Cutting-edge assessment techniques for B cell immune memory: an overview

Abstract

The adaptive humoral immune response depends on B cells. After antigens are processed and presented to naive B cells, these B cells differentiate into either surface immunoglobulin-expressing memory B cells (MBCs) or antibody-secreting plasma cells (PC) and long-lived plasma cells (LLPC). In parallel with the detection of antibodies, the detection of MBCs and LLPC is important to assess the strength and duration of the humoral immune response. The number of antigen-specific cells can be determined by the secretion of antibodies using highly sensitive techniques such as ELISpot or FluoroSpot. Flow cytometry is usually used to determine the antigen-specific MBC, PC and LLPC by binding of antigens to their surface immunoglobulins. In this technique, the antigen is either directly labelled with a fluorochrome or non-covalently linked to labelled streptavidin in a tetramer. The advantage of flow cytometry is that detailed phenotypic analysis of B cell subpopulations can be performed. Single-cell RNA Sequencing provides in-depth information about B-cell receptor diversity, functional states, transcriptomic changes associated with rare cell populations or dynamic cellular processes within the immune system. Additionally, it can lead to the discovery of novel B cell subsets previously unknown. The advantages, disadvantages and potential applications of each technique are thoroughly outlined. This overview of current knowledge on B-cell immune memory highlights some implications for advancing vaccination strategies, autoimmune disease management and personalized medicine.

Keywords:

• B cells
• B lymphocytes subpopulations
• B cellular immune response
• Methods for B cells assessment

Introduction

B-cells are responsible for the formation of antibodies, which participate in the protection of the macroorganism against infections by virus neutralization, activation of complement, opsonization of pathogens, facilitating phagocytosis and antibody-dependent cellular cytotoxicity [1]. B cells, upon encountering foreign antigens, undergo clonal expansion and differentiation into antibody-secreting plasma cells (PCs), culminating in the production of high-affinity antibodies. This process constitutes the primary immune response. However, a fraction of activated B cells transform into long-lived memory B cells (MBCs), which confer immunological memory, leading to a heightened and accelerated secondary response upon re-encounter with the same antigen [1,2].

For instance, in infectious diseases, the generation and persistence of B-cellular immune memory plays an important role in host defense mechanisms. Long-lasting B cellular memory ensures a more efficient and potent immune response upon secondary exposure to a previously encountered pathogen, thus conferring a protective advantage. This phenomenon underlies the principle of vaccination, wherein the immune system is primed to recognize and combat specific pathogens. Understanding the dynamics of B-cellular immune memory is pivotal for the design and optimization of vaccination strategies, thereby enhancing our ability to combat infectious diseases [3]. Conversely, dysregulation in the generation and function of B-cellular immune memory is implicated in autoimmune disorders [4], but the focus of this overview is on infectious agents. Evaluating specific B memory is crucial for several reasons: 1. Long-term protection: long-lived plasma cells (LLPCs) and memory B cells (MBCs) provide prolonged defense, critical for rapidly mutating pathogens like influenza. 2. Predicting durability: assessing specific b memory helps predict immunity duration, informing decisions on booster shots. 3. Vaccination efficacy: understanding B-cell memory aids in assessing vaccine effectiveness, informing antigen and adjuvant selection for optimal immune memory. 4. Personalized medicine: an individualized understanding of B-cell memory enables tailored vaccination and immunotherapy, enhancing immune response efficacy while minimizing adverse effects.

Nowadays, accurate and comprehensive evaluation of B-cellular response plays a pivotal role in the effective management of the SARS-CoV-2 pandemic, providing critical insights into the development of robust and lasting immunity, guiding vaccination strategies, and enhancing our understanding of long-term protection against the virus.

Development of human B cell subpopulations and immune response against infectious antigens and generation of immune memory

Our understanding of the development, maturation and basic functions of B cells has seen considerable advancements since their discovery in 1965 [5]. Several B cell subpopulations have been identified, including B1, B2, and regulatory B cells. B1 cells are formed mainly in the fetal liver and are subdivided into B-1a and B-1b. B1 cells are primarily found in body cavities such as the peritoneal and pleural cavities. One of the important functions of B1 cells is their ability to produce natural antibodies, which can present even in the absence of external stimuli. B1 cells are involved in the maintenance of tissue homeostasis and have been implicated in various immune regulatory processes [6]. The function of regulatory B cells (Bregs) is to suppress immune responses, mainly via the production of the antiinflammatory cytokine IL-10. B-cell tolerance is established at several checkpoints, during B-cell development in the bone marrow (central tolerance) and their maturation and activation in the periphery (peripheral tolerance) [7–9].

