The Ox40/Ox40 Ligand Pathway Promotes Pathogenic Th Cell Responses, Plasmablast Accumulation, and Lupus Nephritis in NZB/W F1 Mice This information is current as of September 29, 2021. Jonathan Sitrin, Eric Suto, Arthur Wuster, Jeffrey Eastham-Anderson, Jeong M. Kim, Cary D. Austin, Wyne P. Lee and Timothy W. Behrens J Immunol published online 10 July 2017 http://www.jimmunol.org/content/early/2017/07/07/jimmun Downloaded from ol.1700608 Supplementary http://www.jimmunol.org/content/suppl/2017/07/07/jimmunol.170060 http://www.jimmunol.org/ Material 8.DCSupplemental Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published July 10, 2017, doi:10.4049/jimmunol.1700608 The Journal of Immunology The Ox40/Ox40 Ligand Pathway Promotes Pathogenic Th Cell Responses, Plasmablast Accumulation, and Lupus Nephritis in NZB/W F1 Mice Jonathan Sitrin,* Eric Suto,† Arthur Wuster,*,‡ Jeffrey Eastham-Anderson,x Jeong M. Kim,{ Cary D. Austin,x Wyne P. Lee,† and Timothy W. Behrens* Ox40 ligand (Ox40L) locus genetic variants are associated with the risk for systemic lupus erythematosus (SLE); however, it is unclear how Ox40L contributes to SLE pathogenesis. In this study, we evaluated the contribution of Ox40L and its cognate receptor, Ox40, using in vivo agonist and antagonist approaches in the NZB 3 NZW (NZB/W) F1 mouse model of SLE. Ox40 was highly expressed on several CD4 Th cell subsets in the spleen and kidney of diseased mice, and expression correlated with disease severity. Treatment of aged NZB/W F1 mice with agonist anti-Ox40 mAbs potently exacerbated renal disease, which was Downloaded from accompanied by activation of kidney-infiltrating T cells and cytokine production. The agonist mAbs also induced activation and inflammatory gene expression in splenic CD4 T cells, including IFN-regulated genes, increased the number of follicular helper T cells and plasmablasts in the spleen, and led to elevated levels of serum IgM and enhanced renal glomerular IgM deposition. In a type I IFN–accelerated lupus model, treatment with an antagonist Ox40:Fc fusion protein significantly delayed the onset of severe proteinuria and improved survival. These data support the hypothesis that the Ox40/Ox40L pathway drives cellular and humoral autoimmune responses during lupus nephritis in NZB/W F1 mice and emphasize the potential clinical http://www.jimmunol.org/ value of targeting this pathway in human lupus. The Journal of Immunology, 2017, 199: 000–000. ystemic lupus erythematosus (SLE) is a multiorgan auto- disappointing, indicating the urgent need to identify and validate immune disease characterized by aberrant cellular and new therapeutic targets. S humoral immune responses. Lupus nephritis (LN), one of One key aspect of SLE pathophysiology is that immune com- the most common and severe clinical presentations of SLE, occurs plexes (ICs), consisting largely of autoantibodies, such as anti- in up to 50% of adults and 70% of children with the disease (1, 2). dsDNA and anti-RNA–binding proteins, together with their cognate Despite decades of effort, most clinical trials for SLE have been Ags, deposit in blood vessels and renal glomeruli, leading to vas- by guest on September 29, 2021 culitis and nephritis [(3), reviewed in Refs. 4, 5)]. IC deposition results in the recruitment of lymphocytes and myeloid cells to *Department of Human Genetics, Genentech, Inc., South San Francisco, CA 94080; †Department of Translational Immunology, Genentech, Inc., South San Francisco, kidney glomeruli, arterioles, and tubular interstitium, which further CA 94080; ‡Department of Bioinformatics and Computational Biology, Genentech, exacerbates renal damage. Inc., South San Francisco, CA 94080; xDepartment of Pathology, Genentech, Inc., South San Francisco, CA 94080; and {Department of Cancer Immunology, Genentech, Recent genome-wide association studies indicate that many Inc., South San Francisco, CA 94080 immune-related pathways contribute to human SLE, and .50 ORCID: 0000-0002-5388-7413 (C.D.A.). genetic loci are now associated with disease risk (6). Understanding Received for publication April 27, 2017. Accepted for publication June 7, 2017. how these loci predispose to disease is critical for understanding This work was supported by Genentech, Inc. disease etiology and for advancing therapeutic hypotheses. Ox40 J.S. conceptualized, designed, performed, and analyzed experiments and wrote ligand (Ox40L; TNFSF4) was originally identified as an SLE risk the manuscript; E.S. designed and performed experiments; A.W. and J.E.