title: Fluorescent target array T helper assay: a multiplex flow cytometry assay to measure antigen-specific CD4+ T cell-mediated B cell help in vivo.
abstract: CD4(+) T cells play a central role in regulating the immune response. Their effector function is commonly assessed by their capacity to secrete cytokines detected by ELISPOT and intracellular cytokine staining. However, one aspect of their effector function that is often overlooked is their ability to help activation of cognate B cells directly, a process that is initiated through the engagement of their T cell-receptor (TCR) with cognate peptide presented on major histocompatibility complex class II (MHC-II) molecules by B cells. Here we report a method to monitor CD4(+) T cell-mediated B cell help in vivo using a multiplex high throughput assay. This assay utilizes a fluorescent target array (FTA), which is composed of lymphocytes labeled with numerous (>200) unique fluorescence signatures that can be delineated in a single recipient animal based on combination labeling with the three vital dyes carboxyfluorescein diacetate succinimidyl ester (CFSE), CellTrace Violet (CTV) and Cell Proliferation Dye eFluor 670 (CPD). By pulsing different B cell populations in a FTA with titrated amounts of cognate MHC-II binding peptides, CD4(+) T cell help could be assessed by measuring induction of the B cell activation markers CD69 and CD44 by antibody labeling and flow cytometry. We call this the "FTA T helper assay", and have found it to be a robust and sensitive assay to measure CD4(+) T cell helper activity across a multitude of peptide-pulsed B "target" cells in real time in vivo. Furthermore, the technique can be used simultaneously with the FTA killing assay that measures cytotoxic T cell function, to provide a comprehensive tool for measuring both CD4(+) and CD8(+) T cell activity during an immune response in vivo.
authors.affiliation: Department of Immunology, John Curtin School of Medical Research, Australian National University, Canberra, ACT 2601, Australia. ben.quah@anu.edu.au
authors.forename: Benjamin J C
authors.initials: BJ
authors.lastname: Quah
authors.forename: Danushka K
authors.initials: DK
authors.lastname: Wijesundara
authors.forename: Charani
authors.initials: C
authors.lastname: Ranasinghe
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Parish
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000276:immunology*; D015703:Antigens, CD / Q000276:immunology / Q000378:metabolism; D000945:Antigens, Differentiation, T-Lymphocyte / Q000276:immunology / Q000378:metabolism; D001402:B-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D003602:Cytotoxicity, Immunologic / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D000949:Histocompatibility Antigens Class II / Q000276:immunology / Q000378:metabolism; D018960:Hyaluronan Receptors / Q000276:immunology / Q000378:metabolism; D037181:Lectins, C-Type / Q000276:immunology / Q000378:metabolism; D008213:Lymphocyte Activation / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000276:immunology; D015203:Reproducibility of Results; D013388:Succinimides / Q000737:chemistry; D000099192:CD69 Antigens
chemical_list: D000941:Antigens; D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D005452:Fluoresceins; D005456:Fluorescent Dyes; D000949:Histocompatibility Antigens Class II; D018960:Hyaluronan Receptors; D037181:Lectins, C-Type; D010455:Peptides; D011948:Receptors, Antigen, T-Cell; D013388:Succinimides; D000099192:CD69 Antigens; C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester