Regulatory mechanisms governing Th17 cell effector identity and plasticity

Duke University, Durham NC

Investigators

Abstract

PROJECT SUMMARY T helper (Th) cells are CD4-expressing T lymphocytes that specialize into several subclasses to support distinct immune responses. Among this diversity, IL-17A-producing inflammatory Th17 cells stand out as unique by virtue of their high level of inherent plasticity. Indeed, this subset that normally functions in mucosal immunity against bacteria and fungi can easily adopt features of other T helper subsets when environmental conditions change. While this feature can be advantageous towards clearance of infections, dysregulated Th17 cell function has been implicated in numerous autoimmune conditions, including inflammatory bowel disease, and multiple sclerosis. Moreover, Th17 cell plasticity exhibited in the context of inflammatory disease takes on Th1-like traits, such as expression of IFN and T-bet, that drive increased pathology. In previous work, we identified AP-1 factor JunB as a critical regulator of Th17 cell identity that both activates the Th17 cell program while restraining inappropriate plasticity to the Th1 lineage fate. In our current funded studies, we extended the scope of JunB function to a related type 3 lineage—IL-17A-producing  T (T17) cells—where it similarly limits type 1 effector conversion in the context of infection-induced intestinal inflammation. The conservation of JunB function among type 3 lineage T cells underscores the central importance of the AP-1 regulator as a gatekeeper of type 1 plasticity. While much is known about the signals and transcription factors that drive Th17 cell differentiation, the mechanisms controlling Th17 cell flexibility are less understood. We hypothesize that reversible silencing of alternative effector programs governs the Th17 cell poised state and facilitates type 1 effector plasticity in response to environmental triggers. Here, we exploit the function of JunB as a transcriptional repressor of alternative fates to probe the cis and trans silencing regulatory network that governs Th17 cell type 1 effector conversion. To this end, in Aim 1, we will define the silencer regulatory network governing Th17 cell effector flexibility. For this, we will use a novel in vivo high throughput reporter assay for large-scale identification of silencer cis elements with regulatory roles in Th17 cell plasticity induced by intestinal Citrobacter rodentium infection. We will validate candidates using CRISPR/Cas9 deletion in vitro and in mice, including a novel JunB-dependent silencer Ifng CNS+18 that limits IFN expression in Th17 cells. In Aim 2, we will apply advanced proteomics approaches to characterize activating and repressive JunB protein complexes in Th17 cells. For this, we will use JunB TurboID proximity-labeling and affinity purification of total and chromatin-associated complexes in combination with CRISPR/Cas9 screening to identify and validate novel coregulators of Th17 cell plasticity. Taken together, the proposed work will fill an important gap in knowledge concerning the cis and trans molecular mechanisms governing Th17 cell identity and plasticity.