Parallel analysis of multiple human memory CD4+ T‐cell subsets within antigen‐specific responses using cell proliferation dyes

• Published in: Immunology and cell biology 2023-02, Vol.101 (2), p.171-178
• Main Authors: Cook, Laura, Zaunders, John, Seddiki, Nabila, Bockel, David, Kelleher, Anthony D, Munier, C. Mee Ling
• Format: Article
• Language: English
• Subjects: AIM assay; Antigens; Antigens - metabolism; antigen‐specific response; CD25 antigen; CD4 antigen; CD4-Positive T-Lymphocytes; Cell activation; Cell growth; Cell lineage; Cell Proliferation; Cell surface; cell proliferation; Coloring Agents - metabolism; Dyes; Forkhead Transcription Factors - metabolism; Humans; Immunological memory; Kinases; memory; Short Communication; Surface markers; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory; Tregs; Tumor necrosis factor
• ISSN: 0818-9641; 1440-1711; 1440-1711
• DOI: 10.1111/imcb.12606

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 ex vivo CD4+ T‐cell populations prior to any activation using well defined ex vivo lineage surface marker combinations. These populations were memory non‐Tregs, CD39+ Tregs and CD39neg Tregs, although any three memory CD4+ 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 ex vivo 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+ 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+ memory T‐cell subsets to recall responses. Activation‐induced marker (AIM) assays enable rapid detection of antigen‐specific T cells. However, cellular activation changes cell phenotypic marker expression preventing the determination of cell subsets within these responses. This approach allows up to three unique cell populations to be tracked within AIM assays and subsequent proliferation assays.