Journal: Journal of Clinical Investigation

Article Title: FOXP3+ Tregs require WASP to restrain Th2-mediated food allergy

doi: 10.1172/jci85129

Figure Lengend Snippet: Figure 2. Spontaneous sensitization to food antigens and food allergy in Was–/– mice. (A) Comparative analysis of total sIgE and IgG1 levels in 3-month- old WT BALB/c (open circles, n = 7) and Was–/– mice (gray circles, n = 9) of mixed genders. (B) IgE and IgG1 reactivity against the 5 main (% w/w) chow components as determined by ELISA in 1:30 (IgE) or 1:1,000 (IgG1) diluted serum samples. (C) Loading of WT bone marrow–derived mast cells with serum of food-allergic (FA sens) or non–food-allergic (FA non-sens) Was–/– mice compared with no-serum control (left panel). Appearance of surface LAMP-1 as a marker of mast cell degranulation after stimulation with antigen extracts from conventional chow (CCh), elemental chow (ECh), or PBS (–). (D) Intestinal mast cell expansion as determined by chloroacetate esterase staining of jejunal cross-sections (×20, with digital magnification to ×50 shown in window) and quantification per 4 high-power fields (4hpf) in WT (n = 7) and Was–/– mice (n = 8). (E) Serum levels of mast cell protease 1 (MCPT1) determined by ELISA. (F) Effect of 7-day treatment with elemental diet on serum MCPT1 in Was–/– mice (n = 11). Spearman’s rank correlation between cumulative anti- food IgE titers of mice and response to allergen elimination defined as ΔMCPT1. (G) Effect of oral rechallenge with 12.5 mg soy protein extract on body tem- perature and serum MCPT1 after 4 hours. Symbols represent individual mice and error bars depict SEM. *P < 0.05, **P < 0.01, ***P < 0.001 as determined by 2-tailed Student’s t test. NS, not significant; ND, not detectable. Results in C are representative of 2 independent experiments. Equivalent results were obtained in a cohort of WT and Was–/– mice on the 129SvEv background.

Article Snippet: Capture antibodies: anti–mouse IgE (Southern Biotech, catalog 1110-01) and anti–mouse IgG1 (Southern Biotech, catalog 1070-01).

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Control, Marker, Staining

Journal: Journal of Clinical Investigation

Article Title: FOXP3+ Tregs require WASP to restrain Th2-mediated food allergy

doi: 10.1172/jci85129

Figure Lengend Snippet: Figure 3. Commensals are dispensable for spontaneous sensitization to food in Was–/– mice but shape the isotype composition of the humoral anti- food response. (A) Comparison of total IgE and total IgG1 serum levels in 4- to 6-month-old WT (open circles, n = 5) or Was–/– (gray circles) on the 129SvEv background that were housed under either SPF (n = 10) or GF (n = 14) conditions. (B) Food-specific IgE and IgG1 for the 5 main chow constituents in IgE (serum dilution 1:100) or IgG1 (1:5,000) from SPF (n = 8) and GF (n = 7) Was–/– mice. (C) Comparison of serum MCPT1 levels. (D) Comparison of cumulative anti-food titers of IgE, IgG1, IgG2a (1:1,000), IgG2b (1:1,000), IgG3 (1:200), and IgA (1:5,000) in SPF (n = 8) and GF (n = 7) Was–/– animals. Symbols represent individual mice and error bars depict SEM. *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant as determined by 2-tailed Student’s t test. Results are shown from sera obtained from mice from ≥ 3 independent cohorts.

Article Snippet: Capture antibodies: anti–mouse IgE (Southern Biotech, catalog 1110-01) and anti–mouse IgG1 (Southern Biotech, catalog 1070-01).

