Work

Analysis of Antigen‐Specific Human Memory B Cell Populations Based on In Vitro Polyclonal Stimulation

Year: 2020

Type: article

Abstract: Antigen-specific memory B cell (MBC) populations mediate the rapid, strong, and high-affinity secondary antibody responses that play a key role in combating infection and generating protective responses to vaccination. Recently, cell staining with fluorochrome-labeled antigens together with sequencing methods such as Drop-seq and CITE-seq have provided information on the specificity, phenotype, and transcriptome of single MBCs. However, characterization of MBCs at the level of antigen-reactive populations remains an important tool for assessing an individual's B cell immunity and responses to antigen exposure. This is readily performed using a long-established method based on in vitro polyclonal stimulation of MBCs to induce division and differentiation into antibody-secreting cells (ASCs). Post-stimulation antigen-specific measurement of the MBC-derived ASCs (or the secreted antibodies) indicates the size of precursor MBC populations. Additional information about the character of antigen-reactive MBC populations is provided by analysis of MBC-derived antibodies of particular specificities for binding avidity and functionality. This article outlines a simple and reliable strategy for efficient in vitro MBC stimulation and use of the ELISpot assay as a post-stimulation readout to determine the size of antigen-specific MBC populations. Other applications of the in vitro stimulation technique for MBC analysis are discussed. The following protocols are included. © 2020 Wiley Periodicals LLC Basic Protocol 1: Polyclonal stimulation of memory B cells using unfractionated PBMCs Alternate Protocol: Stimulation of small PBMC numbers using 96-well plates with U-bottom wells Basic Protocol 2: ELISpot assay for enumeration of memory B cell-derived antibody-secreting cells.

Source: Current Protocols in Immunology

DOI: 10.1002/cpim.109

Authors: Phuong Nguyen‐Contant, A. Karim Embong, David J. Topham, Mark Y. Sangster

Institutions: University of Rochester Medicine, University of Rochester

Language: English

Topic: T-cell and B-cell Immunology

Subfield: Immunology

Field: Immunology and Microbiology

Domain: Life Sciences

SDG: Good health and well-being

Open Access status: green

Funders: National Science Foundation, National Institute of Allergy and Infectious Diseases