Project 2 explores the paradigm-shifting hypothesis that the epithelium is at the heart of two fundamentally different molecular phenotypes of severe asthma (SA). In the 1st, bronchial epithelial cells (BEC)s respond to initial environmental stimuli to drive an innate intrinsic phenotype. In the 2nd, BEC cell death pathways intersect with CD8 T-cell immune processes to drive an immune interactive phenotype. This hypothesis is based on published, submitted and preliminary data across Project 1 and Project 2 which identify two immune cell phenotypes of severe asthma, one independent of T-cells and another, highly associated with them, in particular with CD8 cells. Similar levels of Type-2 (T2) biomarkers/genes are present in both. In published data from the previous cycle, epithelial growth and repair modules more strongly associated with asthma severity than T2 modules, suggesting T2 immunity alone does not drive severity. Consistent with gene expression, poor wound repair of asthmatic BECs in vitro was reported, with associated loss of a proliferative capacity. The tethered mucin MUC4b, increased in asthma, lowers BEC proliferation, while increasing secretion. BEC gene clustering mirrors Project 1 with T-cell- and non-T-cell associated SA phenotypes, each with similar levels of T2 biomarkers. The innate intrinsic/Non-T-cell SA cluster associates with innate NF-kB and apoptotic pathways potentially through Toll like receptors (TLR)s. The immune interactive/CD8 cluster associates with IL-12 and -18, interferons and upregulation of inflammasome and pyroptosis pathways through the 17q12-21 gene, GSDMB. Thus, interactions between BEC genetic/epigenetic risks, innate and adaptive immunity and cell death pathways may contribute to these distinct phenotypes, with differing treatment responses. Using cutting edge technologies of scRNAseq, advanced bioinformatics and mechanistic in vitro and in vivo models, we will refine the molecular phenotypes identified in the 1st cycle and link them with cell profiles identified in Project 1. We propose 3 aims: 1) Identify the mechanisms for and functional implications of an innate intrinsic epithelial phenotype using fresh and cultured healthy and asthmatic BECs 2) Evaluate the role of GSDMB in the development of a IL-18/CD8-associated immune interactive phenotype and its functional implications using fresh and cultured healthy and asthmatic BECs 3) Integrate immune-inflammatory phenotypes with epithelial molecular phenotypes, as the penultimate effort to link cell types, mechanisms and compartments to rigorously refine phenotypes. These studies will improve the understanding of molecular phenotypes in relation to cell death, allowing Identification of novel precision therapeutic targets to eventually confirm underlying endotypes.