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Meri K. Tulic, BSc, PhD, David Andrews, MD, Maxine L

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1 Changes in thymic regulatory T-cell maturation from birth to puberty: Differences in atopic children 
Meri K. Tulic, BSc, PhD, David Andrews, MD, Maxine L. Crook, BSc, Adrian Charles, MD, Michelle R. Tourigny, PhD, Redwan Moqbel, PhD, FRCPath, Susan L. Prescott, MD, PhD  Journal of Allergy and Clinical Immunology  Volume 129, Issue 1, Pages e4 (January 2012) DOI: /j.jaci Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Enumeration of thymic Treg cells with age in atopic and nonatopic children. A, FOXP3+CD25+ Treg cell expression in the thymus from a neonate (middle panel) or a 6-year-old child (right panel). Total thymocytes were initially gated on CD4+ T cells and then on CD127lo/− cells. The left panel shows staining with the isotype control in the thymus from the 6-year-old child. B, Percentage of Treg cells as the proportion of total thymocytes in the left panel and the proportion of total CD4+ T cells in the right panel. C, FOXP3 mRNA expression in nonatopic (white bars) and atopic (gray bars) children with age (n = 10 per age group). Results are expressed as box-and-whisker plots. ∗P < .05 and ∗∗P < .001 versus nonatopic age-matched control subjects. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Thymic Treg cell function in atopic and nonatopic children. A and B, Effects of adding increasing numbers of Treg cells to effector T-cell (Teff) cultures on IL2 mRNA expression (Fig 2, A) and IL-13 protein production (Fig 2, B) in nonatopic (white bars, n = 10) or atopic (gray bars, n = 10) children less than 6 months of age (left panel) or more than 5 years of age (right panel). Results are expressed as box-and-whisker plots. ∗P < .05 and ∗∗P < .001 versus Treg cell ratio 1:0 (SEB stimulated without Treg cells). C, Immunofluorescence imaging showing individual channels in grayscale depicting staining for thymic CD4+ T cells (panel 2), IL-13+ cells (panel 3), and their colocalization (merge image in panel 4, with CD4 as red, IL-13 as green, and colocalization as yellow immunostaining), which is reduced with addition of Treg cells in nonatopic children (panel 5). Panel 1 is the hematoxylin and eosin stain of the same area used for immunofluorescence. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Maturation of TSLP with age and its effect on Treg cell suppression in atopic and nonatopic children. A, TSLP immunoreactivity in the medulla (left panel) and Hassall corpuscles (middle and right panels) of neonatal thymic tissue from nonatopic subject. The insert in the middle panel is stained with isotype control antibody. B, Number of TSLP-immunoreactive cells in nonatopic (white bars, n = 20) and atopic (gray bars, n = 20) children with age (n = 10 per age group). C, Effect of blocking TSLP on Treg cell–induced suppression of effector T-cell function in nonatopic (white bars, n = 10) or atopic (gray bars, n = 10) children less than 6 months of age (left) or more than 5 years of age (right). Results are expressed as box-and-whisker plots. ∗P < .05 and ∗∗P < .001. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig E1 Thymic cytokine maturation in atopic and nonatopic children. Cytokine expression in nonatopic (white bars) or atopic (gray bars) children less than 6 months (group 1: n = 10), 6 to 12 months (group 2: n = 10), 1 to 5 years (group 3: n = 10), or greater than 5 years (group 4: n = 10) after PHA stimulation. Results are expressed as box-and-whisker plots. ∗P < .05 versus group 1 for each cytokine. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig E2 Turnover of thymic Treg cells with age in atopic and nonatopic children. A, CD4+CD25+CD127lo/−Ki67+ Treg cell proliferation in the thymus from a neonate (middle panel) or 6-year-old child (right panel). Total thymocytes were initially gated on CD4+ T cells and then on CD127lo/− cells. The left panel shows staining with the isotype control in the thymus of 6-year old child. B, Percentage of Treg cells (CD4+CD25+CD127lo/−Ki67+ cells) undergoing turnover in nonatopic (white bars, n = 20) and atopic (gray bars, n = 20) children with age (n = 10 per age group). Results are expressed as box-and-whisker plots. ∗P < .05 and ∗∗P < .001 versus nonatopic age-matched control subjects. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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