Figure 3. HpBARI suppresses Alternaria-induced innate immune responses.

Alternaria allergen (10 μg) and HpBARI (10 μg) were intranasally administered and mice culled 24 hr later. Eosinophils were enumerated in the BAL (A) and lung (B), and intracellular cytokine staining carried out for IL-13 in ICOS⁺lineage^–CD45⁺ lung ILC2s (C). IL-5 was measured in cell-free BAL supernatants (D). Data representative of 2 repeat experiments, each with 3–4 mice per group. Error bars show SEM.

Figure 3—figure supplement 1. C-terminus tagged HpBARI shows abrogated suppressive activity, while no suppression is seen with control protein administration.

Alternaria allergen (20 μg), N-terminal tagged HpBARI (HpBARI_N), C-terminal tagged HpBARI (HpBARI_C) or control protein (HpAChE) (10 μg of each recombinant protein) were intranasally administered and mice culled 24 hr later. (A–B) Eosinophils numbers in the BAL (A) or lung (B), measured by flow cytometry. (C–D) Intracellular cytokine staining carried out for IL-5 (C) and IL-13 (D) in ICOS⁺lineage^–CD45⁺ lung ILC2s. (E) Representative flow cytometry plots of IL-5 vs IL-13 on gated ICOS⁺lineage^–CD45⁺ cells. (F) Geometric mean fluorescence intensity of ST2 on ICOS⁺lineage^–CD45⁺ lung ILC2s. (G) Representative flow cytometry plots of ST2 on gated ICOS⁺lineage^–CD45⁺ cells. Data representative of at least two repeat experiments, each with 3–4 mice per group, apart from Alt+HpAChE group, which is from a single experiment. Error bars show SEM.

Figure 3—figure supplement 2. Gating strategy for lung ILC2s.

A single cell suspension of lung cells was prepared, stimulated for 4 hr in PMA, Ionomycin and Brefeldin A, and stained for flow cytometry. Gating strategy for a lung sample from an Alternaria-treated positive control sample shown. IL-5, IL-13 and ST2 staining in ICOS⁺lineage^–CD45⁺ ILC2s from this experiment shown in Figure 3—figure supplement 1E and G.