Identification of Novel CD4+ T Cell Subsets in the Target Tissue Of
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Identification of Novel CD4+ T Cell Subsets in the Target Tissue of Sjögren's Syndrome and Their Differential Regulation by the Lymphotoxin/LIGHT Signaling Axis This information is current as of October 2, 2021. Scott Haskett, Jian Ding, Wei Zhang, Alice Thai, Patrick Cullen, Shanqin Xu, Britta Petersen, Galina Kuznetsov, Luke Jandreski, Stefan Hamann, Taylor L. Reynolds, Norm Allaire, Timothy S. Zheng and Michael Mingueneau J Immunol 2016; 197:3806-3819; Prepublished online 7 Downloaded from October 2016; doi: 10.4049/jimmunol.1600407 http://www.jimmunol.org/content/197/10/3806 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2016/10/06/jimmunol.160040 Material 7.DCSupplemental References This article cites 46 articles, 8 of which you can access for free at: http://www.jimmunol.org/content/197/10/3806.full#ref-list-1 Why The JI? Submit online. by guest on October 2, 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 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. The Journal of Immunology Identification of Novel CD4+ T Cell Subsets in the Target Tissue of Sjo¨gren’s Syndrome and Their Differential Regulation by the Lymphotoxin/LIGHT Signaling Axis Scott Haskett, Jian Ding,1 Wei Zhang,1 Alice Thai, Patrick Cullen, Shanqin Xu, Britta Petersen, Galina Kuznetsov, Luke Jandreski, Stefan Hamann, Taylor L. Reynolds, Norm Allaire, Timothy S. Zheng, and Michael Mingueneau Despite being one of the most common rheumatologic diseases, there is still no disease-modifying drug for primary Sjo¨gren’s syndrome (pSS). Advancing our knowledge of the target tissue has been limited by the low dimensionality of histology techniques and the small size of human salivary gland biopsies. In this study, we took advantage of a molecularly validated mouse model of pSS to characterize tissue-infiltrating CD4+ T cells and their regulation by the lymphotoxin/LIGHT signaling axis. Novel cell Downloaded from subsets were identified by combining highly dimensional flow and mass cytometry with transcriptomic analyses. Pharmacologic modulation of the LTbR signaling pathway was achieved by treating mice with LTbR-Ig, a therapeutic intervention currently being tested in pSS patients (Baminercept trial NCT01552681). Using these approaches, we identified two novel CD4+ T cell subsets characterized by high levels of PD1: Prdm1+ effector regulatory T cells expressing immunoregulatory factors, such as Il10, Areg, Fgl2, and Itgb8, and Il21+ effector conventional T cells expressing a pathogenic transcriptional signature. Mirroring these observations in mice, large numbers of CD4+PD1+ T cells were detected in salivary glands from Sjo¨gren’s patients but not in http://www.jimmunol.org/ normal salivary glands or kidney biopsies from lupus nephritis patients. Unexpectedly, LTbR-Ig selectively halted the recruitment of PD12 naive, but not PD1+, effector T cells to the target tissue, leaving the cells with pathogenic potential unaffected. Altogether, this study revealed new cellular players in pSS pathogenesis, their transcriptional signatures, and differential dependency on the lymphotoxin/LIGHT signaling axis that help to interpret the negative results of the Baminercept trial and will guide future therapeutic interventions. The Journal of Immunology, 2016, 197: 3806–3819. rimary Sjo¨gren’s syndrome (pSS) is characterized by a frequent extraglandular involvement and increased risk for B cell lymphocytic infiltration of the lacrimal and salivary lymphoma (reviewed in Refs. 1, 2). Th1 cells are thought to be the by guest on October 2, 2021 P glands, leading to xerophtalmia and xerostomia, as well as predominant CD4+ T cell subset infiltrating human biopsies (3, 4), although cytokine profiling identified secretion patterns, such as those characterized by IL-10 and TGF-b production, which are Immunology Research, Biogen, Cambridge, MA 02142 compatible with other effector or regulatory T cell (Treg) subsets 1J.D. and W.Z. contributed equally to this work. (4–6). Accordingly, markers of Th17 cells (7, 8), Tregs (9–11), ORCIDs: 0000-0001-9853-5289 (S. Haskett); 0000-0002-5689-1784 (L.J.); 0000- and T follicular helper (Tfh) cells (6, 12) were detected in pSS 0002-6941-0900 (T.L.R.). biopsies, but the relative importance of each of these subsets in Received for publication March 9, 2016. Accepted for publication September 11, pSS pathogenesis and their relationships remain unknown. 