,1 Elizabeth W. Grogan,*,1 Marcus Norris,* Alexander Steinkasserer,† Henrique Lemos,‡ Andrew L. Mellor,‡ Irina Y. Tcherepanova,* Charles A. Nicolette,* and Mark A. DeBenedette* The transmembrane protein CD83, expressed on APCs, B cells, and T cells, can be expressed as a soluble form generated by alternative splice variants and/or by shedding. Soluble CD83 (sCD83) was shown to be involved in negatively regulating the immune response. sCD83 inhibits T cell proliferation in vitro, supports allograft survival in vivo, prevents corneal transplant rejection, and attenuates the progression and severity of autoimmune diseases and experimental colitis. Although sCD83 binds to human PBMCs, the specific molecules that bind sCD83 have not been identified. In this article, we identify myeloid differentiation factor-2 (MD-2), the coreceptor within the TLR4/MD-2 receptor complex, as the high-affinity sCD83 binding partner. TLR4/MD-2 Downloaded from mediates proinflammatory signal delivery following recognition of bacterial LPSs. However, altering TLR4 signaling can atten- uate the proinflammatory cascade, leading to LPS tolerance. Our data show that binding of sCD83 to MD-2 alters this signaling cascade by rapidly degrading IL-1R–associated kinase-1, leading to induction of the anti-inflammatory mediators IDO, IL-10, and PGE2 in a COX-2–dependent manner. sCD83 inhibited T cell proliferation, blocked IL-2 secretion, and rendered T cells unre- sponsive to further downstream differentiation signals mediated by IL-2. Therefore, we propose the tolerogenic mechanism of action of sCD83 to be dependent on initial interaction with APCs, altering early cytokine signal pathways and leading to T cell http://www.jimmunol.org/ unresponsiveness. The Journal of Immunology, 2017, 198: 2286–2301. uman CD83, identified as a 45-kDa type 1 membrane malignancies, whereby higher sCD83 plasma levels correlated with glycoprotein of the Ig superfamily of receptors, is expressed shorter treatment-free survival of these patients (11, 12). Released H as a membrane-bound form and a soluble form (1, 2). The sCD83 from mature DCs infected with CMV leads to inhibition of transmembrane form of CD83 is expressed on mature dendritic T cell proliferation (13), and neonatal DC infection with either Gram- cells (DCs), B cells, macrophages, activated T cells and T regulatory negative or -positive bacteria releases sCD83, resulting in suppression cells, and thymic epithelial cells (3, 4). The expression on DCs is of allergic responses (14). These observations prompted several by guest on September 29, 2021 involved in the activation of T cell–mediated immune responses groups to develop recombinant sCD83 proteins (15–17) to evaluate (5–8); however, a soluble form of CD83, generated by splice the immunosuppressive activity of sCD83 for therapeutic use in variants or by shedding, inhibits T cell proliferation (9). Soluble models of autoimmunity and transplantation. Preparations derived CD83 (sCD83) released from tumor cells blocks CD4+ and CD8+ from expression of the soluble portion of CD83 lacking the trans- T cell proliferation (10). Additional studies revealed that sCD83 is membrane domain used in in vivo models of murine and rat kidney present in elevated concentrations in a number of hematological and heart allograft transplantation prevented organ transplant rejec- tion (18–20). Moreover, sCD83 induced tolerance in skin allograft *Research Department, Argos Therapeutics, Inc., Durham, NC 27704; †Cancer Immunol- transplants (21). In vitro evaluations showed that recombinant CD83 ogy, Department of Immune Modulation, University Hospital Erlangen, University of protein preparations inhibits T cell proliferation in vitro (22–24). ‡ Erlangen-Nuremberg, D-91052 Erlangen, Germany; and Inflammation and Tolerance Moreover, recombinant sCD83 protein reduced the symptoms as- Program, Cancer Center, Georgia Regents University, Augusta, GA 30912 1 sociated with models of experimental autoimmune encephalomy- J.M.H. and E.W.-G. contributed equally to this work. elitis (25) and murine experimental colitis (26) and, when applied ORCIDs: 0000-0001-7187-6729 (A.S.); 0000-0002-9553-6160 (A.L.M.); 0000-0002- topically, prevented corneal graft rejection (27). 2285-9778 (C.A.N.). The underlying mechanism by which sCD83 mediates its reg- Received for publication May 6, 2016. Accepted for publication January 13, 2017. ulatory properties is not clear. The extracellular domain of CD83 is This work was supported by research funding from Argos Therapeutics, Inc. (to J.M.H., E.W.G., M.N., I.Y.T., C.A.N., and M.A.D.). A.S. was supported by Deutsche reported to bind to monocytes and DCs (24, 28), thus positioning Forschungsgemeinschaft Grant DFG-SFB1181 Project B3. A.L.M. was supported CD83 at the interface between innate and adaptive immunity. Pub- by the National Institute of Allergy and Infectious Diseases/National Institutes of lished reports suggest that the extracellular domain of CD83 fused Health (Grant AI103347) and the Carlos and Marguerite Mason Trust. to Ig interacts with a 72-kDa glycosylated protein involved in cell Address correspondence and reprint requests to Dr. Mark A. DeBenedette, Argos Therapeutics, Inc., 4233 Technology Drive, Durham, NC 27704. E-mail address: adhesion (29); however, this was not investigated further or con- [email protected] firmed by other investigators. Recently, it was indicated that CD83 The online version of this article contains supplemental material. may act in a homotypic way (30); however, the investigators failed to Abbreviations used in this article: DC, dendritic cell; IRAK, IL-1R–associated ki- demonstrate a clear biophysical interaction and, therefore, identifi- nase; MD-2, myeloid differentiation factor-2; MFI, mean fluorescence intensity; cation of the sCD83 counterreceptor(s) remains elusive. Identifying sCD83, soluble CD83; TBS-T, TBS with 0.01% Tween 20. the biologically relevant cell surface receptor(s) for sCD83 would This article is distributed under The American Association of Immunologists, Inc., greatly expand our understanding of how sCD83 mediates inhibition Reuse Terms and Conditions for Author Choice articles. of T cell activation, alleviates symptoms of autoimmune diseases, Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 and induces tolerance in the allograft transplant setting. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600802 The Journal of Immunology 2287 In this study, we identified the cell surface binding partner for Materials and Methods sCD83 as myeloid differentiation factor-2 (MD-2), the coreceptor Preparation of recombinant sCD83 protein associated with the TLR4 signaling complex. Furthermore, we The extracellular domain of human CD83 (20–145 aa) was PCR amplified, provide evidence that the expression of CD14 and CD44 on and expression of sCD83 was induced in Escherichia coli and purified as monocytes is a necessary component of this unique complex of previously described (16, 24, 52). Briefly, sCD83 was purified using a receptors. The major role for TLR4 is recognition of pathogen- GSTrap 5-ml column (Amersham Pharmacia Biotech). The GST–sCD83– associated molecular patterns, specifically LPS, from Gram- containing fractions were loaded onto an anion exchange column, and negative bacteria, which serves as a strong inducer of innate proteins were separated by three different linear salt gradients. The GST– sCD83 fusion protein was incubated with thrombin to separate the sCD83 immunity (31–34). LPS signaling through TLR4 requires the protein from GST and loaded onto glutathione Sepharose 4B columns. The coreceptors CD14 and MD-2