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.

journal_name

Blood

journal_title

Blood

authors

Franz B,May KF Jr,Dranoff G,Wucherpfennig K

doi

10.1182/blood-2011-03-341917

subject

Has Abstract

pub_date

2011-07-14 00:00:00

pages

348-57

issue

2

eissn

0006-4971

issn

1528-0020

pii

blood-2011-03-341917

journal_volume

118

pub_type

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