Sex Chromosomes and Gonadal Sex Interactions in Airway and Immune Responses to Allergen Challenge

SUPPLEMENTAL MATERIAL: Supplementary Figure 1. Representative gating of unstained lung control samples. Sequential plots show the gating hierarchy used to define baseline fluorescence and exclude debris and doublets in unstained lung single-cell suspensions. Live/dead discrimination (Zombie NIR) and CD45 expression were used to identify total leukocytes. The final Siglec-F vs. CD11b plot illustrates background fluorescence used to set negative gates for alveolar macrophage and eosinophil populations, serving as the reference for FMO and fully stained samples.Supplementary Figure 2. Representative gating strategy and FMO controls for lung immune-cell profiling. Sequential plots illustrate the gating hierarchy used to identify major lung leukocyte subsets from Four-Core-Genotype (FCG) mice after PBS or HDM exposure. Doublets and debris were excluded, and live CD45⁺ Zombie NIR⁻ cells were analyzed for AMs, eosinophils, dendritic-cell subsets, T and B cells, NK, and NKT cells. FMO controls defined positive gates and compensated spectral overlap. Gates were adjusted to account for biologic shifts such as Siglec-F down-regulation in AMs during inflammation. Supplementary Figure 3A-B. Representative gating of Siglec-F⁺ populations and B/pDC subsets across PBS- and HDM-treated Four-Core-Genotype (FCG) mice. (A) Representative Siglec-F gating across PBS- and HDM-treated samples. Flow-cytometry plots from individual FCG mice show gating of AMs and eosinophils based on Siglec-F and CD11b expression. Fluorescence-minus-one (FMO) controls defined positive boundaries. Reduced Siglec-F intensity in AMs following HDM challenge illustrates expected down-regulation during inflammation, confirming accurate gate adjustment across samples. (B) Representative gating of B cells and plasmacytoid dendritic cells (pDCs). Plots show gating of CD45R/B220⁺ B cells and CD11c⁺ B220⁺ MHC II⁺ pDCs from individual PBS- and HDM-treated FCG mice. FMO controls defined positive boundaries, and consistent gating ensured accurate quantification of these subsets across all samples. Supplementary Figure 4A-B. Representative gating of CD4⁺/CD8⁺ T-cells and NK/NKT cell subsets. Plots from individual PBS- and HDM-treated FCG mice show gating for T-cell (A) and NK/NKT (B) populations. FMO controls defined positive gates. HDM exposure increased CD4⁺ and CD8⁺ T-cell frequencies, while NK and NKT cell levels remained stable across treatments. Supplementary Figure 5A-B. Representative gating of interstitial macrophages and dendritic-cell subsets. Flow-cytometry plots from individual PBS- and HDM-treated FCG mice show gating of interstitial macrophages (CD64⁺ MHC II⁺) (A), CD11b⁺ DCs (A), and CD103⁺ DCs (B). FMO controls defined positive boundaries, and consistent gating ensured accurate quantification across samples.Supplementary Figure 6A-B. Representative gating of monocytes and neutrophils. Plots show gating of undifferentiated monocytes (CCR2⁺ Ly6C⁺) (A) and neutrophils (CCR2⁻ Ly6G⁺) (B) from PBS- and HDM-treated Four-Core-Genotype mice. FMO controls defined positive boundaries. HDM challenge produced minimal changes in these populations, confirming consistent gating across samples.Supplementary Figure 7A-D: Airway hyperresponsiveness in the FCG mice following 5 weeks HDM or PBS exposure. Assessing the influence of sex chromosome and gonadal sex in airway hyperresponsiveness using the lung parameter, Resistance (Rrs) in the FCG mice after Methacholine (doses-3.13, 12.5, 25, 50,100mg/mL) challenge following exposure to PBS or HDM in mice with XYM (A), XYF (B), XXF (C) or XXM (D) genotypes. Data represented as mean ± SD of at least 4-10 mice per treatment group. Linear mixed-effects models with random intercepts for individual animals were used to analyze repeated MCh doses. Independent and interaction effects of treatment, dose, and biological sex variables were tested; pairwise EMMs were adjusted by Tukey’s method. *p