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

Allyssa Phelps, Diego Pazos-Castro, Francesca Urselli, Emily Grydziuszko, Olivia Mann-Delany, Allison Fang, Tina D. Walker, Rangana Talpe Guruge, Jaime Tome-Amat, Araceli Diaz-Perales, Susan Waserman, Jim Boonyaratanakornkit, Manel Jordana, Justin J. Taylor, Joshua F. E. Koenig

Published: 2024-01-19 DOI: 10.1038/s41596-023-00930-8

flow cytometry

enrichment

antigen tetramers

B cell repertoire

fluorescent probe

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Extended

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 . ](https://www.nature.com/articles/s41596-023-00930-8/figures/11)

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.

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Introduction
Materials
Procedure
Troubleshooting
Timing
- Part 1: tetramer production: ~9 h
- Part 2: tetramer enrichment and use: 4–6.5 h
Anticipated results
- Reporting summary
Acknowledgements
Extended Data Fig. 1 Antigen monomer and tetramer staining.
Extended Data Fig. 2 Antigen-specific B cell tracing by adoptive transfer of tetramer enriched cells.
Extended Data Fig. 3 Examples of successful and failed blots to determine biotinylation ratio.
Extended Data Fig. 4 Antigen tetramer enrichment from other tissues.
Extended Data Fig. 5 Example antigen tetramer reagents.
Extended Data Fig. 6 Additional information for Fig.

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

Extended

[ Extended Data Fig. 6 Additional information for Fig. 3 . ](https://www.nature.com/articles/s41596-023-00930-8/figures/11)

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