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Nitric oxide induces human CLA+CD25+Foxp3+ regulatory T cells with skin-homing potential  Cunjing Yu, PhD, Amanda Fitzpatrick, PhD, Duanduan Cong, MMedSci,

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Presentation on theme: "Nitric oxide induces human CLA+CD25+Foxp3+ regulatory T cells with skin-homing potential  Cunjing Yu, PhD, Amanda Fitzpatrick, PhD, Duanduan Cong, MMedSci,"— Presentation transcript:

1 Nitric oxide induces human CLA+CD25+Foxp3+ regulatory T cells with skin-homing potential 
Cunjing Yu, PhD, Amanda Fitzpatrick, PhD, Duanduan Cong, MMedSci, Chengcan Yao, PhD, Jinah Yoo, MB BS, Andrew Turnbull, MB BS, Jürgen Schwarze, MD, Mary Norval, PhD, Sarah E.M. Howie, PhD, Richard B. Weller, MD, Anne L. Astier, PhD  Journal of Allergy and Clinical Immunology  Volume 140, Issue 5, Pages e6 (November 2017) DOI: /j.jaci Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 NO induced de novo human CLA+ Treg cells. CD4+ T cells were activated with anti-CD3/CD28 in the presence of NOC-18 (100 μM) for 5 days. A, Expression of CD25+Foxp3+CD127− cells and Foxp3 (n = 6). B, CLA expression on Treg cells and Teff cells. C, Percentage of NOC-18–induced Treg cells in the presence of various concentrations of the sGC inhibitor ODQ, normalized to control for each donor (n = 3). D, Percentage of CD25+Foxp3+CD127− cells and CLA expression on CD25+Foxp3+CD127− cells after addition of NOC-18 to purified CD4+CD25− T cells. E, NO-induced Treg cells (NO-CD4) were cocultured with prelabeled autologous CD4+ T cells (Resp) or physically separated from Resp cells using transwells. F, Proliferation of Resp cells cocultured with NO-Treg cells with antibodies blocking PD-1 and its ligand PD-L1. Representative of 2 donors. P values were calculated using the Wilcoxon paired t test or using the Friedman test followed by Dunns posttest for multiple comparisons. Mean ± SEM are represented. Ctrl, Control. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 UVB induced NO production and Treg cells in vivo. A, Twenty-four hours after irradiation of the forearm of healthy volunteers (n = 5), cutaneous microdialysis was performed in the presence of L-NAME (n = 5, mean ± SEM), an NO synthase inhibitor, or its inactive enantiomer D-NAME (n = 3). Blood flow results are normalized against initial blood flow. B, Levels of dialysate nitrite concentration (as a measure of NO, triangles) were plotted against blood flow (arbitrary units) as a measure of erythema (black circles). Figure is representative of 2 subjects. Nitrite was at the lower level of detection of the apparatus and could not be combined for analysis. C, A cohort of patients with AD was treated by phototherapy (Table E1). The improvement in disease for each donor was assessed by SCORAD (SCORing Atopic Dermatitis) before and after phototherapy, and correlated with the Treg:Teff cells ratio. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig E1 NO promoted CLA+ human Treg cells. Purified peripheral CD4+ T cells were activated with plate-bound anti-CD3/CD28 in the presence of the NO-donating compound NOC-18 (100 μM) for 5 days before analysis by flow cytometry. A, T-cell viability and CD25 expression (% and MFI) (mean ± SEM, n = 7). B, Representative plots showing expression of CD25+Foxp3+CD127− cells after addition of NOC-18. C, Representative plots showing expression of CLA on CD25+Foxp3+CD127− cells after addition of NOC-18. D, CD4+ T cells were activated in the presence of NOC-18 for various amounts of time before replacing the culture medium without NOC-18, and CLA expression on Treg cells measured after 5 days (mean ± SEM, n = 2). E, Representative FACS plot showing CD25 versus CD127 before (left panel) and after (right panel) CD25+ depletion. F, CD4+ T cells were stained with the proliferation dye ef670 before being activated with plate-bound anti-CD3/CD28 in the presence of 100 μM NOC-18 for 5 days. Proliferation of Treg cells and Teff cells was then analyzed by flow cytometry and normalized to control (n = 3). Ctrl, Control. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig E2 NO-induced CD25+Foxp3+CD127− Treg cells inhibited the proliferation of autologous CD4+ T cells in a dose-dependent manner. CD4+ T cells were activated with plate-bound anti-CD3/CD28 in the presence of 100 μM NOC-18 for 5 days (NO-CD4 Treg cells). Autologous CD4+ T cells were used as responder cells (Resp) and cocultured with NO-CD4 Treg cells at different ratios for 4 days. Twenty percent of cells had a CD25+Foxp3+CD127− Treg-cell phenotype after addition of 100 μM NOC-18 for 5 days. Therefore, to achieve a responder cells:Foxp3+ cells ratio at 1:1 and 1:2, responder cells were cultured with NO-CD4 at 1:5 and 1:10 ratios. A, Representative FACS plot showing proliferation of responder cells cultured with NO-CD4 T cells. B, Mean ± SEM data for proliferation and CD25 expression for 6 donors. P values were calculated by the Friedman test followed by Dunns posttest. C, CD25hi cells were isolated from NO-CD4 Treg cells (NO-Treg cells). Representative FACS plot showing CD25 versus Foxp3 before and after isolation. D, NO-Treg cells were cocultured with autologous responder CD4+ T cells (Resp) at different ratios for 4 days and proliferation of autologous CD4+ T cells was analyzed by flow cytometry. Representative of 3 donors. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig E3 NO-induced Foxp3+ Treg cells required cell-cell contact for their suppressive function. A, CD4+ T cells were activated with anti-CD3/CD28 in the presence of 100 μM NOC-18 for 5 days (NO-CD4), and used as suppressor cells in coculture with ef670-labeled autologous CD4+ T cells or physically separated from autologous CD4+ T cells using transwells. Representative FACS plots showing proliferation of autologous CD4+ T cells. B, Culture supernatants from CD4 T cells cultured with or without 100 μM NOC-18 for 5 days were mixed with fresh RPMI at different ratios and transferred to ef670-labeled autologous CD4 T cells. Proliferation was analyzed by flow cytometry (n = 3, mean ± SEM). C, CD25hi cells (NO-Treg cells) were isolated from total CD4+ T cells activated with 100 μM NOC-18 for 5 days. NO-Treg cells were cocultured with autologous CD4+ T cells in the presence of antibodies blocking CTLA-4. Representative of 2 donors. Ctrl, Control. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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