CRISPR activation and interference screens decode stimulation responses in primary human T cells

Area of Science:

• Immunology
• Molecular Biology
• Genetics

Background:

• Cytokine production regulation in T cells is crucial for immune responses.
• Dysregulation of T cell cytokine production is implicated in autoimmunity, immunodeficiency, and cancer.
• Discovering cytokine regulators in primary human T cells is challenging.

Purpose of the Study:

• To systematically identify gene networks controlling interleukin-2 (IL-2) and interferon-gamma (IFN-γ) production in primary human T cells.
• To develop and apply genome-wide CRISPR activation (CRISPRa) and interference (CRISPRi) screening methods in primary human T cells.
• To reveal how genetic perturbations influence T cell activation and cytokine profiles.

Main Methods:

• Genome-wide CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) screens were performed in primary human T cells.
• Arrayed CRISPRa screens were used for hit confirmation and multiplexed secretome characterization.
• Single-cell RNA sequencing was coupled with CRISPR screening for deep molecular profiling of identified genes.

Main Results:

• CRISPRa and CRISPRi screens successfully identified gene networks regulating IL-2 and IFN-γ production.
• Arrayed CRISPRa confirmed key regulators and revealed altered cytokine secretion profiles.
• Single-cell RNA sequencing elucidated molecular mechanisms by which perturbations modulate T cell states and cytokine expression.

Conclusions:

• Genome-wide CRISPR screens in primary human T cells are effective for discovering cytokine regulators.
• These identified genes can reprogram T cell functions and cytokine production.
• The findings provide insights for designing novel immunotherapies targeting T cell responses.