Altered CD4 ⁺ T cell differentiation in Pik3cd ^E1020K/+ mice in vivo. Supporting data for Fig. 2. (A) Feature plots showing expression of Th2-defining genes Il1rl1, Il4, IL5, and Il13 in scRNAseq analyses of lung CD45⁺ immune cells from the indicated mice. Cells from three mice were analyzed by scRNAseq per genotype and condition. (B) Flow cytometry of lung CD4 T cells (liveCD45⁺TCRβ⁺CD4⁺CD8⁻) measuring GATA3 expression in the indicated populations. n = 9–10 for each group, pooled from two independent experiments. Left: Representative plots showing GATA3 and CD4 expression. Right: Frequencies (left) and cell counts (right) of GATA3⁺ CD4 T cells in the indicated groups. (C–E) Frequencies of (C) IL-13⁺ (liveCD45⁺TCRβ⁺CD4⁺CD8⁻ CD4⁺); (D) IL-17A⁺ (liveCD45⁺TCRβ⁺CD4⁺CD8⁻ CD4⁺), and (E) Foxp3⁺ (Treg) lung CD4 T cells (liveCD45⁺TCRβ⁺CD4⁺CD8⁻ CD4⁺) in the indicated groups, measured by intracellular staining and flow cytometry. n = 9–10 for each group, pooled from two independent experiments. (F and G) Cytokine concentrations (pg/ml) measured from lung homogenates from the indicated mice by Luminex. Th2 cytokines IL-4 and IL-5 are shown in F, and Th1 cytokines GM-CSF and TNFα are shown in G. n = 9–10 for each group, pooled from two independent experiments. (H–L) WT and Pik3cd^E1020K/+ animals were infected intranasally with C. neoformans, and were sacrificed 10 days after infection, and lung tissue was harvested for analysis. n = 6 for each group, pooled from two independent experiments. (H) Experimental design. (I) Cell counts of CD4 T cells (liveCD45⁺TCRβ⁺CD4⁺CD8⁻ CD4⁺) from lungs of the indicated mice. (J) Flow cytometry of lung CD4 T cells (liveCD45⁺TCRβ⁺CD4⁺CD8⁻) measuring GATA3 and Tbet expression in the indicated populations. Left: Representative flow cytometry plots. Right: Frequencies of Tbet⁺ and GATA3⁺ CD4 T cells from the indicated groups. (K) Frequencies of IL-4/5/13⁺ (left), IFNγ⁺ (middle), and IL-2⁺ (right) lung CD4 T cells (liveCD45⁺TCRβ⁺CD4⁺CD8⁻ CD4⁺) in the indicated groups, measured by intracellular staining and flow cytometry. Frequencies of IL-4/5/13⁺ cells were calculated using Boolean (or) gating. (L) Cell counts of eosinophils (liveCD45⁺CD3⁻NK1.1⁻CD19⁻CD11b⁺Ly6G⁻SiglecF⁺) and neutrophils (liveCD45⁺CD3⁻NK1.1⁻CD19⁻CD11b⁺Ly6G⁺) from lungs of the indicated mice. (M–O) Supplemental data for CD4⁺ T cell transfer experiments in Fig. 2, I–L. n = 9–10 mice for each group, pooled from two independent experiments. (M) Cell counts (left) and frequencies (right) of GATA3⁺ CD4 T cells from the indicated groups. (N) Representative flow cytometry plots showing IFNγ and IL-4 expression in lung CD4 T cells (liveCD45⁺TCRβ⁺CD4⁺CD8⁻) from the indicated mice. (O) Cell counts of neutrophils (liveCD45⁺CD3⁻NK1.1⁻CD19⁻CD11b⁺Ly6G⁺) from lungs of the indicated mice. Statistical comparisons were made using unpaired t tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.