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

Fortmann et al., Cell Reports, 2025 introduce Vivo-seq, a way of reading transcription and intracellular phosphorylation from the same single cell, and then use it on developing T helper 17 cells. The finding it produces is that cells carrying phosphorylated ERK1/2 and phosphorylated c-FOS at the same time make the most interleukin 2 and interleukin 17A, which is a claim about two signalling proteins acting together rather than in sequence.

AUMsilence sdASOs from AUM BioTech were used to test that claim by removing the proteins. Five oligonucleotides were designed against each of Fos, Erk1 and Erk2, each against a different region of the transcript, and the five were pooled in equal molar ratio. They were diluted into the culture medium of primary mouse Th17 cells to 1 μM per target, with no transfection reagent, 1 hour before stimulation on culture day 3. Four arms were run in parallel: a scramble control, Fos alone, the two ERK genes together, and all three at once.

Knocking down c-FOS and both ERK genes together gave the largest fall in the proportion of cells making interleukin 2 and interleukin 17A and in the amount each cell made, larger than knocking down either side alone. The same ordering appeared in cytokine secreted into the supernatant, measured across interleukin 2, interleukin 17A, interleukin 17F, tumour necrosis factor alpha and interleukin 10 on a multiplex bead array, with five independent cultures per arm. The cells came from wild type C57BL/6J, Il17a-eGFP and IL2.eGFP-SMARTA mice; the oligonucleotides were used in culture only.