Purification and characterization of antigen-binding virgin and memory B cells

Abstract Studying human antigen-specific memory B cells has been challenging because of low frequencies in peripheral blood, slow proliferation, and lack of antibody secretion. Therefore, most studies have relied on conversion of memory B cells into antibody-secreting cells by in vitro culture. To facilitate direct ex vivo isolation, we generated fluorescent antigen tetramers for characterization of memory B cells by using tetanus toxoid as a model antigen. Brightly labeled memory B cells were identified even 4 years after last immunization, despite low frequencies ranging from 0.01% to 0.11% of class-switched memory B cells. A direct comparison of monomeric to tetrameric antigen labeling demonstrated that a substantial fraction of the B-cell repertoire can be missed when monomeric antigens are used. The specificity of the method was confirmed by antibody reconstruction from single-cell sorted tetramer+ B cells with single-cell RT-PCR of the B-cell receptor. All antibodies bound to tetanus antigen with high affinity, ranging from 0.23 to 2.2 nM. Furthermore, sequence analysis identified related memory B cell and plasmablast clones isolated more than a year apart. Therefore, antigen tetramers enable specific and sensitive ex vivo characterization of rare memory B cells as well as the production of fully human antibodies.

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The splenic autoimmune response to ADAMTS13 in thrombotic thrombocytopenic purpura contains recurrent antigen-binding CDR3 motifs

Blood ◽

10.1182/blood-2014-04-561142 ◽

2014 ◽

Vol 124 (23) ◽

pp. 3469-3479 ◽

Cited By ~ 24

Author(s):

Monica Schaller ◽

Monique Vogel ◽

Karim Kentouche ◽

Bernhard Lämmle ◽

Johanna A. Kremer Hovinga

Keyword(s):

B Cells ◽

Thrombotic Thrombocytopenic Purpura ◽

Memory B Cells ◽

Autoimmune Response ◽

Antibody Repertoire ◽

Antigen Binding ◽

Thrombocytopenic Purpura ◽

Specific Memory ◽

Key Points

Key Points The spleen harbors ADAMTS13-specific memory B cells following acute acquired TTP. The splenic anti-ADAMTS13 antibody repertoire is characterized by a set of unique and novel CDR3 motifs, 4 shared by 2 patients.

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Characterising the Phenotypic Diversity of Antigen-Specific Memory B Cells Before and After Vaccination

Frontiers in Immunology ◽

10.3389/fimmu.2021.738123 ◽

2021 ◽

Vol 12 ◽

Author(s):

M. Christian Tjiam ◽

Sonia Fernandez ◽

Martyn A. French

Keyword(s):

B Cells ◽

B Cell ◽

Phenotypic Diversity ◽

Binding Capacity ◽

Booster Vaccination ◽

Cell Receptor ◽

Memory B Cells ◽

Cell Phenotype ◽

Antigen Binding ◽

B Cell Subsets

The diversity of B cell subsets and their contribution to vaccine-induced immunity in humans are not well elucidated but hold important implications for rational vaccine design. Prior studies demonstrate that B cell subsets distinguished by immunoglobulin (Ig) isotype expression exhibit divergent activation-induced fates. Here, the antigen-specific B cell response to tetanus toxoid (TTd) booster vaccination was examined in healthy adults, using a dual-TTd tetramer staining flow cytometry protocol. Unsupervised analyses of the data revealed that prior to vaccination, IgM-expressing CD27+ B cells accounted for the majority of TTd-binding B cells. 7 days following vaccination, there was an acute expansion of TTd-binding plasmablasts (PB) predominantly expressing IgG, and a minority expressing IgA or IgM. Frequencies of all PB subsets returned to baseline at days 14 and 21. TTd-binding IgG+ and IgA+ memory B cells (MBC) exhibited a steady and delayed maximal expansion compared to PB, peaking in frequencies at day 14. In contrast, the number of TTd-binding IgM+IgD+CD27+ B cells and IgM-only CD27+ B cells remain unchanged following vaccination. To examine TTd-binding capacity of IgG+ MBC and IgM+IgD+CD27+ B cells, surface TTd-tetramer was normalised to expression of the B cell receptor-associated CD79b subunit. CD79b-normalised TTd binding increased in IgG+ MBC, but remained unchanged in IgM+IgD+CD27+ B cells, and correlated with the functional affinity index of plasma TTd-specific IgG antibodies, following vaccination. Finally, frequencies of activated (PD-1+ICOS+) circulating follicular helper T cells (cTFH), particularly of the CXCR3-CCR6- cTFH2 cell phenotype, at their peak expansion, strongly predicted antigen-binding capacity of IgG+ MBC. These data highlight the phenotypic and functional diversity of the B cell memory compartment, in their temporal kinetics, antigen-binding capacities and association with cTFH cells, and are important parameters for consideration in assessing vaccine-induced immune responses.

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Multimeric antibodies from antigen-specific human IgM+ memory B cells restrict Plasmodium parasites

Journal of Experimental Medicine ◽

10.1084/jem.20200942 ◽

2021 ◽

Vol 218 (4) ◽

Author(s):

Christopher D. Thouvenel ◽

Mary F. Fontana ◽

Jason Netland ◽

Akshay T. Krishnamurty ◽

Kennidy K. Takehara ◽

...

Keyword(s):

Plasmodium Falciparum ◽

B Cells ◽

Human Memory ◽

Memory B Cells ◽

B Cell Receptors ◽

Antigen Binding ◽

High Avidity ◽

Linear Epitope ◽

High Affinity ◽

Antibody Function

Multimeric immunoglobulin-like molecules arose early in vertebrate evolution, yet the unique contributions of multimeric IgM antibodies to infection control are not well understood. This is partially due to the difficulty of distinguishing low-affinity IgM, secreted rapidly by plasmablasts, from high-affinity antibodies derived from later-arising memory cells. We developed a pipeline to express B cell receptors (BCRs) from Plasmodium falciparum–specific IgM+ and IgG+ human memory B cells (MBCs) as both IgM and IgG molecules. BCRs from both subsets were somatically hypermutated and exhibited comparable monomeric affinity. Crystallization of one IgM+ MBC-derived antibody complexed with antigen defined a linear epitope within a conserved Plasmodium protein. In its physiological multimeric state, this antibody displayed exponentially higher antigen binding than a clonally identical IgG monomer, and more effectively inhibited P. falciparum invasion. Forced multimerization of this IgG significantly improved both antigen binding and parasite restriction, underscoring how avidity can alter antibody function. This work demonstrates the potential of high-avidity IgM in both therapeutics and vaccines.

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