Lung ILC2s isolated from naïve WT mice also display high levels of TCR constant region transcripts. (A) Naïve lung ILC2s from adult WT mice were isolated based on CD45⁺Lin^–Thy1.2⁺CD127⁺CD25⁺γδTCR^– surface markers with 2 rounds of cell sorting (red gate). Lung tissues were pooled from 8 to 9 sex- and age-matched mice for optimal yield. (B) Fluorescence-activated cell sorting (FACS) plots showing enriched lung ILC2 population before downstream PCR analysis. The purified population was tested for potential γδT cell contamination. (C) RT-PCR was performed on both WT and Rag1^−/− ILC2s to verify the expression of TCR constant region genes seen in cecal ILC2s. WT CD3⁺ splenocytes (Spl^–CD3ε⁺ WT) were used as a positive control and fibroblasts cultured from fibro/adipogenic progenitors were used as a negative control for sterile TCR mRNA expression. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) primers were used as a loading control. (D) scRNA-seq analysis of independently sorted lung CD45⁺Lin^–RORa^+/− cells. ILC2 cluster was identified based on ILC2-specific gene expression (Areg, Il7r, Rora, Gata3) and a relative expression heatmap of TCR constant genes was created to complement the RT-PCR data. FSC, forward scatter; SSC, side scatter.