Results: 4

1.
Figure 2

Figure 2. Paucity of iTreg cells results in Th2 inflammation in the gastrointestinal tract. From: Extrathymically generated regulatory T cells control mucosal Th2 inflammation.

a, Percent of CD4+ cells producing IL-4, IL-13 and IL-5 in 3-month-old mice.
b, Percent of Foxp3 CD4+ cells that were Gata3+ in 3-month old mice.
c, Concentration of IgE and IgA in serum, determined by enzyme linked immunosorbent assay (ELISA).
All data are representative of three or more independent experiments with ≥ 3 mice per group. Error bars represent SD; *, **, and *** indicate p <0.05, 0.01 and 0.001, respectively, as calculated by students’ T-test.

Steven Z. Josefowicz, et al. Nature. 2012 February 8;482(7385):395-399.
2.
Figure 3

Figure 3. iTreg cells deficiency leads to Th2 type gastrointestinal pathology and altered microbial communities. From: Extrathymically generated regulatory T cells control mucosal Th2 inflammation.

a, Body weights of 9–12 (left) or 2.5-month-old individually housed (right) CNS1 and littermate control mice (n ≥ 12).
b, Plasmacytic enteritis (arrowhead) in CNS1-deficient mice revealed by H&E staining of small intestine from 9–12-month-old CNS1 (bottom and right) and littermate control mice (top). An early crypt abscess is indicated (asterisk). Data represent ≥ 20 mice.
c, Percent of Foxp3+ CD4+ cells expressing Gata3+ in 3-month old mice.
d, Percent of total 16S rRNA gene sequences of the Firmicutes and Bacteroidetes phyla in stool from individually housed CNS1 (n=9) and WT (n=6) littermate mice. Mean values +/− SEM are shown.
All data are representative of three or more independent experiments with ≥ 3 mice per group. Error bars represent SD; *, **, and *** indicate p <0.05, 0.01 and 0.001, respectively, as calculated by students’ T-test. Scale bars = 150 um.

Steven Z. Josefowicz, et al. Nature. 2012 February 8;482(7385):395-399.
3.
Figure 4

Figure 4. Unprovoked asthma-like airway pathology in CNS1-deficient mice. From: Extrathymically generated regulatory T cells control mucosal Th2 inflammation.

a, Representative H&E-stained lung section form CNS1 (top) and WT (bottom) mice. The CNS1 lung has marked peribronchiolar inflammation (arrowhead). The reduced lumen (L) contains mucus produced by the hyperplastic respiratory epithelium (E) Arrows indicate reactive (top) and normal (bottom) endothelium. Bottom right hand corner insets are higher magnification of boxed regions and bar indicates smooth muscle thickness. Top right inset (KO) demonstrates eosinophilic crystals. Asterisk marks acidophilic macrophages.
b, PAS/AB staining highlighting mucus-producing goblet cells (dark blue-purple)
c, Trichrome staining illustrating lung fibrosis (blue staining).
d, Arginase-1 staining of lungs from CNS1 and WT mice. “A” indicates airway; an acidophilic crystal is marked by the arrowhead.
e, Chitinase 3-like 3 (C3l3) staining of lungs from CNS1 and WT mice at 10× amplification (top) and 60× magnification of lungs from CNS1 mice demonstrating robust C3l3 expression within acidophilic macrophages (bottom).
f, Lung resistance (left) and compliance (right) of CNS1 and wild type littermate control mice after exposure to methacholine.
Data representative of 2 independent experiments with ≥ 4 mice per group. Error bars represent SD; *, **, and *** indicate p <0.05, 0.01 and 0.001, respectively, as calculated by students’ T-test. Scale bars = 100 um.

Steven Z. Josefowicz, et al. Nature. 2012 February 8;482(7385):395-399.
4.
Figure 1

Figure 1. Impaired iTreg cell generation and altered composition of the peripheral Treg cell population in CNS1-deficient mice. From: Extrathymically generated regulatory T cells control mucosal Th2 inflammation.

a, Relative contribution of CNS1-deficient (GFP+) and -sufficient (GFP) cells to the Foxp3+ thymocyte subset in 4-day-old CNS1+/− female mice.
b, Induction of Foxp3 in Foxp3 TN cells FACS sorted from CNS1 or Foxp3GFP mice stimulated in vitro with TGFβ, IL-2, anti-CD3 and anti-CD28.
c, Percent Foxp3+ cells in the spleen, lymph node (LN), mesenteric lymph nodes (MLN), Peyer’s patches (PP) and cells from the small and large intestine lamina propria (SI and LI) of 6–9 month old CNS1 or control mice.
d, Percent of transferred CNS1 or CNS1+ CD25CD44lowCD45.2+OTII+ cells that induced Foxp3 following administration of OVA in water for 6 days.
e, Stability of Foxp3 expression in iTreg cells. FACS sorted GFP+ or GFP cells from Foxp3eGFP-Cre-ERT2 mice were transferred with GFP or GFP+ cells, respectively, from Ly5.1 Foxp3GFP mice into TCRβδ-deficient recipients. Mice received tamoxifen at 1 (left) or 5 weeks (right) post transfer and stability of Foxp3 expression among YFP-labeled cells was assessed after 4 weeks. All data are representative of two or more independent experiments with n ≥ 3. Error bars represent SD; *, **, and *** indicate p <0.05, 0.01 and 0.001, respectively, as calculated by students’ T-test.

Steven Z. Josefowicz, et al. Nature. 2012 February 8;482(7385):395-399.

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