Abstract
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
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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.
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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.
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Data availability
All data are represented within the paper. Raw data files are available upon request. The corresponding authors have repositories of antigens used for antigen tetramer construction, which can be shared upon request. They are also willing to assist in construction of novel antigen tetramers, if contacted.
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Acknowledgements
We thank J. SoRelle (University of Texas Southwestern) for carefully reviewing and providing suggestions on the manuscript. We thank M. K. Jenkins (University of Minnesota) for supporting the initial development of these protocols and for the inclusion of Extended Data Fig. 1g. We thank J. Carter and D. Galloway (Fred Hutchinson Cancer Center) for providing supernatant containing GST. We thank the McMaster Flow Cytometry Core, H. Liang and M. Subapanditha for access to flow cytometers and experimental support. We thank M. S. Miller (McMaster University) and M. Larche (McMaster University) for providing recombinant RBD. The laboratories of M.J. and J.F.E.K. are funded by the Schroeder Foundation, Food Allergy Canada, ALK-Abello A/S, the Canadian Allergy Asthma and Immunology Foundation, the Zych family and the Satov family. A.P. is funded by an Ontario Graduate Scholarship and The Eva Eugenia Lillian Cope Scholarship. D.P.-C. was funded by Universidad Politécnica de Madrid and Banco Santander with predoctoral and travel Programa Propio grants. J.T.-A. was funded by Severo Ochoa Program (Production of Plant and Human health-relevant proteins in Super-Green Biofactories: PCD-UPM/7/2022). The Centre for Plant Biotechnology and Genomics was granted ‘Severo Ochoa’ Distinctions of Excellence by the Spanish Ministry of Science and Innovation (SEV-2016-0672 and CEX2020-000999-S). J.J.T. has been funded by the National Institutes of Health (R01AI122912, R01AI158728, R01AI167009), The Hartwell Foundation, Vir Biotechnology, Fred Hutch Cancer Center and generous donors. J.B. was supported by a Fast Grants award, and J.B. and J.J.T. were supported by a Fred Hutchinson Cancer Center COVID pilot award.
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Conceptualization: A.P., D.P.-C., J.J.T. and J.F.E.K. Methodology: A.P., D.P.-C., J.J.T. and J.F.E.K. Formal analysis: A.P., D.P.-C., J.B., J.J.T. and J.F.E.K. Investigation: A.P., D.P.-C., F.U., E.G., O.M.-D., A.F., T.D.W., R.T.G., J.T.-A., A.D.-P., J.B. and J.F.E.K. Resources: J.T.-A., A.D.-P. and S.W. Visualization: A.P., D.P.-C., J.J.T. and J.F.E.K. Funding acquisition: J.T.-A., A.D.-P., S.W., M.J., J.J.T. and J.F.E.K. Supervision: J.J.T. and J.F.E.K. Writing — original draft: A.P., D.P.-C., J.J.T. and J.F.E.K. Writing — review and editing: A.P., D.P.-C., F.U., J.B., M.J., J.J.T. and J.F.E.K.
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J.F.E.K. and M.J. receive funding from ALK Abello A/S. M.J. is an advisor for ALK Abello A/S. All other authors declare no competing interests.
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Key references using this protocol
Taylor, J. J. et al. J. Exp. Med. 209, 2065–2077 (2012): https://doi.org/10.1084/jem.20112272
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Extended data
Extended Data Fig. 1 Antigen monomer and tetramer staining.
a) Representative plot of OVA-FITC monomer-stained samples from the mesenteric lymph nodes of mice sensitized with 4 gavages of OVA/CT. b) Isotype of OVA-FITC monomer-binding B cells from unimmunized mice. c) Representative plot of OVA-PE tetramer-stained samples from the spleen and mesenteric lymph nodes of unimmunized and OVA/Alum immunized mice. d) Comparison of SA-PE and OVA PE tetramer staining in unimmunized mice. e) Representative plot of control and antigen tetramer staining of spleen and mesenteric lymph nodes from unimmunized mice. f) Isotype of OVA PE tetramer-binding B cells from unimmunized mice. g) Demonstration of exclusion of backbone-binding B cells using a control tetramer. Comparison of unimmunized mice, mice immunized intraperitoneally with OVA/CFA, PE/CFA, and SA/CFA. H) RBD PE and RBD APC tetramer staining of anti-PE, anti-APC, anti-SA, and anti-RBD loaded compensation beads.
Extended Data Fig. 2 Antigen-specific B cell tracing by adoptive transfer of tetramer enriched cells.
a) Schematic of the experiment. b) Representative plots and quantification of control and OVA tetramer staining of spleen and mesenteric lymph nodes of recipient µMT mice 9 days after OVA/Alum immunization. c) Quantification of serum OVA-specific IgE and IgG1 antibodies by ELISA. d–f) Evaluation of phenotype of OVA-specific B-lineage cells in the enriched fraction of OVA/Alum-immunized µMT recipients. * p<0.05 by two-way ANOVA. Error bars represent SEM. Panel a created with BioRender.com.
Extended Data Fig. 3 Examples of successful and failed blots to determine biotinylation ratio.
Sample blots of a) target biotinylation ratio, b) over-biotinylated antigen, c) under-biotinylated antigen.
Extended Data Fig. 4 Antigen tetramer enrichment from other tissues.
Evaluation of OVA tetramer binding in the unenriched, enriched and flow through fractions and evaluation of phenotype and isotype of OVA-specific B cells within the enriched fraction. a) OVA tetramer enrichment from unimmunized mice or mice sensitized with 4 gavages of OVA/CT. Small intestines from 5 mice were pooled prior to enrichment. b) OVA tetramer enrichment from unimmunized mice or mice immunized with an intraperitoneal injection of OVA/Alum. Bone marrow from the hind legs of one mouse is represented on each plot.
Extended Data Fig. 5 Example antigen tetramer reagents.
Enrichment of antigen tetramer-binding B cells from the spleen and mesenteric lymph nodes of unimmunized mice and those intraperitonally immunized with (a) Beta lactoglobulin (BLG), (b) Alt a 1, (c) Ara h 1, (d) SARS-CoV-2 spike RBD.
Extended Data Fig. 6 Additional information for Fig. 3.
a) Evaluation of phenotype of OVA-specific switched and IgE-expressing B cells from OVA/Alum immunized Verigem mice at 7 days post-immunization. b) Concatenated plot of intracellular IgE staining from 3 mice sensitized by 4 gavages of OVA/CT. Data was collected at day 6 after the last gavage. c) Extracellular IgG1 staining of OVA tetramer-binding B cells from unimmunized and OVA/Alum immunized mice. d) Intracellular IgG1 staining with and without blocking surface IgG1 with an unlabeled antibody.
Supplementary information
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Supplementary Tables 1–3, Text and Methods.
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Programmed Excel worksheet that performs calculations for this protocol.
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Phelps, A., Pazos-Castro, D., Urselli, F. et al. Production and use of antigen tetramers to study antigen-specific B cells. Nat Protoc 19, 727–751 (2024). https://doi.org/10.1038/s41596-023-00930-8
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DOI: https://doi.org/10.1038/s41596-023-00930-8
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