Production and use of antigen tetramers to study antigen-specific B cells

• Published in: Nature protocols Vol. 19; no. 3; pp. 727 - 751
• Main Authors: Phelps, Allyssa, Pazos-Castro, Diego, Urselli, Francesca, Grydziuszko, Emily, Mann-Delany, Olivia, Fang, Allison, Walker, Tina D., Guruge, Rangana Talpe, Tome-Amat, Jaime, Diaz-Perales, Araceli, Waserman, Susan, Boonyaratanakornkit, Jim, Jordana, Manel, Taylor, Justin J., Koenig, Joshua F. E.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01-03-2024; Nature Publishing Group
• Subjects: 631/1647/664/1364; 631/250/1619/40; 631/250/2152/2153; Analytical Chemistry; Antibodies; Antigens; B-Lymphocytes; Binding; Biological Techniques; Biomedical and Life Sciences; Biotin; Biotinylation; Cell activation; Computational Biology/Bioinformatics; Control equipment; Design of experiments; Enrichment; Epitopes; Experimental design; Flow cytometry; Flow Cytometry - methods; Fluorescent indicators; Immunology; Instrumentation; Life Sciences; Lymphocytes B; Medical research; Microarrays; Organic Chemistry; Probes; Protocol; Streptavidin
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/s41596-023-00930-8

B cells generate antibodies that provide protection from infection, but also cause pathology in autoimmune and allergic conditions. Antigen-specific B cells can be detected by binding their surface antibody receptors with native antigens conjugated to fluorescent probes, a technique that has revealed substantial insight into B cell activation and function. This protocol describes the process of generating fluorescent antigen tetramer probes and delineates a process of enriching large samples based on antigen-specificity for high-resolution analyses of the antigen-specific B cell repertoire. Enrichment of tetramer-binding cells allows for detection of antigen-specific B cells as rare as 1 in 100 million cells, providing sufficient resolution to study naive B cells and IgE-expressing cells by flow cytometry. The generation of antigen tetramers involves antigen biotinylation, assessment of biotin:antigen ratio for optimal tetramer loading and polymerization around a streptavidin–fluorophore backbone. We also describe the construction of a control tetramer to exclude B cells binding to the tetramer backbone. We provide a framework to validate whether tetramer probes are detecting true antigen-specific B cells and discuss considerations for experimental design. This protocol can be performed by researchers trained in basic biomedical/immunological research techniques, using instrumentation commonly found in most laboratories. Constructing the antigen and control tetramers takes 9 h, though their specificity should be assessed before experimentation and may take weeks to months depending on the method of validation. Sample enrichment requires ~2 h but is generally time and cost neutral as fewer cells are run through the flow cytometer.
Key points
The protocol describes the design of antigen-specific tetramer probes for the enrichment and identification of rare antigen-specific B cells. The selected antigen is first biotinylated in a biotin:antigen ratio <1:1 and then assembled around a streptavidin fluorescent backbone.
Isolation of antigen-specific B cells relies on the accurate design of control tetramers that do not share any known cross-reactive epitopes with the target antigen.
Antigen-specific B cells constitute a small proportion of the total B cell population, making identification for downstream analysis challenging. Here, this is achieved by pairing fluorescent antigen tetramer probes with a sensitive enrichment approach.