Description:
<jats:title>Abstract</jats:title><jats:p>Activation induced marker (AIM) assays are being used increasingly to measure antigen‐specific T‐cell responses, but this activation can alter cell lineage defining phenotypic markers. We aimed to extend the utility of AIM assays to enable pre‐activation defined cell populations to be tracked and quantified within T‐cell memory responses. We sorted three <jats:italic>ex vivo</jats:italic> CD4<jats:sup>+</jats:sup> T‐cell populations prior to any activation using well defined <jats:italic>ex vivo</jats:italic> lineage surface marker combinations. These populations were memory non‐Tregs, CD39<jats:sup>+</jats:sup> Tregs and CD39<jats:sup>neg</jats:sup> Tregs, although any three memory CD4<jats:sup>+</jats:sup> T‐cell populations able to be isolated by cell surface markers could potentially be tracked. These cells were labeled with three distinct fluorescent cell proliferation dyes (CFSE, CellTrace Violet and Cell Proliferation Dye eF670) and then all autologous PBMCs were reconstituted maintaining <jats:italic>ex vivo</jats:italic> cell ratios and CD25/OX40 AIM assays performed with CMV and HSV antigens. This approach enabled tracking of pre‐defined cell populations within antigen stimulated responses using both activation marker and cell proliferation readouts. We confirmed that although CD39<jats:sup>+</jats:sup> Tregs comprise a substantial proportion of AIM assay responses, they do not make substantial contributions to the proliferative response. This extends the utility of AIM assays to enable parallel analysis of the relative contribution of several CD4<jats:sup>+</jats:sup> memory T‐cell subsets to recall responses.</jats:p>