Single-cell gain-of-function mapping reveals latent regulatory programs governing CD8⁺ T cell fate

Area of Science:

• Immunology
• Cell Biology
• Genetics

Background:

• CD8⁺ T cells are crucial for host defense and tumor immunity, exhibiting diverse differentiation states.
• Understanding regulatory programs is key, but loss-of-function studies miss emergent activities from ectopic transcription factor expression.
• Novel methods are needed to explore transcription factor functions outside their native contexts in T cells.

Purpose of the Study:

• To develop and apply a single-cell gain-of-function (scGOF) sequencing platform for in vivo mapping of transcription factor overexpression in CD8⁺ T cells.
• To uncover novel regulatory activities of transcription factors, including those normally silenced or temporally restricted, within T cell differentiation.
• To engineer T cell states for improved immune responses in infection and cancer models.

Main Methods:

• Developed single-cell gain-of-function (scGOF) sequencing (scGOF-seq), a multiplexed platform for in vivo analysis.
• Enforced expression of canonical, lineage-silenced, and temporally restricted transcription factors in antigen-specific CD8⁺ T cells.
• Applied scGOF-seq in immunocompetent models of infection and cancer.

Main Results:

• Ectopic expression of developmental regulators (NANOG, SOX2, OCT4, GATA2) reshaped T cell differentiation; NANOG promoted stemness and accumulation during chronic infection.
• Sustained cMyc overexpression induced a stem-like, effector-featured CD8⁺ T cell state with enhanced metabolism, reduced exhaustion, and >5,000-fold antigen-dependent expansion.
• scGOF-seq identified cooperating transcription factor modules that enhanced cMyc-driven programs and improved anti-tumor responses in solid tumors.

Conclusions:

• Systematic gain-of-function perturbation via scGOF-seq is a powerful framework for discovering latent and temporally constrained regulatory activities in CD8⁺ T cells.
• NANOG and cMyc possess significant potential for reprogramming T cell function, offering new avenues for immune-state engineering.
• This approach can guide the development of enhanced T cell therapies for infectious diseases and cancer.