# Integration of phospho-signaling and transcriptomics in single cells reveals distinct Th17 cell fates

## TL;DR

This paper introduces a new method called Vivo-seq that captures both gene activity and signaling in single cells, revealing how Th17 immune cells develop and change.

## Contribution

Vivo-seq is a novel platform that simultaneously captures transcriptional and phospho-signaling data in single cells.

## Key findings

- Dual phosphorylation of ERK1/2 and c-FOS is required for maximum IL-2 and IL-17 production in Th17 cells.
- Early IL-2 production imprints Th17 cells for enhanced maintenance or transdifferentiation during restimulation.
- A hyperactivated Th17 state is linked to early IL-2 production and affects functional plasticity.

## Abstract

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.

Vivo-seq allows the simultaneous capture of transcription and intracellular phosphorylation in droplet-based scRNA-seq. Using this approach in Th17 differentiation, we identify dual phosphorylation of ERK1/2 and c-FOS as required for maximum IL-2 production and associated with enhanced Th17 maintenance or transdifferentiation depending on subsequent cytokine signals received during antigenic restimulation.