Reproducible single-cell annotation of programs underlying T cell subsets, activation states and functions
T cells recognize antigens and induce specialized gene expression programs (GEPs), enabling functions like proliferation, cytotoxicity and cytokine production. Traditionally, different T cell classes are thought to exhibit mutually exclusive responses, including T<sub>H</sub>1, T<sub>H</sub>2 and T<sub>H</sub>17 programs. However, single-cell RNA sequencing has revealed a continuum of T cell states without clearly distinct subsets, necessitating new analytical frameworks. Here, we introduce T-CellAnnoTator (TCAT), a pipeline that improves T cell characterization by simultaneously quantifying predefined GEPs capturing activation states and cellular subsets. Analyzing 1,700,000 T cells from 700 individuals spanning 38 tissues and five disease contexts, we identify 46 reproducible GEPs reflecting core T cell functions including proliferation, cytotoxicity, exhaustion and effector states. We experimentally demonstrate new activation programs and apply TCAT to characterize activation GEPs that predict immune checkpoint inhibitor response across multiple tumor types. Our software package starCAT generalizes this framework, enabling reproducible annotation in other cell types and tissues.
Authors
- Kamil Slowikowski (ORCID: https://orcid.org/0000-0002-2843-6370)
- Yuriy Baglaenko (ORCID: https://orcid.org/0000-0002-9020-6824)
- Pardis C. Sabeti (ORCID: https://orcid.org/0000-0002-9843-1890)
- Michelle L. Curtis (ORCID: https://orcid.org/0000-0002-6843-4839)
- Yu Zhao (ORCID: https://orcid.org/0000-0003-2632-7530)
- Kathryn Weinand (ORCID: https://orcid.org/0000-0001-8491-7251)
- Aparna Nathan (ORCID: https://orcid.org/0000-0002-5975-2851)
- Soumya Raychaudhuri (ORCID: https://orcid.org/0000-0002-1901-8265)
- Dylan Kotliar (ORCID: https://orcid.org/0000-0002-7968-645X)
- Deepak A. Rao (ORCID: https://orcid.org/0000-0001-9672-7746)
- Ryan Agnew
Institutions
- Broad Institute (US)
- Brigham and Women's Hospital (US)
- Cincinnati Children's Hospital Medical Center (US)
- Howard Hughes Medical Institute (US)
- Harvard University (US)
- Massachusetts General Hospital (US)
- Harvard–MIT Division of Health Sciences and Technology (US)
- Center for Systems Biology (US)
- University of Cincinnati Medical Center (US)
Publication Details
- Journal
- Nature Methods
- Published
- 2025-09-01
- DOI
- https://doi.org/10.1038/s41592-025-02793-1
- Citations
- 9
- Primary Topic
- CAR-T cell therapy research
- Type
- article
- Field-Weighted Citation Impact
- 9.41
Funders
- Howard Hughes Medical Institute
- National Human Genome Research Institute
- National Institute of Allergy and Infectious Diseases
- National Institute of Arthritis and Musculoskeletal and Skin Diseases