| Autor: |
Franz B; Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, Boston, MA 02215-5450, USA., May KF Jr, Dranoff G, Wucherpfennig K |
| Jazyk: |
angličtina |
| Zdroj: |
Blood [Blood] 2011 Jul 14; Vol. 118 (2), pp. 348-57. Date of Electronic Publication: 2011 May 06. |
| DOI: |
10.1182/blood-2011-03-341917 |
| Abstrakt: |
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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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