Regulatory T Cell Numbers in Inflamed Skin Are Controlled by Local Inflammatory Cues That Upregulate CD25 and Facilitate Antigen-Driven Local Proliferation This information is current as of October 4, 2021. Alison C. Billroth-MacLurg, Jill Ford, Alexander Rosenberg, Jim Miller and Deborah J. Fowell J Immunol 2016; 197:2208-2218; Prepublished online 10 August 2016; doi: 10.4049/jimmunol.1502575 Downloaded from http://www.jimmunol.org/content/197/6/2208 Supplementary http://www.jimmunol.org/content/suppl/2016/08/10/jimmunol.150257 Material 5.DCSupplemental http://www.jimmunol.org/ References This article cites 69 articles, 29 of which you can access for free at: http://www.jimmunol.org/content/197/6/2208.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on October 4, 2021 • 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 Regulatory T Cell Numbers in Inflamed Skin Are Controlled by Local Inflammatory Cues That Upregulate CD25 and Facilitate Antigen-Driven Local Proliferation Alison C. Billroth-MacLurg,* Jill Ford,* Alexander Rosenberg,† Jim Miller,* and Deborah J. Fowell* CD4+Foxp3+ regulatory T cells (Tregs) are key immune suppressors that regulate immunity in diverse tissues. The tissue and/or inflammatory signals that influence the magnitude of the Treg response remain unclear. To define signals that promote Treg accumulation, we developed a simple system of skin inflammation using defined Ags and adjuvants that induce distinct cytokine milieus: OVA protein in CFA, aluminum salts (Alum), and Schistosoma mansoni eggs (Sm Egg). Polyclonal and Ag-specific Treg accumulation in the skin differed significantly between adjuvants. CFA and Alum led to robust Treg accumulation, with >50% of all skin CD4+ T cells being Foxp3+. In contrast, Tregs accumulated poorly in the Sm Egg–inflamed skin. Surprisingly, we found no Downloaded from evidence of inflammation-specific changes to the Treg gene program between adjuvant-inflamed skin types, suggesting a lack of selective recruitment or adaptation to the inflammatory milieu. Instead, Treg accumulation patterns were linked to differences in CD80/CD86 expression by APC and the regulation of CD25 expression, specifically in the inflamed skin. Inflammatory cues alone, without cognate Ag, differentially supported CD25 upregulation (CFA and Alum > Sm Egg). Only in inflammatory milieus that upregulated CD25 did the provision of Ag enhance local Treg proliferation. Reduced IL-33 in the Sm Egg–inflamed environment was shown to contribute to the failure to upregulate CD25. Thus, the magnitude of the Treg response in inflamed tissues is http://www.jimmunol.org/ controlled at two interdependent levels: inflammatory signals that support the upregulation of the important Treg survival factor CD25 and Ag signals that drive local expansion. The Journal of Immunology, 2016, 197: 2208–2218. D4+Foxp3+ regulatory T cells (Tregs) are important im- factors controlling the accumulation, size, and function of the Treg mune sentinels that orchestrate the control of homeo- compartment in specific tissues will be important as strategies for C static and infectious immune activation. Studies have therapeutic manipulation of Treg advance. elegantly defined Treg differentiation, homeostasis, phenotype, Increases in Treg number are seen in a variety of infected and and suppressive function using Tregs within lymphoid tissues (1). autoimmune tissues (12–17). Local Treg proliferation in non- by guest on October 4, 2021 However, recent observations suggest that there is a substantial lymphoid tissues has been observed (15, 18, 19) and can be linked degree of peripheral reshaping of Treg phenotype and function in part to cognate Ag expression (13, 20). Costimulatory signals depending on the tissue location and inflammatory milieu (2–4). from CD28 and CTLA-4, important for Treg development and Tregs in distinct peripheral tissues express specific gene programs homeostasis (21, 22), also tune Ag-induced Treg proliferation and that appear to facilitate tailored local function (5, 6), whereas the function in the periphery (23–27). IL-2 provides a potent survival type of inflammation can also drive Treg functional specialization signal to Tregs (28–31) and has recently become a primary target (7–11). The relative contribution of location and inflammation to for regulating Treg number in vivo (32–34). A deficiency in IL-2 Treg dynamics