Inflammatory bowel diseases (IBD) cause substantial mortality and morbidity. Current treatments that block
pathological inflammatory responses have improved clinical outcomes in some patients but they are not highly
effective in modifying disease progression or preventing relapses. Hence, there is a large unmet need to develop
novel therapeutic targets. Genome-wide association studies (GWAS) offer an unbiased approach to identify
therapeutic targets in relevant immune cell types such as CD4+ T cells that play key roles in IBD pathogenesis.
Because T cells are quiescent in the absence of extrinsic stimulation, it is not possible to fully examine the effects
of disease-risk variants on functionally relevant effector genes under resting conditions. To identify IBD-risk
genes in activated CD4+ T cells, we performed the first large-scale single-cell eQTL study on activated CD4+ T
cells. We found that reduced expression of Leukocyte-specific protein 1 (LSP1), specifically in activated CD4+
T-cell subsets such as TH1 and TH17 cells, was associated with the risk of IBD. In this R01 proposal, we will
investigate how reduced levels of LSP1 influences the differentiation and function of CD4+ T cells to drive disease
pathogenesis, and will test the hypothesis that LSP1 plays a key role in restraining the re-programming of CD4+
T cells into a more pathogenic cell state in IBD patients.
In Aim 1, we will determine the functional IBD-risk variants that reduce LSP1 expression in CD4+ T cells. We will
employ luciferase reporter assays to determine functional variants in the enhancers and promoter of LSP1,
perform CRISPR-mediated editing of prioritized IBD-risk eQTLs to define causal variants, and perform CRISPRi
and ChIP assays to determine functional enhancers, relevant up-stream regulators and whether the functional
LSP1 eQTLs directly perturb the binding of key transcription factors that modulate LSP1 expression.
In Aim 2, we will determine the role of LSP1 in reprogramming of CD4+ T cells into the pathogenic state observed
in IBD. To determine whether LSP1 influences the differentiation and pathogenic function of CD4+ T cells from
healthy and IBD donors, we will reduce and increase LSP1 levels and assess the effects on CD4+ T-cell
activation, apoptosis, proliferation and proinflammatory cytokine production. In an adoptive T cell transfer model
of colitis, we will compare the ability of Lsp1- sufficient (wild-type) and Lsp1-deficient CD4+ T cells in driving
colonic inflammation and pathogenic TH1 and TH17 differentiation. We will determine whether reducing Lsp1
expression in CD4+ T cells enhances their pathogenicity in mouse models of colitis, thus implicating an important
T cell-intrinsic role for LSP1 in IBD pathogenesis.
Overall, this study, examining the function, expression, activation and regulation of LSP1 in CD4+ T cells, will
provide important mechanistic insights into the genetic basis of risk for inflammatory bowel disease.