PROJECT SUMMARY TECH DEV PROJECT The goal of the Technology Development (Tech Dev) Project is to develop approaches and tools that address the needs of The Jackson Laboratory-Icahn School of Medicine at Mt. Sinai Cooperative Center on Human Immunology (JAX-ISMMS CCHI) and that also advance the capabilities of the broader scientific community to tackle fundamental mechanistic questions regarding human lung immunity and human immune-epithelial cell interactions. Major questions related to lung immune function remain unanswered—such as the cell-to-cell interactions between immune and lung epithelial cells that shape responses to foreign agents, and how age- dependent changes in splicing and epigenetic modifications impact tissue immunity and inflammation. A significant technical barrier to studying human immune-lung dynamics is the sheer complexity of the human lung and the dynamic interactions between the lung epithelium and resident immune cells such as macrophages, dendritic cells, and T cells. This complexity cannot be easily modeled in animal systems or using deceased human lung tissue. To surmount these challenges, the Tech Dev Project will focus on three aspects to further increase complexity and utility of an innovative human tissue platform which we have pioneered in our current Center: establish three-dimensional (3D) bioprinted ventilated and vascularized models of the lung and upper respiratory environment for investigating the functional lung-immune interactome during exposure to viral agents (Aim 1); develop improved methods for viral infection and co-culture of immune cells in lung models (Aim 2); and develop methods for, and perform, editing of airway epithelial cells, virus, and commensal bacteria for inclusion in lung models (Aim 3). Epithelial editing will entail development of tools to alter isoform usage (for Project 1), epigenetic modifications (for Project 2), and generating conditional alleles in primary human airway epithelial cells and induced-pluripotent stem cells (iPSCs). For viral editing, we will engineer various reporter influenza viruses to enable tracking of active versus history of infection. For bacterial editing, we will alter production of candidate immune modulating metabolites from commensal bacteria (for Project 2) by perturbing their associated metabolic pathways either by using our CRISPRi toolkit or via overexpression. Each of these Aims addresses a specific unmet need for the Center and will enable us to study human lung immunity within a dynamic and physiologically relevant microenvironment and to interrogate specific cell types and molecular pathways predicted to respond to viral infections. Through these efforts, the JAX-ISMMS CCHI will be equipped to address previously inaccessible questions related to lung-immune dynamics, towards a more mechanistic understanding of lung immune function.