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Published on: April 10, 2018
Fluorescently labeled dengue viruses as probes to identify antigen-specific memory B cells by multiparametric flow
1Division of Infectious Disease and Immunology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
Insights
Researchers developed fluorescently labeled dengue virus (DENV) probes to detect DENV-specific memory B cells in blood. This method allows for the ex vivo analysis of these crucial immune cells in dengue-experienced individuals.
Area of Science:
- Immunology
- Virology
Background:
- Memory B cells are critical for adaptive immunity but are present at low frequencies in peripheral blood.
- Detecting dengue virus (DENV)-specific memory B cells ex vivo is challenging due to a lack of suitable reagents.
Purpose of the Study:
- To investigate the use of fluorescently labeled DENV as probes for detecting antigen-specific memory B cells.
- To enable the ex vivo characterization of DENV-specific memory B cells in individuals with prior DENV exposure.
Main Methods:
- Alexa Fluor dye-labeled DENV was created, yielding viable and storable virus.
- A precise gating strategy and methods to reduce non-specific binding were employed.
- DENV-binding B cells (DENV+ B cells) were sorted and stimulated in vitro.
Main Results:
- Fluorescently labeled DENV successfully identified a small population of DENV-binding B cells in dengue-immune individuals.
- Sorted DENV+ B cells from immune donors, but not naive donors, secreted DENV-binding antibodies after stimulation.
- The labeled DENV probes remained viable and could be stored long-term.
Conclusions:
- Alexa Fluor dye-labeled DENVs are effective reagents for detecting and characterizing memory B cells specific to DENV.
- This approach facilitates the ex vivo study of DENV-specific memory B cells, advancing research in dengue immunity.
Abstract:
Low frequencies of memory B cells in the peripheral blood make it challenging to measure the functional and phenotypic characteristics of this antigen experienced subset of B cells without in vitro culture. To date, reagents are lacking to measure ex vivo frequencies of dengue virus (DENV)-specific memory B cells. We wanted to explore the possibility of using fluorescently labeled DENV as probes to detect antigen-specific memory B cells in the peripheral blood of DENV immune individuals. Alexa Fluor dye-labeled DENV yielded viable virus that could be stored at -80°C for long periods of time. Using a careful gating strategy and methods to decrease non-specific binding, we were able to identify a small frequency of B cells from dengue immune individuals that bound labeled DENV. Sorted DENV(+) B cells from immune, but not naïve donors secreted antibodies that bound DENV after in vitro stimulation. Overall, Alexa Fluor dye-labeled DENVs are useful reagents to enable the detection and characterization of memory B cells in DENV immune individuals.
