Katherine G. MacDonald, BSc, Nicholas A. J

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Regulatory T cells produce profibrotic cytokines in the skin of patients with systemic sclerosis Katherine G. MacDonald, BSc, Nicholas A.J. Dawson, BSc, Qing Huang, PhD, James V. Dunne, MBBch, Megan K. Levings, PhD, Raewyn Broady, MBChB Journal of Allergy and Clinical Immunology Volume 135, Issue 4, Pages 946-955.e9 (April 2015) DOI: 10.1016/j.jaci.2014.12.1932 Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Cytokine production profile of Treg cells in blood of patients with SSc. PBMCs from patients with SSc or control subjects were stimulated with PMA and ionomycin, and expression levels of CD3, CD8, CD4, IFN-γ, IL-4, IL-13, IL-17, FVD, and FOXP3 were measured by flow cytometry. Shown are representative (left) and cumulative (right) data comparing the proportion of cytokine-producing FOXP3+ Treg cells (A) or FOXP3− Tconv cells (B). Each symbol in the graph represents data from 1 subject. Numbers in dot plots represent the averages ± SEMs (n = 20 control subjects and n = 36 patients with SSc). Journal of Allergy and Clinical Immunology 2015 135, 946-955.e9DOI: (10.1016/j.jaci.2014.12.1932) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Treg cells in affected skin of patients with SSc produce TH2 cytokines. Four-millimeter punch biopsy specimens from affected skin of patients with limited or diffuse SSc or skin from healthy control subjects were cultured as explants. After 3 weeks, nonadherent cells were collected, stimulated with PMA and ionomycin, and stained for CD3, CD8, CD4, IFN-γ, IL-4, IL-13, IL-17, FOXP3, and FVD. Shown are representative (left) and cumulative (right) data comparing the proportion of cytokine-producing FOXP3+ Treg cells (A and B) or FOXP3− Tconv cells (C). Each symbol in the graph represents data from 1 subject. Numbers in dot plots represent averages ± SEMs (n = 13 control subjects and n = 19 patients with SSc). *P < .05 and ***P < .001. Journal of Allergy and Clinical Immunology 2015 135, 946-955.e9DOI: (10.1016/j.jaci.2014.12.1932) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Blood of patients with SSc has an increased proportion of skin-homing TH2-like Treg cells. PBMCs from patients with SSc and control subjects were stained for CD3, CD4, CD8, CXCR3, CCR4, CCR6, CCR10, CLA, and FOXP3 and analyzed by flow cytometry. A, Proportions of TH1 (CXCR3+), TH2 (CXCR3−CCR4+CCR6−), and TH17-like (CXCR3−CCR4+CCR6+CCR10−) cells within FOXP3+ cells were determined. B, Proportions of TH2-like (CXCR3−CCR4+CCR6−) cells within CLA+FOXP3+ or CLA+FOXP3− cells were determined. Each symbol in the graph represents data from 1 subject (n = 15 control subjects and 31 patients with SSc). C, Skin-homing (CLA+) and circulating (CLA−) Treg cells (CD25hi127lo) and Tconv cells (CD25lo127hi) were sorted from PBMCs and stimulated for 72 hours. IL-17, IFN-γ, and IL-4 production was measured in supernatants by cytometric bead array. Graphs show means ± SEMs (n = 2 for control subjects and 2 for patients with SSc). *P < .05, **P < .01, and ****P < .0001. Journal of Allergy and Clinical Immunology 2015 135, 946-955.e9DOI: (10.1016/j.jaci.2014.12.1932) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 IL-33 stimulates production of IL-13 from skin Treg cells. A, Fibroblasts from skin of patients with SSc (n = 3) or control subjects (n = 2) were stimulated with or without IL-1β or TNF-α for 24 hours, and quantitative PCR was used to measure IL33 mRNA levels relative to 18s levels. Shown is the fold change relative to the amount in control fibroblasts cultured in media. B, Skin biopsy explants from healthy control subjects were cultured in the presence or absence of rhIL-33. After 3 weeks, the nonadherent cells were collected, stimulated with PMA and ionomycin, and analyzed by means of flow cytometry for CD3, CD8, CD4, IFN-γ, IL-4, IL-13, and FOXP3. The top panel depicts representative plots, and the bottom panel shows the average ± SEM (n = 3). C, Immunofluorescence staining was performed on micrometer-thick sections of skin from control subjects or patients with SSc. Sections were costained for CD3 (green), IL-33 (red), and 4′,6′-diamidino-2-phenylindole (DAPI; blue). Top panels depict representative data at ×10 magnification, with the white insert indicating the location of the image taken at ×40 magnification (n = 3 for control subjects and n = 3 for patients with SSc). Scale bar = 100 μm. *P < .05 and ****P < .0001. Journal of Allergy and Clinical Immunology 2015 135, 946-955.e9DOI: (10.1016/j.jaci.2014.12.1932) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Skin-localized Treg cells express ST2. A, Expanded CD4+ T cells from skin biopsy specimens from control subjects (n = 1) and patients with SSc (n = 3) were rested overnight and then stained for FVD, CD4, FOXP3, and