Regulatory T Cell Numbers in Inflamed Skin Are Controlled by Local Inflammatory Cues That Upregulate CD25 and Facilitate Antigen

Home , HAVCR2, Regulatory T cell

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 absence 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, 2 mg/mouse i.p. (41). Treg accumulation in Materials and Methods the inflamed ear was assessed day 7 postimmunization. Mice Statistical analysis + Female wild-type (WT), DO11.10 TCR-transgenic (Tg) , Foxp3-GFP, and All statistical analysis was done in GraphPad Prism (GraphPad). Where Thy1.1 BALB/c mice were purchased from The Jackson Laboratory. 4get indicated, Mann–Whitney, Wilcoxon matched-pairs, or two-way ANOVA

BALB/c mice were provided by R. Locksley (University of California, San tests were performed for the data: *p , 0.05, **p , 0.005, ***p , 0.0005. Downloaded from Francisco). All mouse experimentation was reviewed and approved by the University Committee for Animal Resources at the University of Rochester Medical Center. Results Differential Treg accumulation in the skin with distinct Cell purification adjuvant-induced inflammation + CD4 T cells were enriched from spleen and LN by complement lysis of To study the effect of inflammation on Tregs accumulation in the

cells bearing CD8 (3.155), MHC class II (M5/114), and heat-stable Ag http://www.jimmunol.org/ (J11D) as described (38) (American Type Culture Collection). CD25+ and skin, we used adjuvants previously reported to induce distinct type CD252 cells were enriched using an MACS column (Miltenyi Biotec) and 1 or 2 cytokine responses (42–46). Foxp3-GFP mice were im- sorted (FACSAria; BD Biosciences) for KJ126+ Foxp3-GFP+CD62L+ munized intradermally with OVA protein in PBS, CFA, Alum, or 2 2 2 CD44 naive Tregs and KJ126+Foxp3-GFP CD62L+CD44 naive Tconv. with Sm Egg. Seven days postimmunization, cells in the skin and + + + TCR-Tg cell adoptive transfer dLN were assayed for CD4 Tconv and CD4 Foxp3 Treg populations by flow cytometry. Inflammation led to significant in- A total of 3–3.5 3 105 naive Thy1.1+DO11.10+ Tregs and 3–3.5 3 105 + + + + creases in the number and frequency of Foxp3 Tregs in the skin, naive Thy1.1 /1.2 DO11.10 Tconv were injected i.v. together, or alone, + into congenic Thy1.2+ WT BALB/c mice 24 h prior to immunization. At representing more than half of all CD4 T cells under certain inflammatory conditions (Fig. 1A–C). The Treg magnitude dif- day 7 postimmunization, single-cell suspensions from ear and LN were as- by guest on October 4, 2021 sessed by flow cytometry. feredwiththeadjuvant:CFAandAlumdrivingstrongTreg numbers and Sm Egg–induced inflammation supporting signifi- Immunizations cantly lower Treg numbers (Fig. 1A, 1B). Treg frequencies were WTor 4get mice were immunized intradermally in the ear with OVAprotein consistently elevated in the inflamed tissues compared with their (20 mg/ear) and the adjuvants CFA, Alum, or Sm Egg (750 eggs/ear). In respective dLNs (Fig. 1C). Indeed, adjuvant-induced changes in some experiments, the contralateral ear was immunized with keyhole limpet hemocyanin (KLH) protein (5 mg/ear) and adjuvants. For the Treg frequency were not seen in the dLN (Fig. 1C), consistent peptide pulse, mice were treated with 40 mg OVA peptide i.v. in 200 ml with the local cutaneous milieu modulating Treg accumulation. PBS 6 h prior to harvest. Adjuvant effects on T cell accumulation in the skin appear to be Treg-specific because the number of Tconv were not compromised Flow cytometry in the Sm Egg–inflamed conditions (Fig. 1D). Ears were treated for 30 min at 37˚C with 1 mg activated Collagenase Dispase To determine if this accumulation pattern was seen with Ag- (Roche) and deactivated using EDTA. Single-cell suspensions of skin and specific Tregs, we transferred naive OVA-specific Foxp3-GFP LN were stained with Abs from eBioscience unless otherwise stated: Live- + + 2 + + Dead Aqua (Lifetech), CD45 (clone 30-F11; BD Biosciences), CD4 (clone DO11.10 Tregs (CD4 CD62L CD44 Foxp3 ) and Tconv (CD4 + 2 2 GK1.5; BD Biosciences), Thy1.1 (clone H1S51), Thy1.2 (clone 53-2.1; BD CD62L CD44 Foxp3 ) into WT hosts 24 h prior to immuniza- Biosciences), CD25 (clone PC61.5), CD127 (clone A7R34), FcER1 (clone tion. DO11.10+ Tregs accumulated to high numbers in the CFA MAR-1), T1/ST2 (clone DJ8; Molecular BioSystems), CD49b (clone DX5), and Alum-inflamed skin, with a lower number in Sm Egg–inflamed Gr-1 (clone RB6-8C5; BioLegend), CD11b (clone M1/70; BioLegend), and Foxp3 (clone FJK-16S) using Foxp3 fix/perm reagents. For BrdU, mice skin (Fig. 2A, 2B), leading to a striking dominance of Ag-specific received 1 mg BrdU i.p. in PBS 4 h prior to harvest. Cells were stained with Tregs in the inflamed skin. The input ratio of Treg to Tconv (1:1) BrdU APC Staining kit (BD Biosciences). was maintained in the dLN and spleen but markedly elevated in the skin with CFA and Alum-induced inflammation (Fig. 2C). As Cytokine measurements seen with the polyclonal response, the Sm Egg–induced inflam- Whole ear homogenate or 1 3 106 LN cells was stimulated overnight with mation failed to support DO11.10+ Treg accumulation in the skin 2 mmol OVA peptide in ELISPOT plates coated with Ab for IFN-g or IL-4 (Fig. 2A–C). Thus, Treg accumulation in the inflamed skin is as described (39). For cytokines in the ear, the ear was treated for 30 min at 37˚C with Collagenase Dispase and homogenized in 500 ml PBS. Ear robust, and the magnitude of this response is inflammatory context homogenate supernatants were assayed for cytokines with the Milliplex dependent. MAP Mouse Cytokine/Chemokine Magnetic Bead Panel kit (Millipore). The differences in accumulation of Tregs in the skin could come For cytokine blockade, 1 mg anti–IFN-g Ab (XMG 1.2) or anti–IL-4 Ab from the differential recruitment or survival of natural Tregs, dif- (11B11) or PBS was given i.p. on days 6, 7, 8, and 9 postimmunization and ferential loss of Foxp3 in natural Tregs, and/or differential de novo harvested on day 10. For IL-23 cytokine blockade, 100 mg anti–IL-23 p19 + (clone MMp19B2; BioLegend) or IgG control was administered i.p. on generation of Foxp3 -induced Tregs from Tconv precursors. To test days 4, 5, and 6 postimmunization and harvested on day 7. these possibilities, we used sorted naive DO11.10 T cell transfers 2210 Treg ACCUMULATION IN THE INFLAMED SKIN

FIGURE 1. Distinct polyclonal Treg accumulation in the skin with different types of adjuvant-induced inflammation. (A) Representative FACS plots of CD4+Foxp3+ Tregs cells in inflamed skin day 7 after immunization of BALB/c mice with OVA protein in CFA, Alum, or Sm Egg. Numbers in top right quadrant are percentage of CD4+ T cells. (B) Number of Foxp3+CD4+ Tregs in the skin on day 7. Each symbol represents an individual mouse. (C) Frequency of Foxp3+ Tregs within the CD4 compartment in dLN (LN) and skin as in (A). (D) Number of Foxp32CD4+ Tconv in skin on day 7. Four p , p , p , independent experiments, three to four mice per group. Statistics by Mann–Whitney test. * 0.05, ** 0.005, *** 0.0005. Downloaded from from Foxp3-GFP reporter mice to determine inflammation-induced (Fig. 2F). Attempts to determine suppressive Treg function from acquisition of Foxp3 expression by Tconv and the potential loss of the different milieus on a per-cell basis using in vitro suppression Foxp3 by Tregs in the inflamed skin. In all inflammatory settings, assays were technically unsuccessful due to the low Treg numbers the Tregs largely remained Foxp3 positive, and the Tconv largely recovered from the inflamed ear, particularly the Sm Egg–inflamed remained Foxp3 negative (Fig. 2D, 2E). We found no evidence for tissue (Fig. 2A). http://www.jimmunol.org/ greater loss of Foxp3 in the transferred Tregs in the Sm Egg– inflamed skin, where Treg numbers had been seen to be lower, nor a Kinetics of adjuvant-induced differences in Treg accumulation greater gain in Foxp3 expression by Tconv in CFA and Alum, in the skin where Treg numbers were higher (Fig. 2D, 2E). The data are in Kinetic analysis was performed to examine the stability of Treg support of the differential accumulation of natural Tregs rather than accumulation postimmunization. No significant differences be- differential induction of Tregs from Tconv. tween adjuvants were seen in polyclonal Treg skin accumulation To assess the physiological consequence of the differences in early, day 5 postimmunization, suggesting initial Treg seeding Treg numbers in the skin, we determined the relative change in of the inflamed skin is independent of the type of inflammation DO11.10 Foxp32 Tconv accumulation with and without cotrans- (Fig. 3). In contrast, maintenance of elevated Tregs in the inflamed by guest on October 4, 2021 fer of DO11.10 Foxp3+ Tregs. Consistent with there being fewer skin was highly context dependent. CFA and Alum inflammation Tregs in the Sm Egg–inflamed ear, suppression of the Tconv re- supported high frequencies of Tregs in the skin for at least 14 d sponse in the ear was attenuated in the Sm Egg–inflamed ear (Fig. 3A, 3B), whereas the Sm Egg environment failed to enhance