In this review, we describe B2 cell development, primary and secondary immune events, immune memory, and methods for its investigation.

Development of B2 cells

The development and maturation of B2 cells involve several stages, beginning in the bone marrow. B cell precursors develop into pro- and pre-B cells, accompanied by the rearrangement of genes for immunoglobulin light and heavy chains (Figure 1). This developmental process extends to the secondary lymphoid organs. In the bone marrow, B-2 cells undergo a series of differentiations, transitioning from hematopoietic stem cells (HSCs) to multipotent progenitor cells, common lymphoid precursors, and then to progenitor B cells, precursor B cells and immature B cells [8,9]. Immature B lymphocytes then circulate and differentiate into transitional B cells, followed by their conversion into follicular B cells (FOB) and marginal zone B cells (MZB) [8,9]. Upon migration to the lymph nodes and spleen, mature B cells contribute to the formation of corresponding structures in secondary lymphoid organs. During the primary immune response within the lymph nodes, naive B cells are activated and proliferate in B cell follicles. This leads to the differentiation of memory B cells (MBCs), short-lived antibody-secreting plasma cells (ASCs) and germinal center (GC) B cells [10]. GC B cells undergo somatic hypermutation, which increases antibody affinity. After remigration to the light zone of the germinal center, GC B cells differentiate into long-lived plasma cells (LLPC) and memory B cells (MBCs) under the influence of antigens and follicular T-helper cells. The immune memory established after infection relies on plasma cells generated during the acute and convalescent phases to provide serological memory. Long-lasting protection is achieved through the induction of LLPCs and memory B cells [11,12]. Memory B cells respond to reinfection by generating plasma cells with high affinity for the antigen. Continuously circulating memory B cells with mature affinity can readily differentiate into effector cells upon encounter with foreign antigens and can be identified by their B cell receptor (BCR) associated with the membrane-bound immunoglobulin (Ig) [11,12].

Figure 1. Development of human B cells.

The secondary immune response involves the MBCs and LLPCs, resulting in the generation of a rapid and effective immune memory upon re-exposure to antigens. This memory response, dominated by high-affinity antibodies such as IgG, IgA or IgE, depends on the pathogen [12–18]. Examples of individual infections illustrate the duration of immune memory and show the complex processes involved in the development, attenuation and sustained protection provided by the adaptive immune system. Studies have described the important role of B-cell immune memory generated after natural antigen encounter or after immunization in several infectious diseases such as measles, Hepatitis B, cytomegalovirus (CMV) and EBV infection [19–22].

Methods for assessment of B cellular immune response

The above review outlines the importance of B2 cells and B-cell immune memory for adaptive immunity against infectious agents. Therefore, it is necessary to have sensitive and reliable methods to study them. Currently, we have four main groups of methods: (a) detection of specific antibodies in body fluids; (b) identification of single specific B-cell subpopulations by antibody secretion (B-ELISpot, B-FLUOROSpot); (c) identification of specific B-cell subpopulations by BCR by flow cytometry and d) proteomic, transcriptomic genomic analysis of single cells.

The most commonly used method for the detection of specific binding and neutralizing antibodies in serum and plasma produced in response to an infection or immunization is the enzyme-linked immunosorbent assay (ELISA) [23].

Since specific immunoglobulin levels decrease dramatically in the first few months after contact with antigens, other methods are needed to detect B-cell memory, including specific MBCs, PBs, PCs and LLPC.

Identification of single antigen-specific B-cell subpopulations by antibody secretion

First described in 1983, the enzyme-linked immunosorbent spot assay (ELISpot) is one of the most widely used assays for the secretion of molecules from individual immune cells. B-ELISpot assesses antibodies released by individual cells, unlike ELISA, which measures the total concentration of molecules of interest produced by all cells in a sample. In the B-ELISpot method, peripheral mononuclear cells are cultured on a 96-well plate that has been preloaded with a specific capture antigen. The antigen on the surface binds to specific antibodies released by the cells. Then the cells are removed after an appropriate incubation period and the synthesized antibodies are assessed using enzyme or fluorescently labelled secondary antibodies. The addition of substrate makes the product of stimulation visible as a “print” on the plate surface, also known as a spot. Each spot represents a single antibody-secreting cell [24]. The technique is highly sensitive. A limitation of the assay is that it requires the secretion of an antibody by B cells, which restricts the study to a subset of the B cell repertoire, namely ASCs. This can be overcome by prior non-specific in vitro stimulation of the memory B lymphocytes and their conversion into antibody-synthesizing plasma cells. Various stimuli such as IL-2, IL-6, IL-15, IL-10, IL-21, and Toll-like receptor agonists (R878) can be used for this purpose. After in vitro cultivation for 3-11 days, both the antibodies secreted in the supernatant and the number of cells secreting antibodies can be measured. Depending on the stimulation conditions used, different levels of antibody secretion and different frequencies of antibody-secreting cells can be observed [25–27].

