Understanding how immune cells behave and change over time is crucial for treating diseases ranging from infections to cancer. However, while single-cell RNA sequencing (scRNA-seq) has helped scientists map gene activity in individual cells, it misses key pieces of the puzzle, like protein activity, needed to fully understand how cells respond to signals.

A research team led by researchers from the University of Alabama at Birmingham has developed a new technique called Vivo-seq, which addresses this limitation. Vivo-seq combines traditional scRNA-seq with information about protein signaling inside cells. It does this by using a special cell-fixing method that preserves both RNA and phospho-signaling data. This allows researchers to simultaneously analyze gene expression and protein activation levels in the same single cell.

The team applied Vivo-seq to study T helper 17 (Th17) cells, a type of immune cell involved in inflammation and autoimmune diseases. They discovered that when two proteins, ERK1/2 and c-FOS, are activated at the same time, the Th17 cells produce more of the signaling molecules IL-2 and IL-17. This increased IL-2 production early in the cell’s life seems to shape how these cells behave later—making them more adaptable and responsive during future immune challenges.

By revealing how gene expression and protein signaling work together at the single-cell level, Vivo-seq opens new possibilities for tracking immune cell behavior and designing better immunotherapies.