Enhancer-driven gene regulatory networks reveal transcription factors governing T cell adaptation and differentiation in the tumor microenvironment

William D Green¹, Amber Gomez², Alec L Plotkin³, Brandon M Pratt⁴, Emily F Merritt⁵, Genevieve N Mullins⁵, Nancy P Kren⁶, Jennifer L Modliszewski⁷, Vasyl Zhabotynsky⁷, Mark G Woodcock⁸, Jarred M Green⁷, Gabrielle Cannon⁹, Matthew E Pipkin¹⁰, Gianpietro Dotti⁵, Jessica E Thaxton², Yuliya Pylayeva-Gupta⁶, Albert S Baldwin¹¹, John P Morris 4th⁴, Natalie Stanley¹², J Justin Milner¹³

Affiliations

¹ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Center for Nanotechnology in Drug Delivery, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
² Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
³ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Computational Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27559, USA.
⁴ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
⁵ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
⁶ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27559, USA.
⁷ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
⁸ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
⁹ Advanced Analytics Core, Center for Gastrointestinal Biology and Disease, University of North Carolina at Chapel Hill, Chapel Hill, NC 27559, USA.
¹⁰ Department of Immunology and Microbiology, UF Scripps Biomedical Research, University of Florida, Jupiter, FL 33458, USA.
¹¹ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
¹² Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Computational Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Computer Science, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
¹³ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address: justin_milner@med.unc.edu.

PMID: 40425012
PMCID: PMC12240711 (available on 2026-05-26)
DOI: 10.1016/j.immuni.2025.04.030

Enhancer-driven gene regulatory networks reveal transcription factors governing T cell adaptation and differentiation in the tumor microenvironment

William D Green et al. Immunity. 2025.

. 2025 Jul 8;58(7):1725-1741.e9.

doi: 10.1016/j.immuni.2025.04.030. Epub 2025 May 26.

Authors

Affiliations

¹ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Center for Nanotechnology in Drug Delivery, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
² Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
³ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Computational Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27559, USA.
⁴ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
⁵ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
⁶ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27559, USA.
⁷ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
⁸ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
⁹ Advanced Analytics Core, Center for Gastrointestinal Biology and Disease, University of North Carolina at Chapel Hill, Chapel Hill, NC 27559, USA.
¹⁰ Department of Immunology and Microbiology, UF Scripps Biomedical Research, University of Florida, Jupiter, FL 33458, USA.
¹¹ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
¹² Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Computational Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Computer Science, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
¹³ Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address: justin_milner@med.unc.edu.

PMID: 40425012
PMCID: PMC12240711 (available on 2026-05-26)
DOI: 10.1016/j.immuni.2025.04.030

Abstract

Tumor-infiltrating lymphocytes (TILs) with a tissue-resident memory CD8⁺ T cell (Trm) phenotype are associated with improved patient outcomes in solid malignancies. To define programs governing the formation of Trm-like TIL, we performed paired single-cell RNA sequencing and single-cell ATAC sequencing of T cell receptor (TCR)-matched CD8⁺ T cells in models of infection and cancer. Enhancer-driven regulons assembled from multiomic profiling data revealed epigenetic and transcriptional programs regulating the formation of Trm-like TIL in relation to canonical exhausted and memory T cell states. The transcriptional regulator KLF2 repressed the formation of CD69⁺CD103⁺ Trm-like TIL and limited anti-tumor activity. Conversely, sustained expression of the transcription factor BATF enhanced formation of CD69⁺CD103⁺ TIL, contingent upon downregulation of KLF2. Transforming growth factor β (TGF-β) signaling and CD103 expression were necessary for Trm-like TIL formation, but BATF overexpression was sufficient to drive formation of CD69⁺CD103⁺ TIL in TGFBR2-silenced cells. These findings reveal mechanisms of Trm-like TIL differentiation and provide a framework for considering tissue residency in the context of CD8⁺ T cell heterogeneity in the tumor microenvironment.

Keywords: CD8(+) T cells; exhaustion; immune memory; infection; pancreatic cancer; single-cell multiomics; tissue-resident cells; tumor immunology; tumor-infiltrating lymphocytes.

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Conflict of interest statement

Declaration of interests G.D. is a paid consultant for Tessa Therapeutics, Catamaran, and Bellicum Pharmaceuticals and reports receiving commercial research grants from Cell Medica and Bluebird Bio.

References

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Enhancer-driven gene regulatory networks reveal transcription factors governing T cell adaptation and differentiation in the tumor microenvironment

Affiliations

Enhancer-driven gene regulatory networks reveal transcription factors governing T cell adaptation and differentiation in the tumor microenvironment

Authors

Affiliations

Abstract

Conflict of interest statement

References

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full Text Sources

Medical

Molecular Biology Databases

Research Materials