2016. Blocking the LTbR signaling pathway showed clear efficacy in S. Haskett, J.D., and M.M. designed, executed, and analyzed all experiments; W.Z. several preclinical models of pSS (13–15). LTbR signaling is established and optimized the hydrodynamic injection technique; immunohistochem- istry method development was directed by G.K. and T.L.R.; immunohistochemistry important for secondary and ectopic/tertiary lymphoid tissue or- experiments were performed by S.X. and B.P., and immunohistochemistry quantifica- ganization (reviewed in Ref. 16). Specifically, LTbR engagement tion methods were designed and executed by L.J. and S. Hamann; histological analyses by LIGHT or LTab regulates the expression of peripheral lymph and interpretation were conducted by T.L.R.; A.T., P.C., and N.A. performed all tran- scriptomic experiments; S. Haskett and T.L.R. contributed to figure and manuscript node addressin (PNAd) and mucosal vascular addressin cell ad- preparation; and M.M. designed the research study and wrote the manuscript. All hesion molecule 1 on high endothelial venules, which are required authors participated in data and project discussion and reviewed and approved the final version of the manuscript. for efficient immune cell recruitment into tissues (17). Novel technological approaches, such as digital immunohisto- The gene expression data presented in this article have been submitted to the National Center for Biotechnology Information Gene Expression Omnibus under accession chemistry techniques, single-cell repertoire sequencing, and highly number GSE81621. multiparametric mass cytometry, have recently improved our Address correspondence and reprint requests to Dr. Michael Mingueneau, Immunol- knowledge of the target tissue in pSS (18–20). Despite these ad- ogy Research, Biogen, 115 Broadway, Cambridge, MA 02142. E-mail address: vances, our knowledge of the cellular composition of the immune [email protected] infiltrate in pSS patients is still incomplete, partly because the The online version of this article contains supplemental material. number of cells that can be recovered from human lip biopsies Abbreviations used in this article: B6, C57BL/6; CyTOF, cytometry by time-of-flight; eTreg, effector Treg; FC, fold change; FDR, false discovery rate; IEG, intermediate- precludes further dissection of cellular subsets and phenotypes early gene; LGT, lacrimal gland tissue; PNAd, peripheral lymph node addressin; pSS, contributing to each cell lineage and the mechanisms by which primary Sjo¨gren’s syndrome; SLE, systemic lupus erythematosus; Tconv, conventional they infiltrate the target tissue. T cell; Tfh, T follicular helper; Treg, regulatory T cell; VAT, visceral adipose tissue. To circumvent these technical limitations, we took advantage of Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 the NOD mouse, which is one of the best-studied and validated www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600407 The Journal of Immunology 3807 models of pSS (reviewed in Ref. 21). Using a combination of mass nonetheless sufficient to characterize enzyme-sensitive surface marker cytometry, highly multiparametric flow cytometry, and tran- expression. scriptomics, we characterized the kinetics of immune infiltration Mass and flow cytometry in lacrimal gland tissue (LGT) and further dissected the subsets of LGT-infiltrating CD4+ T cells in the target tissue. Interestingly, Single-cell suspensions were stained with 30 metal-conjugated Abs (Table I) and prepared for cytometry by time-of-flight (CyTOF) analysis, LTbR signaling blockade using LTbR-Ig revealed that not all as described (23). For flow cytometry, cells were preincubated with anti- CD4+ T cell subsets are affected equally by this treatment, an CD16/32 Ab (2.4G2; BD Biosciences) and stained with fluorochrome- observation that helps us to understand the negative results of the conjugated Abs (described below). All samples were analyzed using an primary end point in the Baminercept clinical trial and will guide LSR II instrument (BD Biosciences). the design of future therapeutic strategies. Fluorochrome-conjugated Abs The following mAbs specific for murine Ags were purchased from BD Materials and Methods Biosciences, BioLegend, and eBioscience: BUV395-conjugated CD4 (SK3), Mice BV785-conjugated PD1 (29F.1A12), V510-conjugated CD45.2 (104), FITC- conjugated CD19 (1D3), PE-conjugated