Abstract PR008: CD4+ T cell rest and activation is enabled by centralized control of state specific regulatory genes

Abstract The ability of individual cells to maintain a distinct identity and respond to transient environmental signals requires tightly controlled regulation of gene networks. However, how discrete sets of regulators coordinate dynamic gene circuits remains poorly defined. The need for context-dependent regulation is prominent in human CD4+ T cells, where distinct cell lineages must respond to diverse signals to orchestrate effective adaptive immune responses and maintain homeostasis. We performed CRISPR screens in multiple primary human CD4+ T cell contexts to identify regulators that control expression of IL2RA, which is a canonical marker of T cell activation in pro-inflammatory effector T cells (Teffs) and constitutively expressed in anti-inflammatory regulatory T cells (Tregs) where it is required for fitness. Strikingly, the majority of identified regulators are required in discrete cell type and stimulation timepoints, and a subset even had opposite functional effects in different conditions. Using single-cell transcriptomics after pooled perturbation of context-specific screen hits, we characterized factors as regulators of overall rest or activation and constructed state-specific regulatory networks. Upstream of these networks, MED12 – a component of the Mediator complex – serves as a dynamic orchestrator of regulators across conditions, governing both cell type- and stimulation-specific gene expression. We determined that MED12 interacts with histone modifying proteins, affecting chromatin state and expression of genes encoding key state- and lineage-defining factors, including numerous regulators of IL2RA. Importantly, MED12 promotes the expression of several core genes including MYC, rest maintenance factor KLF2, and activation promoting gene GATA3. CRISPR ablation of MED12 blunted the transition from rest to activation and protected T cells from activation-induced cell death, resulting in increased durability of conventional effector T cells. Overall, CRISPR screens performed across primary cell conditions enabled the identification of regulatory circuits required to establish T cell rest and activation, which can be modulated to improve cellular persistence. Citation Format: Maya M Arce, Jennifer Umhoefer, Nadia Arang, Sivakanthan Kasinathan, Jacob W Freimer, Zachary Steinhart, Haolin Shen, Mineto Ota, Anika Wadhera, Minh T.N Pham, Rama Dajani, Dmytro Dorovsky, Yan Yi Chen, Qi Liu, Brian R Shy, Julia Carnevale, Ansuman T Satpathy, Nevan J Krogan, Jonathan K Pritchard, Alexander Marson. CD4+ T cell rest and activation is enabled by centralized control of state specific regulatory genes [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr PR008.