title: A live-cell assay to detect antigen-specific CD4+ T cells with diverse cytokine profiles.
abstract: Recently activated, but not resting, CD4(+) T cells express CD154, providing costimulatory signals to B cells and antigen-presenting cells (APCs). Therefore, de novo CD154 expression after stimulation identifies antigen-specific CD4(+) T cells. Previous assays were limited by the transient nature of surface CD154 expression; we overcame this by including fluorescently conjugated CD154-specific antibody during stimulation. Our assay is fully compatible with intracellular cytokine staining, and can be used for stimulations as long as 24 h. Notably, it is nonlethal, providing a means to purify viable antigen-specific CD4(+) T cells for further analysis. Using this assay, we found that stimulated cells expressing tumor necrosis factor (TNF)-alpha, interleukin (IL)-2 or interferon (IFN)-gamma were predominantly CD154(+). Furthermore, some cells expressing none of these cytokines also expressed CD154, suggesting that CD154 marks cells with other effector functions. For vaccine- or pathogen-specific responses, we found substantial heterogeneity in expression of CD154 and cytokines, suggesting previously unrecognized diversity in abilities of responding cells to stimulate APCs through CD40.
authors.affiliation: ImmunoTechnology Section, Vaccine Research Center, National Institute of Allergy and Infectious Disease, National Institutes of Health, 40 Convent Drive, Room 5509, Bethesda, Maryland 20892, USA.
authors.forename: Pratip K
authors.initials: PK
authors.lastname: Chattopadhyay
authors.forename: Joanne
authors.initials: J
authors.lastname: Yu
authors.forename: Mario
authors.initials: M
authors.lastname: Roederer
mesh_terms: D015704:CD4 Antigens / Q000032:analysis / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology* / Q000378:metabolism*; D023201:CD40 Ligand / Q000032:analysis / Q000378:metabolism; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002478:Cells, Cultured; D016207:Cytokines / Q000032:analysis / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D007118:Immunoassay; D007239:Infections / Q000276:immunology; D014611:Vaccination
chemical_list: D015704:CD4 Antigens; D016207:Cytokines; D023201:CD40 Ligand