title: Simultaneous cytometric analysis of (auto)antigen-reactive T and B cell proliferation.
abstract: The detection and characterization of (auto)antigen-specific lymphocytes, both B and T cells, is essential to investigate immunopathologic mechanisms. Our aim was to perform a CFSE (Carboxyfluorescein diacetate succinimidyl ester)-based cytometric analysis of peripheral blood mononuclear cells (PBMC) proliferating in response to antigenic provocation. CFSE-labeled PBMC were stimulated with a superantigen (SEB), a recall antigen (tetanus toxoid), an allergen (grass pollen) and an autoantigen (nucleosomes) and stained after cultivation with CD4-, CD8- and CD19-antibodies. Proliferated cells were identified cytometrically by the decrease of the CFSE fluorescence intensity due to cell division. Antigen-reactive, proliferated B cells were further analysed phenotypically, antigen-specific proliferated Th cells were further characterized functionally regarding their cytokine secretion pattern after polyclonal restimulation. Using this technique, antigen-specific proliferated B and Th cells were detected even at low frequencies. Analyzing the cytokine secretion pattern of allergen-reactive proliferated Th cells after polyclonal restimulation we found differences in the expression of IL-13 and IL-4 between an atopic and a healthy donor. After stimulation of PBMC from TT-vaccinated donors TT-specific proliferated B cells were detected in high frequencies and showed a plasmablast-typical CD20(low) CD27(high) phenotype with only low frequencies expressing CD138 (= Syndecan-1). Proliferation of nucleosome-reactive Th cells and B cells was observed in both patients and healthy controls. We have optimized here the cytometric analysis of reactive cell proliferation based on CFSE offering various facilities of application on the further characterization of both antigen-specific B and T cells.
authors.affiliation: Universitätsklinikum Charité, Med. Klinik m. S. Rheumatologie und Klinische Immunologie, Berlin, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Schneider
authors.forename: Anne
authors.initials: A
authors.lastname: Bruns
authors.forename: Beate
authors.initials: B
authors.lastname: Moewes
authors.forename: Barbara
authors.initials: B
authors.lastname: Holzknecht
authors.forename: Gerd
authors.initials: G
authors.lastname: Hausdorf
authors.forename: Gabriela
authors.initials: G
authors.lastname: Riemekasten
authors.forename: Andreas
authors.initials: A
authors.lastname: Radbruch
authors.forename: Falk
authors.initials: F
authors.lastname: Hiepe
authors.forename: Andreas
authors.initials: A
authors.lastname: Thiel
mesh_terms: D000942:Antigens, Bacterial / Q000276:immunology; D015703:Antigens, CD / Q000276:immunology / Q000378:metabolism; D001324:Autoantigens / Q000276:immunology; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D002454:Cell Differentiation; D002455:Cell Division; D016207:Cytokines / Q000378:metabolism; D004768:Enterotoxins / Q000276:immunology; D005434:Flow Cytometry; D005452:Fluoresceins; D019085:Fluorescent Antibody Technique, Direct / Q000379:methods*; D006801:Humans; D006967:Hypersensitivity / Q000276:immunology; D008180:Lupus Erythematosus, Systemic / Q000276:immunology; D009707:Nucleosomes / Q000276:immunology; D010641:Phenotype; D006109:Poaceae / Q000276:immunology; D011058:Pollen / Q000276:immunology; D013388:Succinimides; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D013745:Tetanus Toxoid / Q000276:immunology
chemical_list: C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D000942:Antigens, Bacterial; D015703:Antigens, CD; D001324:Autoantigens; D016207:Cytokines; D004768:Enterotoxins; D005452:Fluoresceins; D009707:Nucleosomes; D013388:Succinimides; D013745:Tetanus Toxoid; C031818:enterotoxin B, staphylococcal