Reliable detection of antigen-specific T cells is critical to understand immune responses to infection and vaccination, with translational potential to monitor T cell responses across diverse clinical settings. Activation-induced marker (AIM) assays offer a variety of advantages over methods such as ELISPOT and tetramers but are limited by methodological heterogeneity between research groups and lack of standardized protocols, and AIM assay reproducibility is unclear. Key experimental variables, such as marker selection for CD4+ T cells, CD8+ T cells, and Tregs, and data analysis parameters, including flow cytometric gating and mathematical correction for “background” AIM+ frequencies in unstimulated samples, have not been rigorously studied. To address this, we comprehensively characterized AIM assay variability within and between operators and across multiple research centers, seeking to optimize a standard AIM workflow to enhance reproducibility at both experimental and analytical levels.
