Blood Journal
Leading the way in experimental and clinical research in hematology

Ex vivo characterization and isolation of rare memory B cells with antigen tetramers

  1. Bettina Franz1,
  2. Kenneth F. May Jr.2,
  3. Glenn Dranoff3, and
  4. Kai Wucherpfennig4,*
  1. 1 Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, Boston, MA, United States;
  2. 2 Department of Medical Oncology and Cancer Vaccine Center, Dana-Farber Cancer Institute, Boston, MA, United States;
  3. 3 Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, United States;
  4. 4 Program in Immunology, Harvard Medical School, Boston, MA, United States
  1. * Corresponding author; email: kai_wucherpfennig{at}dfci.harvard.edu

Abstract

Studying human antigen-specific memory B cells has been challenging due to 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, using tetanus toxoid as a model antigen. Brightly labeled memory B cells were identified even four years after last immunization, despite low frequencies ranging from 0.01 to 0.11% of class-switched memory B cells. Direct comparison of monomeric to tetrameric antigen labeling demonstrated that a substantial fraction of the B cell repertoire can be missed by using monomeric antigen. The specificity of the method was confirmed by antibody reconstruction from single cell sorted tetramer+ B cells using single cell RT-PCR of the B cell receptor. All antibodies bound to tetanus antigen with high affinity, ranging from 0.23 nM 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.

  • Submitted March 10, 2011.
  • Accepted April 22, 2011.