FIGURE 2. Ag-specific Treg accumulation in different types of skin inflammation. Sorted Treg (Thy1.1) and Tconv (Thy1.1/1.2) DO11.10+ T cells were adoptively transferredtoBALB/cmice(Thy1.2).Number(A) and frequency (B)ofThy1.1+ DO11.10+ Tregs in the CD4+ compartment in the skin on day 7 after immunization with OVA/CFA, OVA/Alum, or OVA/Sm Egg. (C) Ratio of transferred Tregs to Tconv in spleen, dLN, and skin. Input ratio (1:1) marked with dotted line. Five independent experiments, statistics by Mann–Whitney test. Sorted Foxp32GFP+ Treg (Thy1.1) and Foxp32GFP2 Tconv (Thy1.1/1.2) DO11.10+ T cells were transferred into BALB/c mice (Thy1.2) and the frequency of Foxp3-GFP expression in Tregs (D)andTconv(E) populations assessed in the skin day 7 after immunization with OVA and distinct adjuvants. (F) Relative suppression of DO11.10+ Tconv accumulation in the inflamed ear with and without cotransfer of DO11.10+ Tregs (Tconv number with Treg cotransfer/Tconv number in absence of Treg cotransfer 3 100). Two to three independent experiments. Statistics by Mann–Whitney test. *p , 0.05, **p , 0.005, ***p , 0.0005. The Journal of Immunology 2211

ABC

FIGURE 3. Differential Treg frequencies sustained over time. BALB/c mice were immunized with OVA/CFA, OVA/Alum, or OVA/Sm Egg and cells isolated from the inflamed skin on days 5, 7, 10, and 14. Foxp3+ frequency of CD4+ cells (A) and cell number of polyclonal Foxp3+ Tregs (B) in the skin. (C) Ratio of polyclonal Treg/Tconv number in skin. Data are representative of two independent experiments with three to four mice per group. Statistics by two-way ANOVA, p # 0.001 for adjuvant comparison, and Bonferroni posttest shown for Alum versus Sm Egg. Additional statistics: ***CFA versus Sm Egg day 7, day 10 (A); **CFA versus Sm Egg day 7, day 10 (C). **p , 0.005, ***p , 0.0005. and/or maintain Tregs past day 5 (Fig. 3A, 3B). These changes of IFN-g or IL-4 after the inflammation was established (days resulted in sustained differences in the Treg/Tconv ratio in the skin 6–9 postimmunization) did not change the pattern of Treg (Fig. 3C) and prompted investigation into the signals that regulate accumulation in the skin (data not shown). Therefore, IFN-g Treg accumulation in the inflamed skin. andIL-4playnoroleinthemaintenanceofTregsinthein- flamed skin. Effector cytokines and Treg accumulation Cytokines can directly affect Treg numbers in the steady state Downloaded from Effector cytokines have recently been shown to influence Treg and during microbial or autoimmune inflammation (49). To homing and functional capacity (2, 47). Therefore, we char- further characterize the inflammatory milieu, we assayed for acterized IFN-g and IL-4 effector cytokines and immune cytokine and chemokine induction in the inflamed ear tissue subsets present in our inflammatory models using IL-4 reporter (Fig. 4D, 4E). All inflamed environments upregulated cyto- mice 4get (48), day 7 postimmunization when distinct Treg kines in comparison with naive, unimmunized ear tissue, with

numbers were evident (Fig. 3). The number of OVA-specific similarities between the adjuvants in the availability of IL-12p70, http://www.jimmunol.org/ cells producing IFN-g andIL-4intheskinweredetermined GM-CSF, IL-3, and IL-10 (Fig. 4D) and the chemokine CCL11 by ELISPOT (Fig. 4A, 4B). CFA induced a potent type 1 in- (Fig. 4E). CFA clearly induced a potent proinflammatory cy- flammation, with the lowest IL-4 to IFN-g ratio and the re- tokine and chemokine response with strong upregulation of cruitment of distinct innate subsets dominated by neutrophils TNF-a, IL-6, and IL-1b (Fig. 4D) and all chemokines tested (Fig. 4C). Alum inflammation had a greater frequency of IL-4– including CCL5, CXCL9, and CXCL10 (Fig. 4E). Alum and producing cells and more type 2 innate subsets such as eosino- Sm Egg inflammation shared many similarities consistent phils, basophils, and mast cells (Fig. 4A–C) (44). Consistent with reduced IFN-g and enhanced IL-4 (Fig. 4D, 4E). The with its potent type 2 adjuvant effects (45), Sm Egg inflamma- pattern of cytokine and chemokine expression in the Sm tionwasheavilyskewedtowardatype2responsewiththe Egg–inflamed tissue was either comparable to both CFA and by guest on October 4, 2021 highest IL-4/IFN-g ratio and greatest number of CD4+IL-4– Alum or to Alum but not CFA, despite differences in the ac- enhanced GFP+ Th2 cells and type 2 innate effectors eosino- cumulation of Tregs between Sm Egg and CFA/Alum. Thus, phils, basophils, and mast cells (Fig. 4A–C). However, Ab blockade we did not find a cytokine or chemokine pattern that was

ABC

FIGURE 4. Signature cytokines induced by the different adjuvants. Number (A) and ratio (B) of IFN-g and IL-4 producers in skin day 7 postimmunization by OVA-specific ELISPOT. (C) Frequency and number of CD45+ immune cell subsets from immunized IL-4–GFP reporter mice, day 7: CD4 T cell effectors, CD4+GFP+ or GFP2; basophils/mast cells, CD42GFP+FcERI+DX5+; eosinophils, CD42GFP+Gr-1+CD11bhi; neutrophils, CD42GFP2GR- 1hiCD11b+; monocytes, CD42GFP2GR-1intCD11b+; and NK cells, CD42GFP2DX5+CD11b+. Combined results from three independent experiments. Statistics by two-way ANOVA: p , 0.001 for interaction and p , 0.001 for cell type. (D and E) Cytokine and chemokine levels in skin homogenates from naive skin (NV) or day 7 OVA-immunized skin with CFA, Alum, or Sm Egg. Combined two independent experiments, four mice per group. Statistics by Mann–Whitney test. *p , 0.05, **p , 0.005, ***p , 0.0005. 2212 Treg ACCUMULATION IN THE INFLAMED SKIN consistent with the differential Treg numbers in the inflamed pression in the Tregs in the Sm Egg environment, suggesting dermis. that the Tregs that make it to the Sm Egg–inflamed skin retain functional capacity. Thus, differences in Treg accumulation in Gene expression changes in Foxp3+ Tregs reflect changes in the skin appear not to be controlled by differential recruitment anatomical location but not changes between types of or activation of inflammation-specific Treg subsets. inflammation Transcriptional studies have revealed site-specific Tregs profiles Treg numbers correlate with skin-restricted changes in APC that point to tissue regulation ofTreghomingandfunction(50, expression of CD80/CD86 and Treg expression of CD25 51). To determine if Tregs from the skin display molecular IL-2, IL-7, and IL-15 have all been shown to provide important characteristics that may support distinct tissue accumulation, survival signals to Tregs in tissues (54, 55). IL-2 levels were similar we analyzed Treg gene expression from the inflamed skin (day across all types of inflammation (Fig. 6A). IL-7 and IL-15 were also 7) using custom gene array microfluidics cards containing 186 present in both the CFA and Sm Egg inflammation (Fig. 6A). inflammatory and Treg signature genes (Supplemental Fig. 1) Therefore, Treg numbers were low in the Sm Egg inflammation (51, 52). Genes associated with Th1- or Th2-type inflammation despite available IL-2, IL-7, and IL-15. In contrast, Treg numbers (chemokine receptors, effector molecules, and CD4 transcrip- did correlate with the tissue expression of IL-23 and IL-33, a bal- tionfactors),previouslyassociated with “inflammatory” Tregs ance of these cytokines being important in regulating Treg number (4), were not significantly different between types of dermal in the intestine (37). CFA had a low IL-23/IL-33 ratio, driven by inflammation (Fig. 5A–F), despite major differences in the high levels of IL-33 in the skin, which correlated with increased local cytokine milieu (Fig. 4). Instead, Treg genes were sig- Treg accumulation (Fig. 6A, 6B). In contrast, the Sm Egg adjuvant– nificantly different by anatomical location, between the skin induced limited amounts of IL-33 but high levels of IL-23 resulting Downloaded from and LN (50). Chemokine receptors such as CXCR3, CCR5, and in a high IL-23/IL-33 ratio (Fig. 6A, 6B) and correlating with poor CCR4 were upregulated in all inflammatory conditions relative Treg accumulation in the skin. However, following previously to the LN (Fig. 5A, 5B), and CCR7, important in tissue exit published protocols (40, 41), neither Ab blockade of IL-23, nor (53), was downregulated (Fig. 5B). Similarly, markers of Treg addition of rIL-33 was sufficient to boost Treg numbers in the Sm activation such as CTLA-4, granzyme B, and IL-10 were all Egg–induced inflamed dermis (Supplemental Fig. 2). elevatedintheskin,evenintheSm Egg–induced inflammatory Treg survival is also regulated at the level of CD28 signals (21, http://www.jimmunol.org/ setting where the number of Tregs was significantly reduced 26, 27). Therefore, we next looked at adjuvant-induced changes in (Fig. 5C, 5D). Indeed, for a number of activation markers APC in the skin and dLN. The number of MHC class II+ cells (granzyme B and IL-10), there was a trend toward higher ex- (Fig. 7A) and MHC class II expression levels (Fig. 7B) in the dLN by guest on October 4, 2021

FIGURE 5. Treg phenotype in the distinct inflammatory milieus. (A, C, and E) Gene expression analysis by TLDA of Foxp32GFP+CD4+7-amino- actinomycin D2 Tregs isolated from inflamed skin day 7. LN calibrator shown and normalized to GAPDH. Genes clustered on log2 of 22DDCt (2DD threshold cycle [Ct]) using Euclidean distance and average linkage. Numbered groups represent Tregs isolated from individual experiments. (B, D, and F) Relative gene expression of select genes. Statistics by Mann–Whitney. *p # 0.05 for LN and at least one of the inflamed skin groups, three to five independent experiments. The Journal of Immunology 2213

FIGURE 6. Inflammatory milieu and CD25 expression. (A) Cytokine levels in skin homogenates from naive skin (NV) or day 7 OVA-immunized skin with PBS, CFA, Alum, or Sm Egg. Combined two independent experiments, four mice per group. (B) Ratio of IL-23/IL-33 in the inflamed skin. Statistics by Mann–Whitney test. *p , 0.05, **p , 0.005, ***p , 0.0005. were similar for all adjuvants. MHC class II+ cell numbers were Consistent with a role for CD28 signals in the regulation of Treg also similar in the skin for CFA and Sm Egg (Fig. 7A) despite the expression of CD25 (27), Tregs displayed higher levels of CD25 in marked differences in Treg accumulation. For each type of adju- CFA and Alum-inflamed skin and lower levels in Sm Egg– vant, the MHC class II+ cells in the skin also expressed both inflamed skin (Fig. 7E, 7F). In contrast, similar CD25 expression CD11c and CD11b (data not shown). To assess the potential for levels were seen on Tregs in the dLNs of all inflammatory con- costimulation, we analyzed the expression of the CD28 ligands ditions (Fig. 7F, 7G), correlating with the similar CD80/CD86 CD80 and CD86. We chose these particular molecules because of expression in the LN (Fig. 7D). Moreover, we observed a signif- the link between CD28 signaling in the periphery and Treg sur- icant upregulation of CD25 expression in the CFA and Alum- Downloaded from vival (21, 26, 27). There was a striking difference in the level of inflamed skin compared with the dLN that was not observed CD80 and CD86 on the MHC class II+CD11c+CD11b+ population with Sm Egg inflammation (Fig. 7F). This is in contrast to auto- in the different inflammatory milieus, with APCs in CFA and immune settings in which inflamed nonlymphoid tissues fail to Alum expressing more CD80 and CD86 than APCs in Sm-Egg– support high CD25 expression (19). Interestingly, this phenome- induced inflammation (Fig. 7C, 7D). These differences were not non appears to be dependent on inflammatory cues, as Tregs in

seen in the respective dLNs (Fig. 7C, 7D). Thus, skin-restricted naive skin expressed signiﬁcantly less CD25 than those in the dLN http://www.jimmunol.org/ changes in expression of CD80 and CD86 by MHC class II+ (Fig. 7F) (56). Thus, the ability of Tregs to receive costimulatory CD11c+CD11b+ cells correlate with the changes in Treg accu- signals to maintain CD25 expression in the inﬂamed skin, and mulation (Fig. 1). hence respond to IL-2 for survival, appears to be regulated by the by guest on October 4, 2021

FIGURE 7. Differential expression of CD80/CD86 by tissue APCs correlates with differential expression of CD25 by Tregs. Analysis of APCs in inflamed skin day 7 after immunization of BALB/c mice with OVA protein in CFA, Alum, or Sm Egg. (A) Number of MHC class II+ cells in the inflamed ear and dLN. (B) MHC class II mean fluorescence intensity (MFI). (C) Representative FACS profiles of CD80 and CD86 expression on MHC class II+ CD11c+CD11b+ T cells. (D) CD80 and CD86 MFI. (E) CD25 levels on polyclonal Foxp3+ Tregs in the skin day 7 postimmunization. Gray filled histogram, fluorescence-minus-one; solid line, CD25 expression; numbers, percentage of cells in CD25hi gate. (F) CD25 MFI of all Tregs from LN and skin. Statistics by paired t test between LN and skin; Mann–Whitney test between skin groups. (G) Representative histogram of CD25 levels on Foxp3+ Tregs in LN day 7 postimmunization. *p , 0.05, **p , 0.005. 2214 Treg ACCUMULATION IN THE INFLAMED SKIN type of inflammation, and these signals are limited in the Sm Egg ST2 levels were moderately but significantly reduced in the environment (19). presence of cognate Ag in the Sm Egg milieu, which may contribute to the reduced Treg accumulation. To directly test if Ag CD25 expression is enhanced by inflammation in the absence drives an increased level of CD25 expression in the inflamed skin, of cognate ligand we pulsed KLH-immunized mice with OVA peptide (pOVA) 6 h In studies to address the role of Ag recognition in the accumulation prior to harvest on day 7. Indeed, increased levels of CD25 were of Tregs in the inflamed skin, we observed an interesting level of observed on Tregs after the pOVA pulse in the KLH/CFA- control of CD25 expression independent of Ag. DO11.10 TCR-Tg inflamed skin (Fig. 9C). However, CD25 expression was not sig- Tregs and Tconv were transferred into mice and immunized with nificantly increased in Sm Egg inflammation with additional noncognate Ag KLH and the distinct adjuvants. CD25 levels on pOVA pulse, suggesting that even with an Ag supply, the degree of OVA-specific Tregs were significantly modulated by the type of CD25 expression is controlled in part by the inflammatory milieu local inflammation: CFA and Alum inducing high levels of CD25 (Fig. 8) or by recognition of Ag in the context of CD28 costim- expression on Tregs in the skin in the absence of cognate Ag ulation (Fig. 7). Moreover, Tregs appear specifically sensitive to (Fig. 8A, 8B). Strikingly, the inflamed Sm Egg environment failed the local skin inflammatory environment with respect to CD25 to support CD25 upregulation in the skin (Fig. 8A, 8B). This expression levels, because Tconv upregulated CD25 expres- suggests that specific inflammatory milieus can modulate Treg sion similarly in all inflammatory settings (Fig. 9D). Thus, CD25- CD25 expression levels locally in the tissue, independent of Ag inducing inflammatory cues may be a necessary preconditioning recognition/TCR signaling. In contrast, although IL-33R (ST2) event for Treg Ag receptivity in inflamed tissues. IL-33 is an in- expression by Tregs was also upregulated in the inflamed tissue teresting candidate for the regulation of CD25 expression on Tregs independent of cognate Ag, it was not differentially regulated by in inflamed tissues given its links to local induction of IL-2 (35, Downloaded from the distinct adjuvants (Fig. 8C), despite very different IL-23/IL-33 36). Indeed, rIL-33 administration was sufficient to boost CD25 ratios (Fig. 6B). IL-23 has been postulated to regulate Treg expression in the Sm Egg–induced inflamed dermis to levels seen numbers by inhibiting IL-33 responsiveness via downregulation of with CFA inflammation, but only in the context of cognate Ag ST2 (37). In these studies, we did not see a correlation between (Fig. 9E). Thus, IL-33 alone is not sufficient to regulate CD25 higher IL-23 and reduced ST2 expression. Thus, rather than IL-23 expression in inflamed tissues (KLH group, Fig. 9E) but can being a negative regulator of Treg accumulation in our studies, the synergize with TCR signals to upregulate CD25 (OVA group, http://www.jimmunol.org/ change in IL-33 levels with the different adjuvants may control Fig. 9E). Treg accumulation indirectly via changes in paracrine IL-2 production by dendritic cells (DCs), as previously suggested (35, 36). Ag-driven Treg CD25 expression correlates with enhanced local proliferation Ag boosts Treg CD25 expression, if the inflammatory milieu is To determine the functional consequence of differential CD25 permissive expression on Tregs in the inflamed skin, we assayed for in situ Cognate Ag boosted Treg expression of CD25 in CFA and proliferation using an acute BrdU pulse 4 h prior to harvest. Alum inflammation, but not in Sm Egg inflammation (Fig. 9A). In Consistent with Treg accumulation patterns, DO11.10+ TCR-Tg contrast, cognate Ag did not enhance the level of expression of Tregs displayed robust BrdU incorporation in CFA and Alum by guest on October 4, 2021 ST2 by Tregs in any of inflammatory milieus (Fig. 9B); in fact, inflammation but limited BrdU staining in the Sm Egg skin (Fig. 10A–C). Treg proliferation appeared to be skin restricted, as Treg expansion in the dLN was not different between adjuvants (Fig. 10D). In contrast, TCR-Tg Tconv in the skin displayed similar proliferation patterns in all types of inflammation (Fig. 10E), arguing against a major difference in Ag availability in the different adjuvant milieus. Our data suggest that inflammatory signals can modulate CD25 expression levels, but Ag is required for active Treg proliferation in the inflamed skin. Indeed, BrdU+ cells were enriched for higher levels of CD25 (Fig. 10F), suggesting the combination of distinct inflammatory cues and cognate Ag synergize to promote local Treg accumulation in the inflamed tissue.

