1. Glucocorticoids promote intrinsic human TH17 differentiation
Juliana de Castro Kroner, MSc, Kristin Knoke, MSc, David M. Kofler, MD, Julia Steiger, Mario Fabri, MD 
Journal of Allergy and Clinical Immunology 
Volume 142, Issue 5, Pages e11 (November 2018)
DOI: /j.jaci
Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2. Fig 1
Glucocorticoids promote TH17 differentiation, a process that involves decreasing IL-2 levels. A-I, Bead-activated naive CD4 T cells were differentiated in the absence (media) or presence of 10−7 mol/L dexamethasone (dexa) for 12 days. Bead-untreated cells (---) were also used as controls. Fig 1, A, Expression of TFs was assessed by using qPCR. Fig 1, B and C, IL-17 secretion was assessed by means of ELISA or cytokine bead array after phorbol 12-myristate 13-acetate (PMA)/ionomycin treatment in absolute (Fig 1, B) or per-cell levels (Fig 1, C). Fig 1, D, IL-17+ cells were measured by using intracellular cytokine staining (ICS) after PMA/ionomycin treatment. Fig 1, E, IL-10 was assessed, as in Fig 1, B. Fig 1, F, IL-17+ and IL-10+ cells were comeasured by using ICS, as in Fig 1, D. A representative plot of 4 experiments performed in triplicate is shown. Fig 1, G, IFN-γ, TNF-α, IL-4, and IL-22 were assessed, as in Fig 1, B. Fig 1, H, Relative cytokine changes in ratios. Fig 1, I, IL-2 in third-day supernatants was assessed, as in Fig 1, B. J, Naive CD4 T cells were cultured, as in Fig 1, A, combined or not (---) with anti-IL-2 or anti-IL-2Rα plus IL-2Rβ antibodies. IL-17 and IFN-γ secretion was measured, as in Fig 1, B. K, Naive CD4 T cells were cultured, as in Fig 1, A. IL-2 (6250 pg/mL) was added or not to dexamethasone-treated samples at the beginning of the T-cell culture. IL-17, IFN-γ, and TNF-α secretion was measured, as in Fig 1, B, and data were displayed as cytokine ratios. Bar graphs display means ± SEMs of n assays, each performed in triplicate. Fig 1, A, n = 6-10; Fig 1, B, n = 9; Fig 1, C, n = 9; Fig 1, D, n = 8; Fig 1, E, n = 7; Fig 1, G, n = 6-8; Fig 1, H, n = 6-8; Fig 1, I, n = 9; Fig 1, J, n = 2; and Fig 1, K, n = 5. *P ≤ .05, **P ≤ .01, and ***P ≤ .001.
Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci )
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3. Fig 2
Effect of glucocorticoids on T cell–mediated cutaneous host defense. A, Supernatants from glucocorticoid-differentiated T cells enhance AMP expression in keratinocytes. Human keratinocytes were cultured in growth medium supplemented with day 12 supernatants from dexamethasone-differentiated or non–dexamethasone-differentiated CD4 T cells (1:5). Supernatants were generated, as described for ELISA/cytokine bead array assays in Fig 1. After 24 hours of keratinocyte culture, AMP (solid columns) and cytokine (open columns) expression was assessed by using qPCR. B-E, Glucocorticoids upregulate IL-17/IFN-γ ratios in skin-resident memory T cells and pathogen-specific T cells. Fig 2, B and C, Skin-resident T cells were activated with beads in the presence or absence of 10−8 mol/L dexamethasone (dexa). Fig 2, B, IL-17 and IFN-γ levels were measured by means of ELISA in day 3 supernatants. Fig 2, C, Cytokine data as ratios. Fig 2, D and E, PBMCs were stimulated with heat-killed Candida albicans (CA) or Staphylococcus aureus (SA) in the presence or absence of 10−8 mol/L dexamethasone. Fig 2, D, IL-17 and IFN-γ levels were measured by means of ELISA in day 5 supernatants. Fig 2, E, Cytokine data as ratios. F, Glucocorticoids suppress IL-17 production from psoriatic T cells. PBMCs from healthy donors and patients with psoriasis were stimulated with C albicans with or without 10−8 mol/L dexamethasone. IL-17 and IFN-γ levels were measured by means of ELISA in day 5 supernatants. Bar graphs in Fig 2, A, represent means ± SEMs of 4 to 5 assays with supernatants from diverse donors. Bar graphs in Fig 2, D-F, represent means ± SEMs of n assays. Fig 2, B, n = 1; Fig 2, C, n = 1; Fig 2, D, n = 5; Fig 2, E, n = 5; Fig 2, F, n = 9 patients with psoriasis and 8 healthy donors. *P ≤ .05, **P ≤ .01, and ***P ≤ .001.
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4. Fig E1
T-cell differentiation assay. A, Graphic representation of our T-cell differentiation protocol, as described in the Methods section. CBA, Cytokine bead array; dexa, Dexamethasone; ICS, intracellular cytokine staining; PMA, phorbol 12-myristate 13-acetate. B, FACS analysis of magnetically isolated naive CD4+ T cells used in Fig E1, A. Representative plots of one assay performed in triplicate are shown. Left plots, Forward scatter (FSC) and side scatter (SSC). Right plots, CD4/CD25 costaining and corresponding isotype controls.
