Activated PI3Kδ reshapes the HDM-induced immune response. (A–J) WT and Pik3cd^E1020K/+ animals were sensitized intranasally with HDM extracts (200 μg on days 0 and 7; 50 μg on days 14, 16, and 18) and lungs examined on day 20. (A) Experimental outline. (B) H&E staining of paraffin-embedded lung sections. Top panel: Arrowheads indicate thickening of the alveolar septa; pound signs indicate iBALT. Bottom panel: Short arrows show lymphocytes, long arrows show macrophages, and yellow arrows show eosinophils. (C) CD4 T cell counts (liveCD45⁺TCRβ⁺CD4⁺CD8⁻) measured by flow cytometry. (D) Eosinophil numbers quantified from H&E-stained lung sections. (E) Neutrophil counts (liveLineage⁻CD11b⁺Ly6G⁺) from lungs of the indicated animals. (F) Airway resistance (Rrs) was measured with a flexiVent instrument as cmH2O.s/ml using the indicated concentrations of methacholine. Data in E are representative of two independent experiments with n = 3–4 for each group per experiment. (G) UMAP showing clusters of lung CD45⁺ immune cells analyzed by scRNAseq from naïve WT, naïve Pik3cd^E1020K/+, HDM-treated WT, and HDM-treated Pik3cd^E1020K/+ animals. Each cluster was assigned a cell type using SingleR and supervised analysis of gene expression specific to each cluster (Table S1). Cells from three mice were analyzed per genotype and condition. (H) Individual lung CD45⁺ immune cells identified by scRNAseq analyzed for enrichment of response to IFNγ gene set. Left: UMAP visualization of enrichment P values in lung CD45⁺ immune cells from the indicated mice; P values described in color scale below. Right: Frequencies of cells with indicated magnitudes of enrichment P values. Statistical comparison of all CD45⁺ cells from WT and Pik3cd^E1020K/+ HDM-treated groups was performed (chi-squared test), comparing frequencies of cells with P < 0.0005. (I) HDM-specific IgG2a, IgG3, IgE, and IgG1 from the indicated mice. (J) CCL3, CCL5, and CXCL10 (pg/ml) measured from lung homogenates from the indicated mice by Luminex. Data in C–E, I, and J are from n = 6–10 mice for each group, pooled from two independent experiments. Data in B are representative of two independent experiments. Unless otherwise indicated, statistical comparisons were made using unpaired t tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.