Temporal perturbation of STAT1/2 activity reveals dynamic ligand discrimination of type I interferon signaling

Type-I interferon (IFN-I) subtypes signal through the same IFNalpha receptor (IFNAR), and initiate temporal STAT1/2 activation to orchestrate innate and adaptive immunity. It remains unknown how IFNAR discriminates between subtypes (e.g., IFNalpha and IFNbeta), and how STAT1/2 signaling is affected by time-varying inputs. Here, we utilize our microfluidic system and live-cell imaging to quantify STAT1/2 activation dynamics in a reporter fibroblast model. Population-averaged and single-cell analyses reveal distinct STAT1/2 responses to various IFNalpha and IFNbeta inputs. Upon continuous stimulation, cells show less sensitivity but more sustained responses to IFNalpha over IFNbeta. A short IFNalpha pulse induces nearly homogeneous STAT1/2 dynamics, in contrast to heterogeneous responses in IFNbeta-pulsed cells. Distinct STAT1/2 refractory states emerge upon exposure to repeated IFN-I pulses, while alternating pulse stimulation reveals that IFNbeta can revoke STAT1/2 refractoriness caused by IFNalpha, but not vice versa. These findings highlight the differences between IFNalpha and IFNbeta signaling and how they can elicit distinct temporal cellular behaviors during viral infection. ### Competing Interest Statement The authors have declared no competing interest.