By combining the principles of ELISpot with fluorescence detection, FLUOROSpot assays measure simultaneously several different analytes (such as different cytokines or antibody isotypes) within a single assay. This allows a more comprehensive assessment of immune responses and antibody production compared to traditional ELISpot assays. In a multicolor FluoroSpot assay, cells are plated on a membrane coated with antigen that captures the secreted antibodies. The detected antibodies are labelled with a different fluorescent dye specific for a particular antibody isotype. This makes it possible to detect simultaneously multiple antibody isotypes in a single well [28]. This assay is particularly useful for the assessment of immune memory to infectious agents such as viruses (e.g. hepatitis B, influenza, CMV, EBV) [20, 29,30].

Multiparameter flow cytometry

Flow cytometry is the most common method for analyzing cell subpopulations using laser technology. In flow cytometry, specific fluorescently labeled antibodies identify the expression of intracellular or surface membrane antigens. This makes it possible to determine the absolute number and relative proportion of cell subpopulations, as well as to estimate rare events. For reviews, see [24, 31]. It is characterized by high specificity and sensitivity and allows the analysis of thousands of cells per second, as well as the sorting of cells with certain phenotypic characteristics. Analysis of antigen-specific MBCs and LLPCs is challenging because in peripheral blood they circulate at a very low frequency (<0.01% of the total B-cell pool).

Dividing B cell subpopulations based on expression of CD markers

Individual subpopulations of B cells heterozygous after contact with an infectious agent can be defined based on the expression of a specific set of surface CD markers (Table 1). By multiparametric flow cytometric analysis, circulating B cells can be classified into distinct stages of maturation and differentiation. As previously reviewed [13,32], using CD38 and immunoglobulin IgD as differentiation markers, B cells can be divided into different populations according to their stage of differentiation in lymphoid organs. Depending on CD27 as the main marker of memory B cells, together with the surface expression of IgD, B cells are divided into four distinct subpopulations: IgD + CD27-B cells represent naïve B cells, the expression of CD27 and the loss of surface expression of IgD on B cells is a characteristic of class-switched B cells. B cells expressing CD27 and IgD are characterized as non-class-switched memory B cells or marginal zone-like B cells. Plasmablasts or plasma cells can be identified by increased expression of CD38 and CD27 compared to memory B cells [13,32,33].

Table 1. B cells subpopulations based on their CD markers expression.

B cell subpopulation	CD markers	Location
B-1 cells	CD5, CD11b	Mostly in peripheral blood
B-2 cells	CD19, CD20, CD21, CD22, CD23, CD24	Peripheral blood
Naive B cells	CD19, CD20, CD21, CD22, CD24, IgD, IgM	Peripheral blood, bone marrow
Transitional B cells	CD19, CD20, CD21, CD24, CD38, IgD	Bone marrow, spleen, lymph nodes
Memory B cells
(A) NCSMB cells	CD19, CD20, CD21, CD27, CD24, IgM	Peripheral blood, bone marrow
(B) CSMB cells	CD19, CD20, CD21, CD27, CD24, IgG	Peripheral blood, bone marrow
Marginal zone B cells	CD19, CD21, CD23, CD35	Peripheral blood, spleen
Follicular B cells	CD19, CD20, CD21, CD23, CD35	Peripheral blood, lymph nodes
Germinal center B cells	CD19, CD20, CD23, CD38, CD77	Germinal centers (secondary lymphoid organs)
Plasmablasts	CD19, CD20, CD27, CD38	Peripheral Blood
Plasma cells	CD19, CD38	Bone marrow
Regulatory B cells (Bregs)	CD19, CD24, CD38, CD1d, IL-10, TGF-β	Peripheral blood, lymph nodes, spleen

