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Genome-scale perturb-seq in primary human CD4+ T cells reveals genes regulating T cell programs and human immune traits.

ABSTRACT: CD4+ T cells orchestrate immune responses against infection and cancer, yet a comprehensive map of their gene regulatory networks has been lacking. Perturb-seq enables systematic mapping of such networks, but large-scale application in primary human T cells has been challenging. Here, we developed a scalable, probe-based Perturb-seq platform and performed genome-scale perturbations of all expressed genes across 22 million primary CD4+ T cells from four donors under three stimulation conditions. We demonstrate the utility of this dataset in three ways. First, we identify previously unrecognized regulators of the two important immune cytokines, IL-10 and IL-21. Second, we show that ex vivo perturbation signatures can model T cell states observed in population-scale atlases, revealing regulators of helper T cell polarization and T cell aging. Third, we leverage regulatory relationships to mechanistically explain gene–trait associations in immune-related diseases, nominating genes for uncharacterized immune functions. Thus genome-scale perturb-seq in primary human T cells provides a foundational resource for decoding immune variation in humans and reprogramming T cell states for next-generation precision therapies.

ORGANISM(S): Homo sapiens

PROVIDER: GSE314342 | GEO | 2026/02/06

REPOSITORIES: GEO

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Project description: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 gene regulators coordinate circuits that respond to extracellular cues 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. Here, 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 specific 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. MED12 interacts with the histone modifying complex COMPASS, affecting chromatin state and expression of genes encoding key context- and lineage-specific regulators; including rest maintenance factor KLF2 and activation promoting genes. We demonstrated that CRISPR ablation of MED12 blunted this cell state transition and protected T cells from activation-induced cell death. Overall, this work leverages CRISPR screens performed across primary cell conditions to identify context-specific regulators and dynamic gene circuits required to establish resting and activated T cell states.

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Genome-scale perturb-seq in primary human CD4+ T cells reveals genes regulating T cell programs and human immune traits.

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