ST2. Representative plots show ST2 expression on FOXP3− and FOXP3+ cells from skin of patients with SSc. The graph shows averaged ± SEM data for patients with SSc (n = 3). B, CD4+ enriched T cells from PBMCs from control subjects (n = 6) and patients with SSc (n = 4) were stained as in Fig 5, A. Representative plots show expression of ST2 on FOXP3+ cells from control subjects and patients with SSc. The graph shows averages ± SEMs (n = 4-6). **P < .01. Journal of Allergy and Clinical Immunology 2015 135, 946-955.e9DOI: (10.1016/j.jaci.2014.12.1932) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Gating strategy for identification of cytokine-producing Treg and Tconv cells in PBMCs. A, Cryopreserved PBMCs were thawed and then left unstimulated or stimulated, as described in the Methods section. Cells were stained with the indicated antibodies and gated as live CD3+CD8−CD4+ cells before division into FOXP3+ or FOXP3− subsets. B, Gates for quantification of cytokine production were based on parallel staining of unstimulated (Unstim) and stimulated (Stim) samples. Journal of Allergy and Clinical Immunology 2015 135, 946-955.e9DOI: (10.1016/j.jaci.2014.12.1932) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Gating strategy for identification of Treg and Tconv cells in skin explants. A, Nonadherent cells from biopsy explants were left unstimulated or stimulated, as described in the Methods section. Cells were stained with the indicated antibodies and gated as live CD3+CD8−CD4+ cells before division into FOXP3+ or FOXP3− subsets. B, Gates for quantification of cytokine production were set based on parallel staining of unstimulated (Unstim) and stimulated (Stim) samples. Journal of Allergy and Clinical Immunology 2015 135, 946-955.e9DOI: (10.1016/j.jaci.2014.12.1932) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 IL-10 expression in skin. Cells isolated from matrix culture were stimulated as described in the Methods section and stained for IL-10 in addition to FOXP3, as described in Fig E2. Journal of Allergy and Clinical Immunology 2015 135, 946-955.e9DOI: (10.1016/j.jaci.2014.12.1932) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 Analysis of FOXP3+ cells by patient subtype. Data from patients with SSc were analyzed on the basis of disease duration (early vs late), treatment (receiving immunosuppression at biopsy or 6 months before [RX+]), or type of disease (limited vs diffuse) to determine whether patient subtype affects observations. Shown are the proportion of FOXP3+ cells producing cytokines (A; corresponding to Fig 2, A and B) or expressing blood-homing markers (B; corresponding to Fig 3, A and B). *P < .05. Journal of Allergy and Clinical Immunology 2015 135, 946-955.e9DOI: (10.1016/j.jaci.2014.12.1932) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 Human TH2 cells do not express sustained levels of FOXP3. A, Sorting strategy and phenotype for TH1 (CXCR3+CRTH2−CCR6−) and TH2 (CXCR3−CRTH2+CCR6−) cells sorted from human peripheral blood. Cells were cultured in vitro for 2 weeks and then stimulated for cytokine production and stained for FVD, CD4, IFN-γ, IL-4, IL-13, and IL-17. B, Sorted cells were stimulated through the T-cell receptor, and FOXP3 expression was monitored for 2 weeks. The graph depicts the average percentage of FOXP3+ cells in TH1 and TH2 cells (n = 3 independent experiments), with representative data shown on the right. Journal of Allergy and Clinical Immunology 2015 135, 946-955.e9DOI: (10.1016/j.jaci.2014.12.1932) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E6 Treg cell marker expression on FOXP3+ cells in skin. CD4+ T cells expanded from skin matrix culture were rested 24 hours and stained for the indicated markers. Graphs show the average mean fluorescence intensity (MFI) in gated FOXP3+ and FOXP3− cells (n = 3 control subjects and n = 4 patients with SSc for CD25 and CD127; n = 2 and n = 3 for cytotoxic T lymphocyte–associated antigen 4 [CTLA-4], respectively). Journal of Allergy and Clinical Immunology 2015 135, 946-955.e9DOI: (10.1016/j.jaci.2014.12.1932) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E7 Gating strategy for classification of TH1-, TH2-, and TH17-like Treg cells. A, Cryopreserved PBMCs were thawed and stained with the indicated antibodies. T cells were gated as live CD3+CD8−CD4+ cells before subdividing on the basis of expression of FOXP3 and the indicated homing receptors. B, Representative data comparing the cytokine profiles of TH2 cells sorted through CCR4 (CXCR3+CCR4+CCR6−) or CRTH2 (CXCR3−CRTH2+CCR6−) from human peripheral blood, cultured in vitro for 2 weeks, stimulated for cytokine production, and stained for FVD, CD4, IFN-γ, IL-4, IL-13, and IL-2. Journal of Allergy and Clinical Immunology 2015 135, 946-955.e9DOI: (10.1016/j.jaci.2014.12.1932) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions