title: Ex vivo characterization and isolation of rare memory B cells with antigen tetramers.
abstract: Studying human antigen-specific memory B cells has been challenging because of low frequencies in peripheral blood, slow proliferation, and lack of antibody secretion. Therefore, most studies have relied on conversion of memory B cells into antibody-secreting cells by in vitro culture. To facilitate direct ex vivo isolation, we generated fluorescent antigen tetramers for characterization of memory B cells by using tetanus toxoid as a model antigen. Brightly labeled memory B cells were identified even 4 years after last immunization, despite low frequencies ranging from 0.01% to 0.11% of class-switched memory B cells. A direct comparison of monomeric to tetrameric antigen labeling demonstrated that a substantial fraction of the B-cell repertoire can be missed when monomeric antigens are used. The specificity of the method was confirmed by antibody reconstruction from single-cell sorted tetramer(+) B cells with single-cell RT-PCR of the B-cell receptor. All antibodies bound to tetanus antigen with high affinity, ranging from 0.23 to 2.2 nM. Furthermore, sequence analysis identified related memory B cell and plasmablast clones isolated more than a year apart. Therefore, antigen tetramers enable specific and sensitive ex vivo characterization of rare memory B cells as well as the production of fully human antibodies.
authors.affiliation: Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, Boston, MA 02215-5450, USA.
authors.forename: Bettina
authors.initials: B
authors.lastname: Franz
authors.forename: Kenneth F
authors.initials: KF
authors.lastname: May
authors.forename: Glenn
authors.initials: G
authors.lastname: Dranoff
authors.forename: Kai
authors.initials: K
authors.lastname: Wucherpfennig
mesh_terms: D000818:Animals; D000918:Antibody Specificity; D000941:Antigens / Q000276:immunology* / Q000378:metabolism / Q000494:pharmacology; D001402:B-Lymphocytes / Q000166:cytology* / Q000187:drug effects / Q000276:immunology; D016466:CHO Cells; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007156:Immunologic Memory / Q000276:immunology; D008954:Models, Biological; D055503:Protein Multimerization / Q000276:immunology / Q000502:physiology; D013745:Tetanus Toxoid / Q000138:chemical synthesis / Q000276:immunology / Q000378:metabolism
chemical_list: D000941:Antigens; D013745:Tetanus Toxoid