Discussion The data presented in this study highlight an important role for inflammatory signals in the promotion of extensive Treg accumulation locally in the skin. The inflamed microenvironment of the skin can support the upregulation of CD25 in the absence of cognate Ag and appears to facilitate Ag-dependent local Treg FIGURE 8. Inflammation drives CD25 expression on skin Tregs. (A) proliferation. Distinct inflammatory environments differentially + CD25 expression on adoptively transferred DO11.10 Tregs in the skin day affect the magnitude of the Treg response in this manner. CFA 7 postimmunization with KLH and CFA, Alum, or Sm Egg. Gray filled and Alum adjuvants enhance Treg CD25 expression and robustly histogram, fluorescence-minus-one; solid line, CD25 expression; numbers, support prolonged Treg accumulation in the inflamed skin. In percentage of cells in CD25hi gate. (B) CD25 mean fluorescence intensity (MFI) on all DO11.10+ Tregs in skin and LN on d7. (C) ST2 MFI on contrast, Sm Egg–inflamed skin neither induces CD25 expression DO11.10+ Tregs in the skin and LN on day 7. Data from two independent on Tregs nor promotes local Treg proliferation and accumulation. experiments, three to five mice per group. Statistics by paired t test be- Moreover, Tregs appear to be distinctly regulated by the differ- tween LN and skin; statistics between groups by Mann–Whitney test. *p , ent adjuvant-induced inflammatory milieus because the extent of 0.05, **p , 0.005, ***p , 0.0005. Tconv accumulation was not impacted by the different adjuvants. The Journal of Immunology 2215 Downloaded from

FIGURE 9. Cognate Ag increases Treg accumulation and upregulation of CD25. CD25 mean ﬂuorescence intensity (MFI) (A) and ST2 MFI (B) of all transferred DO11.10+ Tregs in the skin of mice immunized with cognate-Ag (OVA) or noncognate Ag (KLH). KLH data from Fig. 7. (C) CD25 expression on DO11.10+ Tregs in KLH-immunized mice with CFA or Sm egg adjuvants. Mice were pulsed with PBS (+PBS) or pOVA (+pOVA) i.v. 6 h prior to cell harvest. Percentage of Tregs in CD25hi gate. (D) DO11.10+ Tconv transferred alone, CD25 MFI of all Tconv in the skin day 7 postimmunization. (E) rIL-33

(2 mg/ml) was administered i.p. to CFA or Sm Egg–inﬂamed (KLH or OVA) mice on days 4, 5, and 6 postimmunization and tissues analyzed on day 7. http://www.jimmunol.org/ Expression levels for CD25 in the presence or absence of rIL-33 and noncognate Ag (KLH) or cognate Ag (OVA). Data representative of two to three independent experiments. Statistics by t test and Mann–Whitney test. *p , 0.05, **p , 0.005.

This raises the possibility that the magnitude of Treg accumulation did not see differential functional specialization in the distinct in inflamed tissues may be therapeutically regulated independent cytokine milieus in the skin. Instead, the pattern of gene expres- of effects on Tconv. sion appeared anatomically restricted, with an overall increase in How might Treg accumulation be enhanced by the inflammatory Treg effector molecules in the inflamed skin compared with LN. A milieu? Our studies explored a number of possible control points. lack of Treg functional skewing despite very different cytokine

The different patterns of Treg accumulation did not correlate with environments may be explained by our model being one of acute by guest on October 4, 2021 particular inflammatory or growth factor cytokines that have been inflammation and suggests that population-based Treg adaptation implicated in regulating Treg numbers nor did the predominant within tissues may require chronic exposure to distinct inflam- Tconv cytokines IFN-g and IL-4 play a role. We predicted that the matory signals. different inflammatory milieus may select the recruitment or re- Both CFA and Alum-induced inflammation supported the tention of distinct Treg subsets based on current models by which upregulation of CD25 by Tregs, independent of cognate Ag. the polarizing cytokines that drive Tconv differentiation also These adjuvants trigger TLR- and NLR-dependent innate immune modify Treg homing and functional potential (2–4). However, we activation (57) and promote distinct cytokine milieus (Fig. 4).

FIGURE 10. Inflammation-specific local Treg proliferation. (A) Representative plots of BrdU+ expression in DO11.10+ Tregs from the skin day 7 postimmunization. BrdU+ frequency (B) and number of transferred DO11.10+ Tregs (C) in skin, day 7. (D) BrdU+ frequency of transferred DO11.10+ Tregs in dLN day 7. (E) DO11.10 Tconv transferred alone and BrdU+ frequency of Tconv in skin on day 7. (A–E) Data representative of three to four independent experiments. Statistics by Mann–Whitney test. (F) CD25 mean fluorescence intensity (MFI) of BrdU+ and BrdU2DO11.10+ Tregs in the skin, day 7. Data representative of two to three independent experiments. Statistics by paired t test (BrdU+, BrdU2). *p , 0.05, **p , 0.005, ***p , 0.0005. 2216 Treg ACCUMULATION IN THE INFLAMED SKIN

