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Impairment of T-regulatory cells in cord blood of atopic mothers

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1 Impairment of T-regulatory cells in cord blood of atopic mothers
Bianca Schaub, MD, Jing Liu, MD, Sabine Höppler, Severine Haug, Christine Sattler, MD, Anna Lluis, Sabina Illi, PhD, Erika von Mutius, MD, MSc  Journal of Allergy and Clinical Immunology  Volume 121, Issue 6, Pages e13 (June 2008) DOI: /j.jaci Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 The level of mRNA expression was shown as fold difference in gene expression in phytohemagglutinin (PHA; 5 μg/mL), LpA (0.1 μg/mL), peptidoglycan (Ppg; 10 μg/mL), D pteronyssinus 1 (D; 30 μg/mL), and D+L stimulated/unstimulated samples, compared with the housekeeping gene 18S. RNA was prepared as described in Methods. A-D, Quantitative gene expression was assessed using real-time RT-PCR. NA, Cord blood of nonatopic mothers; A, cord blood of atopic mothers. Journal of Allergy and Clinical Immunology  , e13DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Division of effector T cells was shown in percentage, lymphocyte proliferation of effector T-cells in cpm, and cytokine secretion in pg/mL after stimulation and addition of CD4+CD25+ T cells in increasing doses (1:1 to 1:3). Division of effector T cells and lymphoproliferation were measured with CFSE staining and 3H-Thymidine incorporation, cytokine secretion by LUMINEX technology. Cord blood samples of N = 8 nonatopic and 6 atopic mothers were shown (A-F). Journal of Allergy and Clinical Immunology  , e13DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 CD4+CD25 high T cells (ratio of stimulated vs unstimulated cells)
CD4+CD25 high T cells (ratio of stimulated vs unstimulated cells). CD4+CD25 high T cells were significantly higher in LpA/U and peptidoglycan (Ppg)/U samples in CBMCs of nonatopic compared with atopic mothers. CD4+CD25 high T cells were assessed by flow cytometry. Data are shown as means ± SEMs. A, Atopic; NA, nonatopic; U, unstimulated. Journal of Allergy and Clinical Immunology  , e13DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Suppressive capacity of CD4+CD25+ T cells
Suppressive capacity of CD4+CD25+ T cells. CD4+CD25+ T cells significantly suppressed effector T cells after mitogen stimulation in a dose-dependent manner. A, Purity of isolated CD4+CD25+ T cells. B, CFSE staining of effector T cells. C, Unstimulated effector T cells showed no significant T-cell division, 1.8% (CFSE staining). D, Significant T-cell division after phytohemagglutinin (PHA) stimulation (80%). E and F, Dose-dependent suppression of effector T cells through CD4+CD25+ T cells (65.5% and 19.6% T-cell division). A-F, CD4+CD25– and CD4+CD25+ T cells were isolated as described in Methods. CD4+CD25– cells were labeled with CFSE and incubated with irradiated CD3– T cells in coculture without or with increasing ratios of CD4+CD25+ T cells (1:1 to 1:3). The division of CD4+CD25– T cells was measured before and after 3 days of stimulation with 0.8 μg/mL PHA with flow cytometry. Measurement and analysis were performed by using FACScan and CellQuest software. One representative experiment of 14 is shown. Journal of Allergy and Clinical Immunology  , e13DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Suppression of T-cell cytokine secretion of IL-5 and GM-CSF
Suppression of T-cell cytokine secretion of IL-5 and GM-CSF. A and B, Cytokine secretion of effector T cells of GM-CSF but not IL-5 was decreased after suppression through CD4+CD25+ T cells in a dose-dependent manner in cord blood from nonatopic mothers. Isolation and suppression assays were described in Methods; cytokine secretion was assessed by LUMINEX technology. Mean or median (±SEM) values of cord samples of N = 8 nonatopic and 6 atopic mothers are shown. Journal of Allergy and Clinical Immunology  , e13DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 A-F, Correlation of LpA and peptidoglycan (Ppg)–induced secretion of IL-17 and IL-13 in CBMCs of all children and of children without and with atopic mothers. Cytokines (pg/mL) were measured by LUMINEX. Data are shown after log-transformation with regression line and CI for mean predicted values. ln, Log transformation. Journal of Allergy and Clinical Immunology  , e13DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 A-C, CD25+IL-17 positive cells and CD25Foxp3 cells
A-C, CD25+IL-17 positive cells and CD25Foxp3 cells. Intracellular IL-17, Foxp3+ expression, and proliferation after stimulation with phytohemagglutinin (PHA) with and without IL-6 and TGF-β. CBMCs were stimulated, and intracellular IL-17 or Foxp3 was assessed as described in Methods. Proliferation in cpm was measured by 3H-Thymidine incorporation. Data are shown as mean (±SEM) of n = 10 experiments. ∗P > .05. U, Unstimulated. Journal of Allergy and Clinical Immunology  , e13DOI: ( /j.jaci ) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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