Single-cell deep phenotyping of cytokine release unmasks stimulation-specific biological signatures and distinct secretion dynamics.

Cytokines are important mediators of the immune system, and their secretion level needs to be carefully regulated, as an unbalanced activity may lead to cytokine release syndromes. Dysregulation can be induced by various factors, including immunotherapies. Therefore, the need for risk assessment during drug development has led to the introduction of cytokine release assays (CRAs). However, the current CRAs offer little insight into the heterogeneous cellular dynamics. To overcome this limitation, we developed an advanced single-cell microfluidic-based cytokine secretion platform to quantify cytokine secretion on the single-cell level dynamically. Our approach identified different dynamics, quantities, and phenotypically distinct subpopulations for each measured cytokine upon stimulation. Most interestingly, early measurements after only 1 h of stimulation revealed distinct stimulation-dependent secretion dynamics and cytokine signatures. With increased sensitivity and dynamic resolution, our platform provided insights into the secretion behavior of individual immune cells, adding crucial additional information about biological stimulation pathways to traditional CRAs.