title: Development of a cytokine-secreting-based assay for the identification, sorting and transcriptomic analysis of polyfunctional human T cells.
abstract: Polyfunctional T cells that simultaneously produce the cytokines IFN-γ, IL-2 and TNF have been correlated with better clinical outcomes in various diseases. To date, cytokine polyfunctionality within T cells has been exclusively studied by intracellular cytokine staining coupled with flow cytometric analysis. Thus, further downstream interrogation of polyfunctional T cell characteristics such as transcriptomic analysis has not been possible. Here, we report the use of a flow cytometric method based on cytokine secretion assay technology to detect and isolate, for the first time, viable human polyfunctional T cells directly from in vitro stimulated whole blood samples. We demonstrate the successful application of this method to sort polyfunctional T cells obtained from human volunteers, which can be then used for downstream applications such as transcriptomic analysis using RT-qPCR. This assay will facilitate in-depth investigations of T cells with distinct cytokine polyfunctionality, including defining their molecular profile and understanding the mechanisms regulating their generation and function.
authors.affiliation: Molecular Vaccinology Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Australia, School of Medicine, University of Queensland, Brisbane, Australia.
authors.forename: Julie G
authors.initials: JG
authors.lastname: Burel
authors.affiliation: Molecular Vaccinology Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Australia.
authors.forename: Simon H
authors.initials: SH
authors.lastname: Apte
authors.affiliation: Molecular Vaccinology Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Australia, School of Medicine, University of Queensland, Brisbane, Australia.
authors.forename: Denise L
authors.initials: DL
authors.lastname: Doolan
mesh_terms: D000328:Adult; D002469:Cell Separation / Q000379:methods; D016207:Cytokines / Q000096:biosynthesis* / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods*; D020869:Gene Expression Profiling; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis / Q000235:genetics; D007376:Interleukin-2 / Q000096:biosynthesis / Q000235:genetics; D008875:Middle Aged; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016176:T-Lymphocyte Subsets / Q000145:classification* / Q000276:immunology* / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000235:genetics
chemical_list: D016207:Cytokines; C573415:IFNG protein, human; C508594:IL2 protein, human; D007376:Interleukin-2; D012333:RNA, Messenger; C439524:TNF protein, human; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma
keywords: cell sorting; cytokine secretion; novel assay; polyfunctional T cells; transcriptomic analysis