Novel humanized mouse model of mucosal immunity

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PROJECT SUMMARY Although humanized mice have been successfully used for in vivo studies of HIV infection, cancer and immunotherapies, human naïve and adaptive immune responses remain suboptimal, with limited cross-reactivity between murine and human cytokines considered as a key contributing factor. As a result, the human mucosal immune system is not fully developed, and several components are missing, including the presence of human lung epithelial cells in their natural lung environment. To address these research gaps, we propose a combined genetic and cellular editing approach to develop the next generation of humanized mice for studies of human mucosal immunity. Our approach is structured in two aims: in Aim 1, we will construct and credential hNSGF- SGM3-IL6-TSLP-TSLPR mice for human immune cell development. We will generate mice expressing a complete human TSLP receptor and examine engraftment with human hematopoietic progenitor cells (HPCs) by measuring cellular composition of immune cells in the bone marrow, spleen, gut, airways, lungs, and blood. To examine human immune function, we will use a mucosal formulation of influenza vaccine (Flumist) as an immune trigger and antigen source as we have done in the past and analyze cytokine responses in the blood and spleen; human cell migration to mucosal sites, spleen, and bone marrow; and induction of vaccine antigen- specific T and B cells in the spleen. In Aim 2, we will construct and credential humanized airway epithelium in hNSGF-SGM3-IL6-TSLP-TSLPR mice. We will examine the development of human airway epithelium upon transplant of human bronchial epithelial progenitor cells in NSGF-SGM3-IL6-TSLP-TSLPR mice (humanized or not with HPCs from the same donor) by tissue immunofluorescence staining of human-specific targets including HLA class I. To establish the functionality of human airway epithelial cells, we will challenge mice with live influenza virus and measure the induction of species-specific alarmins including IL-33; type I interferon signature in human epithelial cells as a measure of their functionality in vivo, as well as tissue composition and attraction of human immune cells to human airway epithelium. Our hypothesis is that a complete TSLP signaling pathway and human epithelial cells will improve human mucosal immunity in humanized mice by facilitating the crosstalk between stromal cells and immune cells. This model can then be used for i. mucosal vaccine development, and ii. studies of other respiratory viruses including SARS-CoV-2, which requires human-specific receptors to establish infection. Thus, once credentialed, our model will lay the groundwork for future mechanistic studies of mucosal immunity in vivo in the context of genetically variable donors as well as enable studies of mucosal adjuvants, allergens, vaccines, and biologicals.