Genetic variants guiding pathogenicity of colitogenic T cells

SUMMARY ~2.4 million people in the United States have inflammatory bowel diseases (IBDs). While these severe and debilitating diseases have a genetic component, as determined from concordance studies in monozygotic twins, we still know very little about how genetics drives risk and progression for each disease. Genome-wide association studies have identified hundreds of IBD-associated regions of the genome, but there is often tight linkage disequilibrium between causal and non-causal variants in these regions, and most disease-associated genetic variants are in non-coding regions. Thus, determining the variants that promote disease and their effects on disease-relevant cell types is challenging. One cell type that is implicated in IBD is the pathogenic Th17 cell. Th17 cells normally maintain homeostasis of the colonic lamina propria, but in the context of IBD, pathogenic Th17 cells cause unwanted inflammation. Non-coding genetic variants associated with IBD are highly enriched within the accessible chromatin of pathogenic Th17 cells compared to other T cell types. Thus, we hypothesize that disease-causal variants alter cis-regulatory element (CRE) activity in pathogenic Th17 cells that may affect their differentiation and function, making them more pathogenic. We recently discovered that we can identify likely causal variants through testing their allele-specific effects on regulatory region activity in massively parallel reporter assays (MPRAs). Our MPRAs in T and B cell lines and primary T cells identify putative causal variants that are highly cell type and state dependent. Thus, we will perform MPRAs on IBD GWAS variants, identified in East Asian and European individuals, using human pathogenic Th17 cells to discover putatively causal IBD variants across the genome. We will then connect variants to their effects on pathogenic Th17 cell migration and function using CRISPR-interference (CRISPRi) screens in primary T cells. Next, we will use a single-cell screening approach in pathogenic Th17 cells to identify the genes that variant CREs control. We will then determine gene regulatory networks that variant CREs act on to begin to define important disease pathways. In concert with our studies in human T cells, we will use in vivo CRISPRi screens to test conserved variant CRE effects on migration and function of pathogenic Th17 cells in a murine model of IBD, thus exploring additional physiological avenues of variant effects in the in vivo setting. If successful, this work will take the first leap in directly linking hundreds of IBD risk loci to the function of a disease-relevant cell type and it will provide insight into how genetic risk promotes disease. Our findings may therefore identify therapeutically targetable pathways for treatment of IBD.