Temporal variability in healthy donor IFN-γ ELISpot responses reveals a stable IL-7-responsive functional metric for immune phenotyping.

Functional T-cell assays such as the IFN-γ ELISpot are increasingly used for immune phenotyping in infection, cancer, and critical illness. Many studies, including recent work linking low IFN-γ ELISpot responses to adverse outcomes in sepsis, compare patient samples to a single cross-sectional healthy donor baseline, implicitly assuming temporal stability of IFN-γ secretion in health. We conducted a longitudinal study of 9 healthy adults using whole-blood anti-CD3/anti-CD28 ELISpot assays with and without IL-7 co-stimulation. Absolute IFN-γ production varied significantly within individuals over approximately 10 weeks, whereas the ratio of IL-7-augmented to anti-CD3/anti-CD28-only responses remained remarkably stable across all readouts. This IL-7 responsiveness ratio may represent a robust, time-independent functional metric of intact T-cell signaling. These findings demonstrate that absolute IFN-γ ELISpot values in healthy individuals are dynamic and support ratio-based metrics for immune phenotyping applications.