Integration of phospho-signaling and transcriptomics in single cells reveals distinct Th17 cell fates
Single-cell RNA sequencing (scRNA-seq) provides the resolution and scale necessary to identify transcriptional programs but fails to capture post-transcriptional information critical to decipher signaling networks and cellular states. We present Vivo-seq, an innovative platform that integrates scRNA-seq and intracellular cellular indexing of transcriptomes and epitopes by sequencing following cellular fixation with a deep eutectic solvent, which preserves multiple domains of biological information beyond RNA transcripts alone. Vivo-seq enables simultaneous capture of both transcriptional and phospho-signaling states in single cells. Applying this platform to developing T helper 17 (Th17) cells, we find that simultaneous phosphorylation of ERK1/2 and c-FOS leads to maximal interleukin-2 (IL-2) and IL-17 production. Furthermore, we show that early IL-2 production imprints developing Th17 cells for enhanced maintenance or cytokine-dependent transdifferentiation during subsequent antigenic stimulation. By integrating transcriptional and phospho-signaling information at single-cell resolution, we identify a hyperactivated Th17 cellular state associated with early IL-2 production that has downstream consequences on functional plasticity.
Authors
- P. Brent Ferrell (ORCID: https://orcid.org/0000-0003-1140-9154)
- Blake F. Frey (ORCID: https://orcid.org/0000-0003-3003-6904)
- Asif Elahi
- Robert S. Welner (ORCID: https://orcid.org/0000-0003-2498-9469)
- Casey Todd Weaver (ORCID: https://orcid.org/0000-0002-2180-1793)
- Vidya Sagar Hanumanthu (ORCID: https://orcid.org/0000-0001-5744-1446)
- Andrew S. Goldsborough
- Seth D. Fortmann (ORCID: https://orcid.org/0000-0002-9554-8751)
- Maria B. Grant (ORCID: https://orcid.org/0000-0002-6470-0255)
- Awalpreet S. Chadha (ORCID: https://orcid.org/0000-0002-8072-2243)
- Shanrun Liu
Institutions
- University of Alabama at Birmingham Hospital (US)
- University of Alabama at Birmingham (US)
- Sylvester Comprehensive Cancer Center (US)
- O'Neal Comprehensive Cancer Center
- Vanderbilt University Medical Center (US)
- Core Laboratories (United States) (US)
Publication Details
- Journal
- Cell Reports
- Published
- 2025-07-16
- DOI
- https://doi.org/10.1016/j.celrep.2025.116006
- Primary Topic
- T-cell and B-cell Immunology
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- Division of Intramural Research, National Institute of Allergy and Infectious Diseases
- Mark Foundation For Cancer Research
- National Institutes of Health
- Center for AIDS Research, University of Washington
- National Heart, Lung, and Blood Institute
- National Eye Institute
- National Cancer Institute
- National Institute of Allergy and Infectious Diseases
- National Institute of Diabetes and Digestive and Kidney Diseases
- NHLBI Division of Intramural Research