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

Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, Boston, MA 02215-5450, USA.
Blood (Impact Factor: 10.45). 05/2011; 118(2):348-57. DOI: 10.1182/blood-2011-03-341917
Source: PubMed


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.

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    • "In order to design novel vaccines that are able to induce B cell responses focused on the epitopes targeted by these broadly neutralizing antibodies, both new and improved immunogens are needed, as well as a better understanding of the early B cell responses induced by these novel vaccine candidates (Burton et al., 2012). One way to study these early B cell responses is through the use of antigen-probes designed to stain antigen-specific memory B cells (Scheid et al., 2009b; Franz et al., 2011; Kardava et al., 2014). This approach has proven to be very powerful in order to identify the bnAbs described above (Scheid et al., 2009a; Walker et al., 2011; Sundling et al., 2012a). "
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    • "2) TZM-bl from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc [51,55-57]. 3) HIV-1LAI from Dr. Jean-Marie Bechet and Dr. Luc Montagnier [58,59]. "
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    • "However, a large number of rare cell subtypes exist in the human immune system, and it is well established that rare cells play an important role in the immune system (e.g. HIV (Franz et al., 2011), stem cell research (Notta et al., 2011) and cancer (Zimmerlin et al., 2011)). "
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