Therefore, the signals that regulate Treg CD25 expression in a difference in activation thresholds between Treg and Tconv inflamed tissues appear to be induced broadly by disparate mi- could be explained by intrinsic differences in CTLA-4 expression: crobial and inflammatory signals. Nevertheless, the ability of the Tregs express elevated basal levels of CTLA-4, and Treg CTLA-4 inflammatory milieu to support CD25 was selective, with CFA and is often upregulated in nonlymphoid tissues (Fig. 5) (50). This Alum providing such signals but Sm Egg–induced inflammation model would enable efficient effector Tconv responses to proceed lacking. Unexpectedly, the regulation of Treg CD25 expression by at the initiation of infection in the absence of strong Ag-specific inflammation was independent of Ag (Fig. 8). Certain effector Treg restraint. Ag-dependent regulation in this manner may also functions of conventional CD4 and CD8 T cells have been shown explain the failure of Treg control as autoimmunity progresses. to be induced independent of Ag in response to inflammatory The more efficient the Tconv are at destroying cellular sources of cytokines, such as those of the IL-1 family (58–60). Therefore, Ag in the target tissue, the more compromised, in local accumu- similar signals may also regulate Tregs independent of Ag, al- lation and/or expansion, the Treg response in the tissue may be- though IL-1b expression in the inflamed skin did not correlate come. It is important to note that our studies have demonstrated an with its ability to support CD25 (low in both Alum and Sm Egg effect of the inflammatory milieu on the differential accumulation despite the Alum environment supporting CD25 upregulation, of Treg numbers in the skin, but there may also be a differential Fig. 4). More work will be needed to identify possible cytokines, effect of the inflammatory milieu on the intrinsic suppressive or combination of cytokines, that may regulate CD25 expression capacity of such Tregs in the skin. Although our RNA analysis by Tregs independent of Ag. Alternatively, differential availability would suggest a number of the key Treg functional molecules are of costimulatory signals may also promote CD25 expression in- expressed at similar levels in the different settings (CTLA-4, dependent of Ag, as discussed below. Interestingly, for CD8 cells, granzyme B, and IL-10; Fig. 5), it will be interesting to ascer- the inflammatory milieu was shown to enhance CD8 T cell sen- tain if, independent of differences in Treg number, there are ad- Downloaded from sitivity to Ag (61), a mechanism that may be consistent with our ditional differences in the quality of their suppressive properties observations that in the absence of the appropriate inflammatory influenced by the inflammatory milieu. “conditioning” cues (cytokine and/or costimulation), Ag is not There may be additional factors that balance the magnitude of sufficient to rescue Treg accumulation. Treg accumulation in the skin. Recent studies in the intestine The global availability of IL-2 in the tissue is unlikely to be a suggest the balance between IL-23 and IL-33 signals may be major driver of the Treg CD25 expression and accumulation in the critical in the control of local Treg numbers and function (37). IL-33 http://www.jimmunol.org/ skin, as the adjuvanted milieus all resulted in similar IL-2 tissue appears to promote Treg function and accumulation in inflamed levels, although we cannot rule out distinct microanatomical dif- tissues, possibly through promoting DC-induced IL-2 production ferences in available IL-2. Instead, the upregulation of costimu- (35), whereas IL-23 restrains Treg responsiveness to IL-33 (37). latory ligands such as CD80 and CD86 may be a good mechanism Interestingly, the severe hepatic granulomatous inflammation for regulating Treg CD25 expression and/or accumulation in the against schistosome eggs is critically dependent on egg-induced tissue, given the important role of CD28 signaling in Treg pe- IL-23 expression (66, 67). We also see a significant increase in the ripheral homeostasis (18, 21, 26, 27). We found that the relative IL-23/IL-33 ratio in the Sm Egg milieu compared with CFA or expression levels of CD80/CD86 on APCs in the inflamed skin Alum, consistent with the poor Treg accumulation in Sm Egg– by guest on October 4, 2021 correlated with the magnitude of Treg accumulation. Strikingly, inflamed skin. Indeed, we found an interesting synergy between Sm Egg–induced inflammation upregulated CD80/CD86 in the cognate Ag signals and IL-33, for the upregulation of CD25: dLN but failed to support CD80/CD86 expression levels in the rIL-33 boosted CD25 expression on Tregs in the Sm Egg– inflamed skin. This pattern correlates with Sm Egg–inflamed Treg inflamed dermis if cognate Ag was present (Fig. 9E). Thus, re- expansion in the dLN, as previously described (62), and the rel- duced IL-33 availability in the Sm Egg–inflamed skin may func- ative inability of the local Sm Egg microenvironment to support tion to modulate Treg CD25 levels through its role in promoting enhanced Treg numbers. Indeed, infection with the S. mansoni paracrine IL-2 production from DCs (35). Further investigation parasite itself has been shown to limit induction of CD28 ligands into this pathway, using genetic manipulation of key components, or upregulate negative regulators such as programmed cell death is warranted given the unchecked schistosome egg–induced im- ligand-1 on APC, and this may account for the failure of the Sm munopathology (68). Egg–induced inflammatory milieu to support Treg accumulation We show that adjuvant-induced inflammation strongly supports (63, 64). In an autoimmune setting, such a B7-dependent mech- Treg accumulation in the inflamed skin. Inflammation alone can anism may also underlie the marked downregulation of Treg CD25 upregulate Treg CD25 expression locally in the inflamed tissue, and in autoimmune tissues where chronic nonmicrobially induced this appears to prime cells for additional Ag-dependent signals that inflammation may fail to support a perceived threshold level of drive local Ag-specific Treg expansion. The differences in the CD28 ligands necessary for Treg CD25 upregulation and/or magnitude of Treg accumulation in the skin with distinct adjuvants survival. was striking and may have important implications for the control In our studies, cognate Ag played a major role in the further of microbially induced immune pathology as well as the efficacy upregulation of CD25 and in local Treg proliferation, but the effects of vaccination (57). Successful vaccine strategies are currently of Ag remained inflammatory context dependent. The simplest evaluated based on the magnitude of systemic immune memory. explanation for the poor Treg accumulation in the Sm Egg– However, given the growing importance of local resident tissue inflamed skin would have been that the Sm Egg–induced inflam- memory (69), the degree of local Treg accumulation induced by mation led to reduced Ag presentation and hence an inability to specific adjuvants may well modify the establishment of protective locally expand Ag-specific Tregs. However, Ag-specific Tconv immunity. robustly accumulated and proliferated locally in the Sm Egg– inflamed skin similar to both CFA and Alum, indicating that Ag Acknowledgments was not overtly differentially limited by distinct adjuvants. We We thank the members of the Fowell laboratory for helpful discussions on suggest that effector Tconv and Tregs may be differentially sen- the studies. The schistosome eggs were kindly provided by the laboratories sitive to cognate Ag at inflammatory sites or that they may be of Dr. Ed Pearce and Dr. David Artis and originated from Biomphalaria differentially dependent on costimulatory reinforcement (65). Such glabrata snails provided by the National Institute of Allergy and Infectious The Journal of Immunology 2217