in peripheral tissues remains unclear. Defining the within target tissues has been linked to the negative regulation of Tregs in autoimmune settings through downregulation of CD25 expression (19). Less well understood are the positive signals that *Department of Microbiology and Immunology, David H. Smith Center for Vaccine support CD25 expression by Tregs in peripheral tissues and pro- Biology and Immunology, Aab Institute of Biomedical Sciences, University of mote local Treg accumulation. Recent studies suggest IL-33 may † Rochester, Rochester, NY 14642; and Division of Allergy, Immunology and Rheu- be an important factor in promoting Treg expansion in tissues via matology, University of Rochester Medical Center, Rochester, NY 14642 upregulation of local IL-2 (35, 36) and can be counterregulated by ORCID: 0000-0001-7093-4448 (D.J.F.). IL-23 (37). Received for publication December 15, 2015. Accepted for publication July 11, 2016. To study the signals that modulate Treg accumulation at sites This work was supported by National Institutes of Health/National Institute of of inflammation we focused our studies on the skin and assessed Allergy and Infectious Diseases Grants P01 AI02851, R01 AI070826 and HHSN272201200005C (to D.J.F.). Treg accumulation to OVA immunization in the context of different Address correspondence and reprint requests to Dr. Deborah J. Fowell, David H. Smith types of induced inflammation, using distinct Th1 CFA or Th2- Center for Vaccine Biology and Immunology, University of Rochester, 601 Elmwood inducing (aluminum salts [Alum] or Schistosome mansoni Eggs Avenue, Box 609, Rochester, NY 14642. E-mail address: Deborah_Fowell@urmc. [Sm Egg]) adjuvants. We demonstrate distinct variations in Treg rochester.edu accumulation in the skin between inflammatory milieus with ro- The online version of this article contains supplemental material. bust accumulation of Tregs in CFA and Alum-induced skin in- Abbreviations used in this article: Alum, aluminum salt; DC, dendritic cell; dLN, draining lymph node; KLH, keyhole limpet hemocyanin; LN, lymph node; pOVA, flammation but not in Sm Egg–inflamed skin. This was tissue OVA peptide; Sm Egg, Schistosoma mansoni egg; Tconv, conventional CD4+ T cell; specific, observed in the inflamed skin but not the draining lymph Tg, transgenic; TLDA, TaqMan low-density array; Treg, regulatory T cell; WT, wild- node (dLN), and cell type specific, seen for polyclonal and Ag- type. specific Tregs but not conventional CD4+ T cells (Tconv). The Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 differences were not dependent on the primary effector cytokines www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502575 The Journal of Immunology 2209 in the skin, nor did they reflect the selection of particular milieu- TaqMan low-density array specific Treg subsets. Rather, we found the distinct inflammatory cDNA from purified Tregs from ear and LN was isolated using the Cells-To- milieus differentially regulated the expression of CD25 by Tregs CT kit (Ambion Life Technologies). Samples were preamplified using in the skin, independent of Ag. CFA and Alum potently upregu- primer pools specific to custom TaqMan low-density arrays (TLDAs) lated CD25 on Tregs, whereas the Sm Egg milieu failed to support (Supplemental Fig. 1). Amplified samples were loaded onto TLDA cards CD25 upregulation. The upregulation of CD25 correlated with and RT-PCR performed using 7900HT Fast Real-Time PCR System (Applied Biosystems) at the University of Rochester Genomics Center. differences in extent of CD80/CD86 expression by APCs in the Hypoxanthine phosphoribosyltransferase and GAPDH were used as en- skin. However, CD25 upregulation by the inflammatory milieu dogenous controls, and Treg samples from non-dLN were used as the was not sufficient to drive local Treg proliferation, which required calibrator. cognate Ag. Unexpectedly, the effect of Ag was only seen in in- Anti–IL-23 blockade and IL-33 administration flammatory settings that supported CD25 upregulation in the ab- sence of Ag, suggesting that distinct inflammatory cues precondition Anti–IL-23 Ab (BioLegend) or control IgG was administered to immu- Tregs for receptivity to Ag in inflamed tissues. nized mice on days 4, 5, and 6 postimmunization, 100 mg/mouse i.p. (40). rIL-33 (BioLegend) or PBS was administered to immunized mice on days 4, 5, and 6 postimmunization,