Dissecting the genetic networks that control regulatory T cell stability using pooled CRISPR screens

Abstract

Abstract Regulatory T cells (Tregs) are a specialized subset of CD4+ T cells that suppress inflammation to maintain homeostasis and prevent autoimmunity. Treg development and function depend on expression of the master transcription factor Foxp3. While Tregs have been thought to be irreversibly committed to suppressive functions, lineage tracing studies have challenged this by revealing that Tregs can exhibit plasticity. Tregs that lose Foxp3 expression, termed ‘exTregs’, have been shown to acquire cytokine production capabilities of pro-inflammatory effector T cells and exacerbate autoimmunity. However, the gene regulatory programs that promote or disrupt Foxp3 stability in Tregs under various physiological conditions are not well understood. Here we have leveraged improved functional genetic tools, including pooled CRISPR screens, to identify nuclear factors that regulate Treg plasticity. A Foxp3 lineage-tracing reporter mouse model was used to confidently distinguish Foxp3− exTregs from contaminating Foxp3−T effectors. Using HiChIP, we identified Treg and exTreg-specific chromatin loops and systematically assessed their contribution to Foxp3 stability. Furthermore, we investigated transcriptional signatures of distinct exTreg populations residing in the peripheral organs using single-cell RNA sequencing. Collectively, this work helps us better understand the genetic networks that control stable expression of Foxp3 in Tregs as they encounter changing inflammatory environments.