Diseases (NIAID) Schistosomiasis Resource Center of the Biomedical 24. Wing, K., Y. Onishi, P. Prieto-Martin, T. Yamaguchi, M. Miyara, Z. Fehervari, Research Institute (Rockville, MD) through National Institutes of T. Nomura, and S. Sakaguchi. 2008. CTLA-4 control over Foxp3+ regulatory T cell function. Science 322: 271–275. Health/NIAID Contract HHSN272201000005I for distribution through 25. Gogishvili, T., F. Luhder,€ S. Goebbels, S. Beer-Hammer, K. Pfeffer, and BEI Resources. T. Hunig.€ 2013. Cell-intrinsic and -extrinsic control of Treg-cell homeostasis and function revealed by induced CD28 deletion. Eur. J. Immunol. 43: 188–193. 26. Zhang, R., A. Huynh, G. Whitcher, J. Chang, J. S. Maltzman, and L. A. Turka. 2013. Disclosures An obligate cell-intrinsic function for CD28 in Tregs. J. Clin. Invest. 123: 580–593. The authors have no financial conflicts of interest. 27. Tang, Q., K. J. Henriksen, E. K. Boden, A. J. Tooley, J. Ye, S. K. Subudhi, X. X. Zheng, T. B. Strom, and J. A. Bluestone. 2003. Cutting edge: CD28 controls peripheral homeostasis of CD4+CD25+ regulatory T cells. J. Immunol. 171: 3348–3352. References 28. Sakaguchi, S., N. Sakaguchi, M. Asano, M. Itoh, and M. Toda. 1995. Immu- 1. Josefowicz, S. Z., L. F. Lu, and A. Y. Rudensky. 2012. Regulatory T cells: nologic self-tolerance maintained by activated T cells expressing IL-2 receptor mechanisms of differentiation and function. Annu. Rev. Immunol. 30: 531–564. alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes 2. Liston, A., and D. H. Gray. 2014. Homeostatic control of regulatory T cell di- various autoimmune diseases. J. Immunol. 155: 1151–1164. versity. Nat. Rev. Immunol. 14: 154–165. 29. Fontenot, J. D., J. P. Rasmussen, M. A. Gavin, and A. Y. Rudensky. 2005. A 3. Feuerer, M., J. A. Hill, D. Mathis, and C. Benoist. 2009. Foxp3+ regulatory T cells: function for interleukin 2 in Foxp3-expressing regulatory T cells. Nat. Immunol. differentiation, specification, subphenotypes. Nat. Immunol. 10: 689–695. 6: 1142–1151. 4. Campbell, D. J., and M. A. Koch. 2011. Phenotypical and functional speciali- 30. D’Cruz, L. M., and L. Klein. 2005. Development and function of agonist- zation of FOXP3+ regulatory T cells. Nat. Rev. Immunol. 11: 119–130. induced CD25+Foxp3+ regulatory T cells in the absence of interleukin 2 sig- 5. Cipolletta, D., M. Feuerer, A. Li, N. Kamei, J. Lee, S. E. Shoelson, C. Benoist, naling. Nat. Immunol. 6: 1152–1159. and D. Mathis. 2012. PPAR-g is a major driver of the accumulation and phe- 31. Cheng, G., A. Yu, M. J. Dee, and T. R. Malek. 2013. IL-2R signaling is essential notype of adipose tissue Treg cells. Nature 486: 549–553. for functional maturation of regulatory T cells during thymic development. 6. Burzyn, D., W. Kuswanto, D. Kolodin, J. L. Shadrach, M. Cerletti, Y. Jang, J. Immunol. 190: 1567–1575. E. Sefik, T. G. Tan, A. J. Wagers, C. Benoist, and D. Mathis. 2013. A special 32. Wrenshall, L. E., and J. L. Platt. 1999. Regulation of T cell homeostasis by Downloaded from population of regulatory T cells potentiates muscle repair. Cell 155: 1282–1295. heparan sulfate-bound IL-2. J. Immunol. 163: 3793–3800. 7. Koch, M. A., G. Tucker-Heard, N. R. Perdue, J. R. Killebrew, K. B. Urdahl, and 33. Blattman, J. N., J. M. Grayson, E. J. Wherry, S. M. Kaech, K. A. Smith, and D. J. Campbell. 2009. The transcription factor T-bet controls regulatory T cell R. Ahmed. 2003. Therapeutic use of IL-2 to enhance antiviral T-cell responses homeostasis and function during type 1 inflammation. Nat. Immunol. 10: 595–602. in vivo. Nat. Med. 9: 540–547. 8. Chaudhry, A., D. Rudra, P. Treuting, R. M. Samstein, Y. Liang, A. Kas, and 34. Sereti, I., H. Martinez-Wilson, J. A. Metcalf, M. W. Baseler, C. W. Hallahan, A. Y. Rudensky. 2009. CD4+ regulatory T cells control TH17 responses in a B. Hahn, R. L. Hengel, R. T. Davey, J. A. Kovacs, and H. C. Lane. 2002. Long-term Stat3-dependent manner. Science 326: 986–991. effects of intermittent interleukin 2 therapy in patients with HIV infection: charac- 9. Zheng, Y., A. Chaudhry, A. Kas, P. deRoos, J. M. Kim, T. T. Chu, L. Corcoran, terization of a novel subset of CD4(+)/CD25(+) T cells. Blood 100: 2159–2167. P. Treuting, U. Klein, and A. Y. Rudensky. 2009. Regulatory T-cell suppressor 35. Matta, B. M., J. M. Lott, L. R. Mathews, Q. Liu, B. R. Rosborough, B. R. Blazar, http://www.jimmunol.org/ program co-opts transcription factor IRF4 to control T(H)2 responses. Nature and H. R. Turnquist. 2014. IL-33 is an unconventional Alarmin that stimulates 458: 351–356. IL-2 secretion by dendritic cells to selectively expand IL-33R/ST2+ regulatory 10. Linterman, M. A., W. Pierson, S. K. Lee, A. Kallies, S. Kawamoto, T. F. Rayner, T cells. J. Immunol. 193: 4010–4020. M. Srivastava, D. P. Divekar, L. Beaton, J. J. Hogan, et al. 2011. Foxp3+ fol- 36. Morita, H., K. Arae, H. Unno, K. Miyauchi, S. Toyama, A. Nambu, K. Oboki, licular regulatory T cells control the germinal center response. Nat. Med. 17: T. Ohno, K. Motomura, A. Matsuda, et al. 2015. An interleukin-33-Mast cell- 975–982. interleukin-2 Axis suppresses papain-induced allergic inflammation by pro- 11. Chung, Y., S. Tanaka, F. Chu, R. I. Nurieva, G. J. Martinez, S. Rawal, moting regulatory T cell numbers. Immunity 43: 175–186. Y. H. Wang, H. Lim, J. M. Reynolds, X. H. Zhou, et al. 2011. Follicular regu- 37. Schiering, C., T. Krausgruber, A. Chomka, A. Fro¨hlich, K. Adelmann, E. A. Wohlfert, latory T cells expressing Foxp3 and Bcl-6 suppress germinal center reactions. J. Pott, T. Griseri, J. Bollrath, A. N. Hegazy, et al. 2014. The alarmin IL-33 promotes Nat. Med. 17: 983–988. regulatory T-cell function in the intestine. Nature 513: 564–568. 12. Suffia, I. J., S. K. Reckling, C. A. Piccirillo, R. S. Goldszmid, and Y. Belkaid. 38. Fowell, D. J., K. Shinkai, X. C. Liao, A. M. Beebe, R. L. Coffman, D. R. Littman, 2006. Infected site-restricted Foxp3+ natural regulatory T cells are specific for and R. M. Locksley. 1999. Impaired NFATc translocation and failure of Th2 by guest on October 4, 2021 microbial antigens. J. Exp. Med. 203: 777–788. development in Itk-deficient CD4+ T cells. Immunity 11: 399–409. 13. Shafiani, S., G. Tucker-Heard, A. Kariyone, K. Takatsu, and K. B. Urdahl. 2010. 39. Katzman, S. D., and D. J. Fowell. 2008. Pathogen-imposed skewing of mouse Pathogen-specific regulatory T cells delay the arrival of effector T cells in the chemokine and cytokine expression at the infected tissue site. J. Clin. Invest. lung during early tuberculosis. J. Exp. Med. 207: 1409–1420. 118: 801–811. 14. Punkosdy, G. A., M. Blain, D. D. Glass, M. M. Lozano, L. O’Mara, J. P. Dudley, 40. Chen, Y., C. L. Langrish, B. McKenzie, B. Joyce-Shaikh, J. S. Stumhofer, R. Ahmed, and E. M. Shevach. 2011. Regulatory T-cell expansion during chronic T. McClanahan, W. Blumenschein, T. Churakovsa, J. Low, L. Presta, et al. 2006. viral infection is dependent on endogenous retroviral superantigens. Proc. Natl. Anti-IL-23 therapy inhibits multiple inflammatory pathways and ameliorates Acad. Sci. USA 108: 3677–3682. autoimmune encephalomyelitis. J. Clin. Invest. 116: 1317–1326. 15. Taylor, M. D., N. van der Werf, A. Harris, A. L. Graham, O. Bain, J. E. Allen, 41. Kolodin, D., N. van Panhuys, C. Li, A. M. Magnuson, D. Cipolletta, C. M. Miller, and R. M. Maizels. 2009. Early recruitment of natural CD4+ Foxp3+ Treg cells A. Wagers, R. N. Germain, C. Benoist, and D. Mathis. 2015. Antigen- and by infective larvae determines the outcome of filarial infection. Eur. J. Immunol. cytokine-driven accumulation of regulatory T cells in visceral adipose tissue of 39: 192–206. lean mice. Cell Metab. 21: 543–557. 16. O’Connor, R. A., K. H. Malpass, and S. M. Anderton. 2007. The inflamed central 42. Billiau, A., and P. Matthys. 2001. Modes of action of Freund’s adjuvants in nervous system drives the activation and rapid proliferation of Foxp3+ regulatory experimental models of autoimmune diseases. J. Leukoc. Biol. 70: 849–860. T cells. J. Immunol. 179: 958–966. 43. Li, H., S. B. Willingham, J. P. Ting, and F. Re. 2008. Cutting edge: inflamma- 17. Herman, A. E., G. J. Freeman, D. Mathis, and C. Benoist. 2004. CD4+CD25+ T some activation by alum and alum’s adjuvant effect are mediated by NLRP3. regulatory cells dependent on ICOS promote regulation of effector cells in the J. Immunol. 181: 17–21. prediabetic lesion. J. Exp. Med. 199: 1479–1489. 44. Marrack, P., A. S. McKee, and M. W. Munks. 2009. Towards an understanding of 18. Attridge, K., and L. S. Walker. 2014. Homeostasis and function of regulatory the adjuvant action of aluminium. Nat. Rev. Immunol. 9: 287–293. T cells (Tregs) in vivo: lessons from TCR-transgenic Tregs. Immunol. Rev. 259: 45. Everts, B., G. Perona-Wright, H. H. Smits, C. H. Hokke, A. J. van der Ham, 23–39. C. M. Fitzsimmons, M. J. Doenhoff, J. van der Bosch, K. Mohrs, H. Haas, et al. 19. Tang, Q., J. Y. Adams, C. Penaranda, K. Melli, E. Piaggio, E. Sgouroudis, 2009. Omega-1, a glycoprotein secreted by Schistosoma mansoni eggs, drives C. A. Piccirillo, B. L. Salomon, and J. A. Bluestone. 2008. Central role of de- Th2 responses. J. Exp. Med. 206: 1673–1680. fective interleukin-2 production in the triggering of islet autoimmune destruc- 46. Kaplan, M. H., J. R. Whitfield, D. L. Boros, and M. J. Grusby. 1998. Th2 cells tion. Immunity 28: 687–697. are required for the Schistosoma mansoni egg-induced granulomatous response. 20. Betts, R. J., N. Prabhu, A. W. Ho, F. C. Lew, P. E. Hutchinson, O. Rotzschke, J. Immunol. 160: 1850–1856. P. A. Macary, and D. M. Kemeny. 2012. Influenza A virus infection results in a 47. Campbell, D. J. 2015. Control of regulatory T Cell migration, function, and robust, antigen-responsive, and widely disseminated Foxp3+ regulatory T cell homeostasis. J. Immunol. 195: 2507–2513. response. J. Virol. 86: 2817–2825. 48. Mohrs, M., K. Shinkai, K. Mohrs, and R. M. Locksley. 2001. Analysis of type 2 21. Salomon, B., D. J. Lenschow, L. Rhee, N. Ashourian, B. Singh, A. Sharpe, and immunity in vivo with a bicistronic IL-4 reporter. Immunity 15: 303–311. J. A. Bluestone. 2000. B7/CD28 costimulation is essential for the homeostasis of 49. La Cava, A. 2008. Tregs are regulated by cytokines: implications for autoim- the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes. munity. Autoimmun. Rev. 8: 83–87. Immunity 12: 431–440. 50. Burzyn, D., C. Benoist, and D. Mathis. 2013. Regulatory T cells in nonlymphoid 22. Sansom, D. M., and L. S. Walker. 2006. The role of CD28 and cytotoxic T- tissues. Nat. Immunol. 14: 1007–1013. lymphocyte antigen-4 (CTLA-4) in regulatory T-cell biology. Immunol. Rev. 51. Feuerer, M., J. A. Hill, K. Kretschmer, H. von Boehmer, D. Mathis, and 212: 131–148. C. Benoist. 2010. Genomic definition of multiple ex vivo regulatory T cell 23. Schmidt, E. M., C. J. Wang, G. A. Ryan, L. E. Clough, O. S. Qureshi, subphenotypes. Proc. Natl. Acad. Sci. USA 107: 5919–5924. M. Goodall, A. K. Abbas, A. H. Sharpe, D. M. Sansom, and L. S. Walker. 2009. 52. Lazarski, C. A., J. Ford, S. D. Katzman, A. F. Rosenberg, and D. J. Fowell. 2013. Ctla-4 controls regulatory T cell peripheral homeostasis and is required for IL-4 attenuates Th1-associated chemokine expression and Th1 trafficking to suppression of pancreatic islet autoimmunity. J. Immunol. 182: 274–282. inflamed tissues and limits pathogen clearance. PLoS One 8: e71949. 2218 Treg ACCUMULATION IN THE INFLAMED SKIN

53. Debes, G. F., C. N. Arnold, A. J. Young, S. Krautwald, M. Lipp, J. B. Hay, and 62. Taylor, J. J., M. Mohrs, and E. J. Pearce. 2006. Regulatory T cell responses E. C. Butcher. 2005. Chemokine receptor CCR7 required for T lymphocyte exit develop in parallel to Th responses and control the magnitude and phenotype of from peripheral tissues. Nat. Immunol. 6: 889–894. the Th effector population. J. Immunol. 176: 5839–5847. 54. Maloy, K. J., and F. Powrie. 2005. Fueling regulation: IL-2 keeps CD4+ Treg 63. Kane, C. M., L. Cervi, J. Sun, A. S. McKee, K. S. Masek, S. Shapira, cells fit. Nat. Immunol. 6: 1071–1072. C. A. Hunter, and E. J. Pearce. 2004. Helminth antigens modulate TLR-initiated 55. Wuest, T. Y., J. Willette-Brown, S. K. Durum, and A. A. Hurwitz. 2008. The dendritic cell activation. J. Immunol. 173: 7454–7461. influence of IL-2 family cytokines on activation and function of naturally oc- 64. Smith, P., C. M. Walsh, N. E. Mangan, R. E. Fallon, J. R. Sayers, curring regulatory T cells. J. Leukoc. Biol. 84: 973–980. A. N. McKenzie, and P. G. Fallon. 2004. Schistosoma mansoni worms induce 56. Gratz, I. K., H. A. Truong, S. H. Yang, M. M. Maurano, K. Lee, A. K. Abbas, anergy of T cells via selective up-regulation of programmed death ligand 1 on and M. D. Rosenblum. 2013. Cutting Edge: memory regulatory t cells require macrophages. J. Immunol. 173: 1240–1248. IL-7 and not IL-2 for their maintenance in peripheral tissues. J. Immunol. 190: 65. Yan, D., J. Farache, M. Mingueneau, D. Mathis, and C. Benoist. 2015. Imbal- 4483–4487. anced signal transduction in regulatory T cells expressing the transcription factor 57. Maisonneuve, C., S. Bertholet, D. J. Philpott, and E. De Gregorio. 2014. FoxP3. [Published erratum appears in 2016 Proc. Natl. Acad. Sci. USA 113: Unleashing the potential of NOD- and Toll-like agonists as vaccine adjuvants. E256.] Proc. Natl. Acad. Sci. USA 112: 14942–14947. Proc. Natl. Acad. Sci. USA 111: 12294–12299. 66. Rutitzky, L. I., L. Bazzone, M. G. Shainheit, B. Joyce-Shaikh, D. J. Cua, and 58. Raue´, H. P., C. Beadling, J. Haun, and M. K. Slifka. 2013. Cytokine-mediated M. J. Stadecker. 2008. IL-23 is required for the development of severe egg- programmed proliferation of virus-specific CD8(+) memory T cells. Immunity induced immunopathology in schistosomiasis and for lesional expression of 38: 131–139. IL-17. J. Immunol. 180: 2486–2495. 59. Guo, L., G. Wei, J. Zhu, W. Liao, W. J. Leonard, K. Zhao, and W. Paul. 2009. 67. Shainheit, M. G., P. M. Smith, L. E. Bazzone, A. C. Wang, L. I. Rutitzky, and IL-1 family members and STAT activators induce cytokine production by Th2, M. J. Stadecker. 2008. Dendritic cell IL-23 and IL-1 production in response to Th17, and Th1 cells. Proc. Natl. Acad. Sci. USA 106: 13463–13468. schistosome eggs induces Th17 cells in a mouse strain prone to severe immu- 60. Robinson, D., K. Shibuya, A. Mui, F. Zonin, E. Murphy, T. Sana, S. B. Hartley, nopathology. J. Immunol. 181: 8559–8567. S. Menon, R. Kastelein, F. Bazan, and A. O’Garra. 1997. IGIF does not drive 68. Wilson, M. S., M. M. Mentink-Kane, J. T. Pesce, T. R. Ramalingam, R. Thompson, Th1 development but synergizes with IL-12 for interferon-gamma production and T. A. Wynn. 2007. Immunopathology of schistosomiasis. Immunol. Cell Biol. and activates IRAK and NFkappaB. Immunity 7: 571–581. 85: 148–154. 61. Richer, M. J., J. C. Nolz, and J. T. Harty. 2013. Pathogen-specific inflammatory 69. Park, C. O., and T. S. Kupper. 2015. The emerging role of resident mem- milieux tune the antigen sensitivity of CD8(+) T cells by enhancing T cell re- ory T cells in protective immunity and inflammatory disease. Nat. Med. 21: Downloaded from ceptor signaling. Immunity 38: 140–152. 688–697. http://www.jimmunol.org/ by guest on October 4, 2021 Supplementary Figure 1A Inflammation-Specific TLDA: CD4 Inflammation in Tissue!

Gene Symbol Assay ID Gene Symbol Assay ID List of 96 genes: 93 genes of interest and 3 Ahr Mm01291777_m1 IL2 Mm00434255_g1 endogenous controls Anxa4 Mm01183650_m1 IL21 Mm00517640_m1 (GAPDH, HPRT and Bcl-6 Mm00477633_m1 IL21r Mm00600319_m1 Rn18s) are included in Ccr1 Mm01216147_m1 IL22 Mm01226722_g1 a custom designed Ccr2 Mm00438270_m1 IL23p19 Mm01160011_g1 microfulidics card, Ccr4 Mm00438271_m1 IL23r Mm00519942_m1 TLDA (Taqman Low Ccr5 Mm04207879_m1 IL27p28 Mm00461164_m1 Density Array). Gene Ccr6 Mm01700300_g1 IL27r Mm00497254_g1 symbol (right) and Ccr7 Mm01301785_m1 IL2ra Mm00434261_m1 Taqman assay ID for Ccr8 Mm01351703_m1 IL2rb Mm00434264_m1 each gene is listed. ! Ccr9 Mm02620030_s1 IL4 Mm00445259_m1 CD40L Mm00441911_m1 IL4ra Mm00439634_m1 CTLA4 Mm00486849_m1 IL5 Mm00439646_m1 Cxcr3 Mm00438259_m1 IL6 Mm01210733_m1 Cxcr4 Mm01292123_m1 IL6ra Mm00439653_m1 Cxcr5 Mm00432086_m1 IL7r Mm00434295_m1 Cxcr6 Mm00472858_m1 IL9 Mm00434304_m1 Cxcr7 Mm00432610_m1 Irf4 Mm00516431_m1 Ebi-3 Mm00469294_m1 Jun Mm00495062_s1 Eomes Mm01351985_m1 Klf9 Mm00495172_m1 Fos Mm00487425_m1 Lag3 Mm00493071_m1 FoxP3 Mm00475166_g1 Lgal Mm00839408_g1 Furin Mm00440646_m1 Maf Mm02581355_s1 Gapdh Mm99999915_g1 Pdcd1 Mm01285676_m1 Gata3 Mm00484683_m1 Plxna4 Mm00558881_m1 Gﬁ1 Mm00515855_m1 Pparg Mm01184322_m1 Gzmb Mm00442834_m1 Prf1 Mm00812512_m1 havcr2 Mm00454540_m1 Rn18s Mm03928990_g1 HPRT Mm01318747_g1 RORc Mm01261022_m1 ICOS Mm00497600_m1 Sema3A Mm00436469_m1 Ifnar1 Mm00439544_m1 Sema4A Mm00443140_m1 Ifng Mm01168134_m1 Sema4D Mm00443147_m1 Ifngr1 Mm00599890_m1 Sema4F Mm01181883_m1 Igf1r Mm00802831_m1 Sema6D Mm01334437_m1 Igf2r Mm00439576_m1 Sema7A Mm00441361_m1 Ikzf1 Mm01187882_m1 Skp2 Mm00449925_m1 Ikzf2 Mm00496108_m1 Socs2 Mm01247834_m1 Ikzf3 Mm01306721_m1 Tbx21 Mm00450960_m1 Ikzf4 Mm00558871_s1 Tg1 Mm00441729_g1 IL10 Mm01288386_m1 Tgr2 Mm03024091_m1 IL10ra Mm00434151_m1 Tgr3 Mm03053837_s1 IL12p35 Mm00434169_m1 Timd2 Mm00506693_m1 IL12p40 Mm01288994_g1 Tnfr Mm01309635_m1 IL12rb2 Mm00434200_m1 Tnfrsf18 Mm00437136_m1 IL13 Mm00434204_m1 Tnfrsf1b Mm00441889_m1 IL17 Mm00439619_m1 Tnfrsf4 Mm00442039_m1 IL18r Mm00515180_m1 Tnfrsf9 Mm00441899_m1 IL1rl1 Mm01233982_m1 Tnfsf11 Mm00441908_m1 Supplementary Figure 1A Treg-Speciﬁc TLDA: Treg Signature Genes!

Gene Symbol Assay ID Gene Symbol Assay ID List of 96 genes: 93 genes of interest and 3 Adam19 Mm00477337_m1 Melk Mm00487680_m1 endogenous controls, Ahr Mm01291777_m1 Mxd1 Mm00487504_m1 GAPDH, HPRT and Arhgap20 Mm00615839_m1 Ndrg1 Mm00440447_m1 Rn18s) included in a Arhgef12 Mm00804321_m1 Nrp1 Mm00435379_m1 custom designed Bhlhb2 Mm00478593_m1 Nt5e Mm00501910_m1 microfulidics card, Capn3 Mm00482978_m1 Pde3b Mm01271639_m1 TLDA (Taqman Low Cd200 Mm00487740_m1 Pdlim4 Mm00450823_m1 Density Array). Gene CD36 Mm01135198_m1 Penk1 Mm01212875_m1 symbol (right) and Cerk Mm00461054_m1 Phlda1 Mm00456345_g1 Taqman assay ID for Cish Mm00515488_m1 Phlpp Mm01295850_m1 each gene is listed. ! Cpe Mm00516341_m1 Plagl1 Mm00494251_m1 CTLA4 Mm00486849_m1 Plscr1 Mm01228223_g1 Dgat1 Mm00515643_m1 Pmaip Mm00451763_m1 Dnahc12 Mm01210451_m1 Pou2af1 Mm00448326_m1 Dst Mm00467166_m1 Ppm1l Mm00618786_m1 Dusp4 Mm00723761_m1 Prg4 Mm01284582_m1 Ecm1 Mm00514634_m1 Prnp Mm00448389_m1 Eef2k Mm00432996_m1 Ptger2 Mm00436051_m1 Entpd1 Mm00515447_m1 Ptpn13 Mm00478426_m1 Folr4 Mm00491664_m1 Rapgef4 Mm00517216_m1 FoxP3 Mm00475166_g1 Rcn1 Mm00485644_m1 Frmd4b Mm00520190_m1 Rgs1 Mm00450170_m1 Gapdh Mm99999915_g1 Rgs16 Mm00803317_m1 Gata1 Mm01352636_m1 Rhoq Mm00467435_m1 Gbp1 Mm00657086_m1 Rn18s Mm03928990_g1 Gbp3 Mm00497606_m1 Rragd Mm00546741_m1 Gﬁ1 Mm00515855_m1 Serpina3g Mm02602542_m1 Gnaq Mm00492381_m1 Serpinb6b Mm00488413_m1 Gpr15 Mm01346276_g1 Sgk1 Mm00441387_g1 Gpr177 Mm00509695_m1 Sh3brl2 Mm00552919_m1 Gpr83 Mm00439103_m1 Slc22a2 Mm00457295_m1 Hipk2 Mm00439329_m1 Slc22a5 Mm00441468_m1 Hmgn3 Mm00518151_m1 Slc2a3 Mm03053806_s1 HPRT Mm01318747_g1 Socs2 Mm01247834_m1 Igf1r Mm00802831_m1 Spry1 Mm01285700_m1 Igf2r Mm00439576_m1 Stat3 Mm01219775_m1 Ikzf2 Mm00496108_m1 Stx11 Mm01192495_m1 Ikzf4 Mm00558871_s1 Swap70 Mm00486503_m1 Il1rl1 Mm01233982_m1 Tiam1 Mm01170430_m1 IL2ra Mm00434261_m1 Tmem158 Mm02342239_s1 IL2rb Mm00434264_m1 Tmem64 Mm00841719_m1 Itgae Mm00434443_m1 Tnfrsf1b Mm00441889_m1 Itgb8 Mm00623991_m1 Tnfrsf4 Mm00442039_m1 Ih5 Mm00552678_m1 Tnfrsf9 Mm00441899_m1 Klf9 Mm00495172_m1 Tnfsf11 Mm00441908_m1 Klrg1 Mm00516879_m1 Traf1 Mm00493827_m1 Map3k8 Mm00432637_m1 Usp27x Mm02526762_s1 Mdﬁc Mm00612985_m1 Zc3h12c Mm01177353_m1 * * A 1500 n.s. 10 n.s. ** ** n.s. 8 n.s. 1000 6

4 500 Frequency Cell number 2

0 0 Ctrl an Ctrl an Ctrl an Ctrl an IL-23 IL-23 IL-23 IL-23 CFA Sm Egg CFA Sm Egg

B 1500 5

4 1000 3

2 500 * Cell number Frequency 1

0 0 PBS rIL-33 PBS rIL-33 PBS rIL-33 PBS rIL-33 CFA Sm Egg CFA Sm Egg

Supplementary Figure 2. IL-23 and IL-33 manipulations fail to boost Treg accumulation in the Sm Egg-induced inflamed dermis. Sorted DO11.10+ Foxp3+ Tregs (Thy1.1+) and/or DO11.10+ Foxp3- Tconv (Thy1.1/1.2+) cells were transferred to WT Balb/c (Thy 1.2+) mice and immunized with OVA and adjuvant. A) Number and frequency (of CD4) of DO11.10 Foxp3+ Tregs in the inflamed ear, d7 post-immunization, with and without blocking antibody to IL-23 i.p. on days 4, 5, 6. B) Number and frequency (of CD4) of DO11.10 Foxp3+ Tregs in the inflamed ear, d7 postimmunization, with and without rIL-33 i.p. on days 4, 5, 6. Two independent experiments. Statistics by Mann Whitney.