Detection of antigen-specific B cells by the binding of antigen to the BCR

Analysis of antigen-specific B cells by flow cytometry is based on binding to BCR a labeled antigen with an appropriate fluorescent label to allow detection. Fluorochromes can be attached covalently by chemical conjugation to the antigen or attached non-covalently by biotinylation of the antigen [34]. After biotinylation, the antigen is coupled to fluorochrome-conjugated streptavidin to produce a labeled tetramer of the antigen [35]. A significant advantage of tetrameric antigen complexes is that they can be constructed with streptavidin labeled with bright fluorochromes. Such antigenic tetramers allow sensitive identification and isolation of antigen-specific memory B cells by flow cytometry, despite their low frequency. An important consideration is the possibility of nonspecific binding of streptavidin or fluorochrome to polyreactive plasma and B cells. Binding between fluorochromes, linkers or streptavidin and BCRs in humans who have never been exposed to these antigens is usually of low affinity, and these BCRs are usually expressed by naïve and potentially polyreactive B cells. Potential nonspecific binding can be distinguished from specific binding by dual labeling, in which the same antigen is labeled separately with two different fluorochromes [35,36]. Thus, by identifying double-positive B cells is possible to rule out non-specific binding of B cells to the fluorochrome. However, even when tetramers are used by double labeling, streptavidin-specific B cells can contaminate the double-positive population [37]. To avoid completely non-specific reactions of the fluorochrome, streptavidin and linkers, it is recommended to use a “decoy” streptavidin to identify these ‘non-specific’ B cells. Thus, decoy-binding B cells can be excluded from true antigen-specific B cells. Because of the low frequency of memory B cells, it is necessary to reduce the background as much as possible [25,35,36]. One of the greatest advantages of multiparameter flow cytometry is the ability to further characterize antigen-specific B cells by expression of cell surface markers and intracellular staining. In this way, subpopulations of both naïve and memory B-cells and plasma cells can be distinguished. As previously outlined [35], another important advantage of the flow cytometric method is the ability to isolate cells of interest by fluorescence-activated cell sorting (FACS) for subsequent analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling [35,38–40]. The method has practical application for the detection of specific cells in EBV, CMV and SARS-CoV-2 infections [41–43].

Detection of rare subpopulations

Detecting specific MBCs (memory B Cells) and LLPCs (long-lived plasma cells) in peripheral blood can be challenging due to their low frequency. To address this, enrichment or separation methods are employed. Presently, magnetic beads conjugated with specific antibodies are widely used. Negative selection with a cocktail of antibodies (CD2, CD3, CD14, CD16, CD56) and glycophorin A helps initially enrich B-cells by eliminating T-cells, NK-cells, monocytes and others. This process removes most cells that might nonspecifically bind the tetramer [38–40]. The enriched cells are then labeled with specific markers to distinguish individual subpopulations. This approach allows the detection of cells at frequencies as low as 0.01% to 0.0005% in the sample [36].

As previously reviewed [34], recent methods have been developed to enhance sensitivity in detecting rare antigen-specific B cells. Magnetic nanoparticles, conjugated to antibodies targeting the fluorochrome on the antigen of interest, enable the enrichment of antigen-specific B cells before flow cytometry. This is particularly beneficial for identifying rare antigen-specific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts. Magnetic enrichment streamlines the analysis of more cells in a shorter time by concentrating the cells of interest before flow cytometry [35,44].

However, when identifying cells at very low frequencies, inherent background noise in the detection system can be amplified, especially for weak signals. Therefore, crucial considerations include: 1. use of decoys to exclude non-specific B cells, 2. avoid spillover, 3. selection of bright fluorochromes (R-phycoerythrin or allophycocyanin) [35].

Single-cell sequencing

Single-cell sequencing has emerged as a transformative tool for understanding cellular heterogeneity. Specifically, when applied to B cell subpopulations, it unveils crucial insights into the diversity and functional states of these immune cells [45]. ScRNA-seq provides an in-depth view of the transcriptome of individual B cells. This technique is invaluable for several applications in B cell research. It allows for the assessment of B cell receptor (BCR) diversity, shedding light on antigen specificity. Moreover, scRNA-seq enables the identification of various functional states, such as naive, memory and plasma cells, as well as the mapping of their differentiation trajectories. It also provides insights into how different subpopulations of B cells respond to different stimuli or pathogens. The unbiased profiling offered by scRNA-seq is particularly powerful for uncovering rare subpopulations and characterizing their unique functional states.

This information is invaluable for understanding the molecular mechanisms underlying B cell differentiation, antibody production and immune response regulation. Additionally, scRNA-seq enables the exploration of transcriptional regulatory networks governing B cell fate decisions, such as lineage commitment and terminal differentiation. Through integrative analysis with other omics data, such as epigenomics and proteomics, scRNA-seq contributes to a comprehensive understanding of the regulatory landscape shaping B cell behavior in health and disease. Furthermore, the high-throughput nature of scRNA-seq allows for the simultaneous profiling of thousands of individual B cells, enabling the detection of subtle transcriptomic changes associated with rare cell populations or dynamic cellular processes within the immune system [46]. It can lead to the discovery of novel B cell subsets previously unknown to science [47–49]. On the other hand, Single-cell DNA sequencing (scDNA-seq) focuses on sequencing the DNA of individual cells. This technique is instrumental in identifying rare genetic variants or mutations within B cell populations. By analyzing DNA, researchers can pinpoint somatic mutations, critical in processes like affinity maturation. Additionally, it offers the ability to identify genetic variations that may lead to genomic instability in specific B cell subsets. scDNA-seq also aids in revealing clonal relationships and evolution within B cell populations. The sensitivity of this technique is particularly advantageous for detecting rare genetic variants or mutations, providing a comprehensive view of genomic changes within individual B cells. When combined with scRNA-seq, it allows for a holistic understanding of both genomic and transcriptomic alterations [49].

Table 2 compares the advantages and disadvantages of all the presented methods.

Table 2. Summary of advantages and disadvantages of techniques for analysis of B-cell subpopulations.

Method	Advantages	Disadvantages
ELISpot	- Highly sensitive - Single-cell resolution - Quantitative data	- Limited multiplexing - Subjectivity in spot counting - Limited spatial information - Cell viability requirement
FluoroSpot	- Multiplexing capability - Sensitivity and specificity - Single-cell resolution - Quantitative data	- Cost - Complex data analysis - Spot overlapping - Subjectivity in spot counting - Limited spatial information
Flow cytometry for B cell subpopulations	- High throughput - Multi-parameter analysis - Cell phenotype and function - Identification of rare cell subsets - Sorting capabilities—dynamic analysis - Biomarker discovery	- Complexity of instrumentation - Cost and maintenance - Data analysis complexity
MFC with tetramers for specific B cells	- High specificity - Quantitative analysis - Multiparametric analysis - Identification of rare specific B cell populations - Dynamic analysis - Sorting capabilities—compatibility with functional assays	- Tetramer availability - Specificity and affinity of tetramers - Complexity of instrumentation and analysis - Cost and resource intensiveness - Cell viability requirement - Data analysis complexity
Single-cell sequencing	- Provides transcriptomic information at single-cell level - Allows for unbiased discovery of cell populations and states - Enables the study of cellular heterogeneity - Provides information on cell–cell interactions and signaling pathways	- Technically complex and resource-intensive - Requires specialized bioinformatics expertise - Limited sensitivity for low-abundance transcripts - May require a large number of cells for certain protocols

Conclusions

In conclusion, the diverse techniques discussed in this mini-review offer valuable insights into B-cellular immune responses. Each method has distinct strengths and considerations. ELISpot and FluoroSpot provide sensitive, single-cell resolution data. Flow cytometry enables high-throughput, multi-parameter analyses. Multiparameter flow cytometry with tetramers enhances specificity. Single-cell sequencing revolutionizes transcriptomic insights. Choosing the right technique depends on research objectives and available resources. Combining methods can provide a comprehensive view. These advances deepen our understanding and hold promise for vaccine development, autoimmune disease management, and personalized medicine. The establishment and persistence of B cell-facilitated immunological memory are critical components of long-term protection against bacterial and viral pathogens. Investigating the dynamics of memory B-cell populations and their ability to confer persistent immunity is central to shaping vaccination strategies and providing durable protection against pathogens. As research progresses, we expect even greater strides in manipulating B-cell immunity, for example, deepening knowledge of B-cell memory in SARS-CoV-2, can improve pandemic management.

Author contributions

Writing—original draft, MB; review and editing, PG and VR; supervision, HT and MM.

Acknowledgements

This work was supported in part by the following grants: (1) КОВ-3/2021 and (2) КП-06-ПН53/12 Duration of immunological memory after vaccination against COVID-19—cellular and humoral immunity defined by memory T and B cells subsets.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.