Techniques: Comparison

Journal: Journal of Clinical Investigation

Article Title: FOXP3+ Tregs require WASP to restrain Th2-mediated food allergy

doi: 10.1172/jci85129

Figure Lengend Snippet: Figure 4. WASP deficiency in Tregs is sufficient for the development of spontaneous food allergy and results in more severe disease. (A) Comparison of MCPT1 levels in mice with cell type–specific WASP deletions. Mice with conditional deletion of Wasfl/fl alleles in B cells (Wasfl/fl Mb1-Cre), CD11c+ dendritic cells (Wasfl/fl Itgax-Cre) or Tregs (Wasfl/fl Foxp3-Cre) of ≥ 2 months of age, n ≥ 5 per group. (B) Representative H&E (×10 magnification) and chloroacetate esterase (CAE) staining (×20 magnification) of intestinal cross-sections in Wasfl/fl Foxp3-Cre or WasWT Foxp3-Cre littermates on the C57BL/6 background. (C) Comparison of total and soy-specific IgE and IgG1 at 2 months in cohoused WT (open circles, n = 9), Was–/– (gray circles, n = 12), and Wasfl/fl Foxp3-Cre (black circles, n = 9) mice of mixed genders on the C57BL/6 background. (D) Comparison of serum protein and jejunal mRNA expression levels of mucosal mast cell marker MCPT1. Symbols represent individual mice and error bars depict SEM. *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant as deter- mined by 2-tailed Student’s t test (A) or 1-way ANOVA with Tukey’s multiple comparisons test (C and D). BDL, below detection limit. Data in C and D are representative of 2 independent cohorts.

Article Snippet: Capture antibodies: anti–mouse IgE (Southern Biotech, catalog 1110-01) and anti–mouse IgG1 (Southern Biotech, catalog 1070-01).

Techniques: Comparison, Staining, Expressing, Marker

Journal: Journal of Clinical Investigation

Article Title: FOXP3+ Tregs require WASP to restrain Th2-mediated food allergy

doi: 10.1172/jci85129

Figure Lengend Snippet: Figure 6. WASP-deficient FOXP3+ Tregs fail to suppress Th2-type lymphoproliferation in vivo. (A) Quantification by flow cytometry of FOXP3+ Tregs amongst CD4+ T cells obtained from MLNs or PPs of WT (open circles, n = 5), Was–/– (gray circles, n = 6) and Wasfl/fl Foxp3-Cre (black circles, n = 4) mice. (B) Production of IL-2 by CD4+ mesenteric T lymphocytes stimulated with anti-CD3/CD28 ex vivo. Each dot represents the average cytokine production from triplicate cell suspensions from a single mouse. (C) Total CD4+ T cell numbers obtained from MLNs and PPs. (D) Gating strategy of GATA3+ICOS+ Th2-type effector cells within the parent gate of effector memory T cells from MLNs of representative samples, with quantification and statistical testing in the right panels. (E) Fraction of T-bet+ and RORγt+ effector memory T cells. (F) Production of IL-4, IL-13, IFN-γ and IL-17a by CD4+ mesenteric T lymphocytes stimulated with anti-CD3/CD28 ex vivo. Each data point represents the average cytokine production from triplicate cell suspensions from a single mouse. (G) Serum levels of anti-soy specific IgE and IgG1, and MCPT1 in Was–/– mice on the 129SvEv background with either Il4+/+ (gray circles, n = 10 or 15) or Il4–/– alleles (gray squares, n = 8). (H) Anti-soy IgG2b titer as determined by ELISA in 1:1,000 serum dilution. Symbols represent individual mice and error bars depict SEM. *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant as determined by 2-tailed Student’s t test or 1-way ANOVA with Tukey’s multiple comparisons test. In B and F, data were log-transformed prior to statistical testing. Data from 2 pooled experiments (C, G, and H) or representative results from ≥ 2 independent experiments (A, B, and D–F) are shown.

Article Snippet: Capture antibodies: anti–mouse IgE (Southern Biotech, catalog 1110-01) and anti–mouse IgG1 (Southern Biotech, catalog 1070-01).

Techniques: In Vivo, Flow Cytometry, Ex Vivo, Enzyme-linked Immunosorbent Assay, Transformation Assay