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5. Fig E2
Kinetics of IL-17 and IL-10 production by glucocorticoid-differentiated CD4 T cells. Bead-activated naive CD4 T cells were differentiated in the absence (media) or presence of 10−7 mol/L dexamethasone (dexa). Bead-untreated cells (---) were also used as controls. Supernatants were harvested at different time points (specified in the graphic), and cytokine (IL-17 and IL-10) levels were measured by means of ELISA. Data represent means ± SEMs of 3 assays, each performed in triplicate.
Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci )
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6. Fig E3
Dose response of dexamethasone on TH17 induction. Bead-activated naive CD4 T cells were differentiated in the absence (media) or presence of different doses of dexamethasone (dexa) for 12 days. Bead-untreated cells (---) were also used as controls. The frequency of IL-17+ cells was measured by using intracellular cytokine staining after phorbol 12-myristate 13-acetate/ionomycin treatment. Bar graph shows the number of cells as means ± SEMs for one representative assay performed in triplicate. *P ≤ .05 and **P ≤ .01.
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7. Fig E4
Glucocorticoids affect T-cell numbers, proliferation, and viability. A, T cells cultured for the purpose of supernatant analyses (Fig 1) were counted by using Trypan blue exclusion. B, Naive CD4 T cells were carboxyfluorescein succinimidyl ester (CFSE) stained and cultured with activation beads in the absence or presence of 10−7 mol/L dexamethasone (dexa). On day 12, proliferation was assessed as the percentage of CFSE-negative cells. C, Non–CFSE-stained cells cultured as in Fig E4, B, were incubated with 7-aminoactinomycin D (7-AAD) on day 12 for viability analyses. Dead cells were assessed as the percentage of 7-AAD–positive cells, and dot plots show 2 representative assay. Bar graphs represent means ± SEMs of n assays. Fig E4, A, n = 9; Fig E4, B, n = 6; Fig E4, C, n = 3. *P ≤ .05.
Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci )
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8. Fig E5
Vitamins D and A do not regulate TH17 differentiation. Bead-activated naive CD4 T cells were differentiated in the absence (media) or presence of 10−7 mol/L dexamethasone (dexa) for 12 days. Alternatively, cells were stimulated with 10−7 mol/L 25-hydroxyvitamin D (25D), 10−8 mol/L 1,25-dihydroxyvitamin D (1,25D), 10−7 mol/L isotretinoin, or 10−7 mol/L bexarotene separately (A) or combined (B) with dexamethasone. Bead-untreated cells (---) were also used as controls. The frequency of IL-17+ cells was measured by using intracellular cytokine staining after phorbol 12-myristate 13-acetate/ionomycin treatment. Bar graphs display means ± SEMs of n assays, each performed in triplicate. In Fig E5, A, the mean refers to the absolute number of cells (n = 8). In Fig E5, B, the mean refers to the relative number of cells normalized to the media condition (n = 4). *P ≤ .05.
Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci )
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9. Fig E6
Glucocorticoids increase relative levels of T cell–secreted IL-17 and IL-10 but do not affect IL-22 relative levels. Bead-activated naive CD4 T cells were differentiated in the absence (media) or presence of 10−7 mol/L dexamethasone (dexa). Bead-untreated cells (---) were also used as controls. On day 12, cytokine secretion was assessed by means of ELISA after phorbol 12-myristate 13-acetate/ionomycin treatment. Bar graphs display ratios representing relative IL-17 amounts in comparison with IL-4 (A), as well as relative amounts of IL-10 (B) and IL-22 (C) in comparison with IFN-γ, TNF-α, and IL-4. Means ± SEMs refer to 6 to 8 assays. **P ≤ .01 and ***P ≤ .001.
Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci )
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10. Fig E7
Glucocorticoids do not affect T-cell secretion of TH17-polarizing cytokines (IL-1β, IL-6, and TGF-β) but interfere with IL-2. A, Bead-activated naive CD4 T cells were differentiated in the absence (media) or presence of 10−7 mol/L dexamethasone (dexa) for 3 to 12 days, and supernatants were harvested after phorbol 12-myristate 13-acetate/ionomycin treatment. IL-1β, IL-6, and TGF-β were analyzed by using a cytokine bead array. B, Twelve-day supernatants were analyzed for their IL-2 content, as described in Fig E7, A. Bar graphs display means ± SEMs of 4 to 10 assays, each performed in triplicate. *P ≤ .05.
Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci )
Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11. Fig E8
IL-2 regulates TH17 induction by glucocorticoids. Bead-activated naive CD4 T cells were differentiated in the absence (media) or presence of dexamethasone (dexa) for 12 days. IL-2 was added at different concentrations or not to dexamethasone-treated samples at the beginning of the T-cell culture. After phorbol 12-myristate 13-acetate/ionomycin treatment, supernatants were harvested for IL-17, IFN-γ, and TNF-α assessment by using an ELISA or cytokine bead array. Bar graphs display ratios representing relative IL-17 amounts in comparison with IFN-γ and TNF-α. Means ± SEMs refer to 4 assays, each performed in triplicate. *P ≤ .05. ns, Not significant.
Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci )
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12. Fig E9
Characterization of isolated skin T cells. Human skin explants rested on metal grids under proper culture conditions (see the Methods section). After 21 days, cells were flushed from the grids, counted, and costained with CD4/CD8 antibodies or isotype-matched controls for FACS analyses.
Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci )
Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions