title: Enumeration and characterization of virus-specific B cells by multicolor flow cytometry.
abstract: To better characterize B cell responses induced to influenza virus, we developed an assay to directly quantify and characterize virus-specific B cells. We used purified and biotinylated whole virus as well as the major influenza virus surface antigen, hemagglutinin (HA) to label virus-specific B cells induced by immunization of mice with whole influenza virus in adjuvant. Immunization with adjuvant alone caused non-specific binding of whole virus to a large number of B cells in the draining lymph nodes as assessed by flow cytometry. This precluded the use of whole virus as a specific staining reagent. In contrast, staining with bromelain-cleaved purified and biotinylated influenza virus HA identified a small population of B cells (roughly 1%) only in the draining lymph nodes of virus-immunized mice. FACS-purification and subsequent ELISPOT analysis showed that HA-labeled B cells contained the vast majority of virus-specific antibody-secreting cells at day 10 after immunization. Overall, virus-specific antibody-secreting cells comprised roughly 10% of the HA-labeled cells. Using HA-staining in conjunction with 8-color flow cytometry we further demonstrated that close to 90% of the HA-labeled cells were CD19+ IgD- CD23- CD24high CD38low germinal center B cells, many of which had incorporated bromodeoxyuridine, indicating recent cell division in vivo. We conclude that viral HA can be used in conjunction with cell surface and intracytoplasmic stains in multicolor flow cytometry to provide detailed phenotypic and functional information on virus HA-specific B cells.
authors.affiliation: Center for Comparative Medicine, University of California, Davis, County Rd 98, CA 95616, USA.
authors.forename: Virginia P
authors.initials: VP
authors.lastname: Doucett
authors.forename: Walter
authors.initials: W
authors.lastname: Gerhard
authors.forename: Kristina
authors.initials: K
authors.lastname: Owler
authors.forename: Dyan
authors.initials: D
authors.lastname: Curry
authors.forename: Lorena
authors.initials: L
authors.lastname: Brown
authors.forename: Nicole
authors.initials: N
authors.lastname: Baumgarth
mesh_terms: D000818:Animals; D000906:Antibodies / Q000097:blood; D000956:Antigens, Viral / Q000276:immunology*; D016175:B-Lymphocyte Subsets / Q000276:immunology* / Q000821:virology*; D001710:Biotin; D001973:Bromodeoxyuridine; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D018985:Epitopes, B-Lymphocyte / Q000276:immunology*; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000276:immunology* / Q000302:isolation & purification; D009980:Influenza A virus / Q000276:immunology; D008198:Lymph Nodes / Q000166:cytology / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C
chemical_list: D000906:Antibodies; D000956:Antigens, Viral; D018985:Epitopes, B-Lymphocyte; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D001710:Biotin; D001973:Bromodeoxyuridine