Host Immune Composition Complexity Shape Murine IFN-γ ELISpot Readouts 2251432

Introduction ELISpot enables highly sensitive counting of cytokine-secreting cells at single-cell resolution; however, it remains unclear how the host’s immune experience interacts with assay parameters to dictate readouts. We established a mouse strain and microbial experience-tuned workflow for murine IFN-γ ELISpot through joint optimization of cell number, stimulant dose, and capture time with intracellular cytokine staining (ICS) validation. Methods Peripheral blood and splenocytes were harvested from inbred and outbred specific-pathogen free (SPF), or -pathogen experienced (SPExp) mice. Using an in-house IFN-γ ELISpot, we titrated cell input, anti-CD3/CD28 mAb concentration, and incubation time (1-4 or 22 hours). Plates were imaged on an ImmunoSpot S6 Entry (CTL), and spot counts and sizes were quantified. In parallel, IFN-γ production was assessed by ICS and flow cytometry (4-hour incubation with BfA added in the last hour). Results Across SPF mice, spot counts exhibited sigmoidal responses to the number of cells seeded, stimulation dose, and incubation time; the broadest dynamic range was at 4 × 10^5 cells with a 4-hour capture. In SPExp cohorts, just 2 × 10^4 cells maximized signal, and the optimal window shifted with days post last infection. PBMC ELISpot mirrored spleen longitudinally. Spot number aligned best with Ag-experienced CD8+ frequency and IFN-γ GMFI, with additional IFNγ contribution from naïve CD8+, CD4+, and NK cells. Conclusion Thus, titrating cells seeded and capture time to immune cell composition context improves accuracy. SPExp mice reach maximal dynamic range at low inputs, enabling minimally invasive longitudinal PBMC ELISpot. Across cohorts, ELISpot signal aligns with the product of Ag-experienced CD8 frequency and per-cell IFN-γ output by ICS, offering a quantitative bridge between frequency and function.