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Integrating Metabolism and Immunity | EK16


An in vivo forward genetic screen in primary T cells to identify lipid immunometabolic regulatory genes in airway inflammation


Jan 25, 2021 12:00am ‐ Jan 25, 2021 12:00am

Description

An in vivo forward genetic screen in primary T cells to identify lipid immunometabolic regulatory genes in airway inflammation. Arissa C Young (1), Ayaka Sugiura (2), Andrew R. Patterson (2), Jeffrey C. Rathmell (2,3) (1) Department of Medicine, (2) Pathology, Microbiology, and Immunology, (3) Vanderbilt Center for Immunobiology, Vanderbilt University Medical Center, Nashville, TN 37232 Many autoimmune and inflammatory diseases can be driven by an imbalance between T helper cells (Th cells) and T regulatory cells (Treg). Different subsets of Th cells utilize distinct metabolic programs and manipulation of metabolic pathways can affect Th cell differentiation. In this study, we developed a custom CRISPR library to knock out genes involved in lipid metabolism to investigate how this metabolic pathway affects survival and proliferation of murine Th cells and to identify potential immunometabolic mechanisms of allergic airway disease. Genes involved in lipid metabolism or known to regulate these pathways were selected and a custom retroviral CRISPR library was generated. Ten non-targeted controls and two positive controls (Rheb and Tsc2) were also included. CD4+ T cells were isolated from OT-II Cas9 transgenic mice and activated with ovalbumin peptide. After two days CD4+ T cells were transduced with pooled retrovirus at a M.O.I. < 0.3 to ensure each cell had only one gene targeted. Transduced cells were collected on day 3 from each biologic replicate to serve as a baseline for comparison. The remaining transduced CD4+ T cells were adoptively transferred into Rag -/- recipient mice, which were subsequently sensitized and challenged with intranasal ovalbumin on days 4, 6, 8 and 10. DNA was collected from CD4+ T cells isolated from the lungs on the following day (day 11). Guide frequencies at baseline (day 3) and in the inflammatory lung T cell population (day 11) were determined by sequencing and the Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK) method was used to analyze the sequencing results. Guides that were significantly enriched in the inflammatory lung T cell population compared to baseline were genes that, when knocked out, conferred a selective advantage, whereas guides that were significantly depleted were genes essential for survival and proliferation. The most significantly depleted genes across all biologic replicates were Acetyl-CoA carboxylase 1 (Acaca), Stearoyl CoA desaturase 1 (Scd1) and Mitochondrial trans-2-enoyl-coenzyme A reductase (Mecr), demonstrating a critical role for these genes in T cell-mediated lung inflammation. In future studies, we plan to explore the effect of these genes, and Mecr in particular, on Th cell differentiation and function in depth through single knock out CRISPR experiments in both in vivo and in vitro systems. Email: arissa.c.young.1@vumc.org

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