-A. gene by Vyse and colleagues (7); however, only modest progress analyzed data; J.M.K. supervised and generated reagents; C.D.A. designed, per- has been made in understanding its role in SLE pathogenesis. formed, and analyzed experiments and edited the manuscript; W.P.L. supervised and designed experiments; and T.W.B. supervised and wrote and edited the Ox40L, and its cognate receptor, Ox40 (TNFRSF4), are mem- manuscript. bers of the TNFR superfamily. There is significant evidence that The RNA sequencing data presented in this article have been submitted to the this receptor/coreceptor pair is responsible for regulating adaptive National Center for Biotechnology Information Gene Expression Omnibus (https:// T cell immune responses (reviewed in Refs. 8–11). In brief, www.ncbi.nlm.nih.gov/geo/) under accession numbers GSE99645 and GSE99646. Ox40L expression is largely restricted to APCs, whereas Ox40 is Address correspondence and reprint requests to Dr. Jonathan Sitrin, Genentech, Inc., OMNI Human Genetics, 133003-4, MS93B, 1 DNA Way, South San Francisco, CA expressed primarily on activated or Ag-experienced Th cells and 94080. E-mail address: [email protected] regulatory T cells (Tregs). Ox40 promotes Th cell survival (12– The online version of this article contains supplemental material. 14), cytokine production (15), and memory cell formation and Abbreviations used in this article: cat. no., catalog number; GC, germinal center; IC, recall responses (16, 17). Ox40 may also have an important role in immune complex; IHC, immunohistochemistry; IPA, Ingenuity Pathway Analysis; regulating the balance of effector T cell, Th cell, and Treg func- LN, lupus nephritis; nRPKM, normalized read per kilobase gene model per million total reads; NZB/W, NZB 3 NZW; Ox40L, Ox40 ligand; PAS, periodic acid–Schiff; tions (18, 19). RNASeq, RNA sequencing; SLE, systemic lupus erythematosus; Tconv, Foxp32 Follicular helper T (Tfh) cells make up a specific helper cell conventional helper T; Tfh, follicular helper T; Treg, regulatory T cell. subset that promotes humoral immune responses and has been This article is distributed under The American Association of Immunologists, Inc., implicated in lupus pathogenesis (20, 21). Tfh cells express Ox40, Reuse Terms and Conditions for Author Choice articles. as well as other functional and identifying markers (e.g., CXCR5 Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 and ICOS), and produce cytokines, such as IL-21. Disruption of www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700608 2 Ox40 SIGNALING EXACERBATES MURINE LUPUS NEPHRITIS Tfh cells or their key functional molecules ameliorate lupus chromatography (S200; both from GE Healthcare) to .95% purity by symptoms in animal models (22). For example, in Roquin-deficient SDS–gel electrophoresis, ,5% aggregate (analytical size-exclusion chro- , mice that develop lupus-like autoimmunity due to derepressed matography multi-angle static light scattering; Wyatt Technology), and 0.5 EU/mg. Functional binding of Ox40:Fc was confirmed using a cell line ICOS expression and Tfh cell activity (23), deletion of ICOS, SAP expressing murine Ox40L. (a signaling molecule required for germinal centers [GCs]), or Bcl6 For IFN-a–accelerated lupus studies, mouse IFN-a5 was cloned into a (a transcription factor required for Tfh cell development) disrupted pShuttleX vector and transferred into an Ad5 viral vector (Ad5–IFN-a)by Tfh cells and disease in these animals (24, 25). In addition to ICOS, the Baylor College of Medicine Vector Development Lab (Houston, TX). Twelve- to thirteen-week-old NZB/W F1 animals were randomized into Ox40 was recently found to be a direct target of the Roquin re- treatment groups, and serum was collected prior to treatment. A single i.v. pressor protein family (26), highlighting the possibility that the injection of the Ad5–IFN-a vector (1.2 3 108 PFU) was given 3 d prior to Ox40 pathway might contribute to the disease phenotypes in those treatment with Ox40:Fc or a control Ig s.c. at a dose of 20 mg/kg, twice a animals. week, for