B Cell Overview

B cells, also known as B lymphocytes, are a type of white blood cell that plays an essential role in the adaptive immune response [1]. B cells produce high-affinity antibodies, generate immunological memory, act as antigen-presenting cells, and secrete cytokines including CCL22, CCL17, IL-2, IL-4, IL-6, IFN-gamma, TNF-alpha, GM-CSF, IL-10, TGF-β1, IL-35. Memory and plasma B cells produce antibodies including immunoglobulin (Ig) IgM, IgG, and IgE. This page describes the development of mature B cell types and tools to study B cells including cell culture, immunoassays, and cell markers for immunophenotyping.

B cell development in specific organs and niche environments is coordinated by expression of transcription factors [2, 3, 4, 5, 6, 7]. Each cell fate depends on the activation and silencing of certain B cell genes and this developmental state can be assayed by the expression of surface and intracellular markers. Most B cell development takes place in the bone marrow (BM) followed by mature cell development in secondary lymphoid organs (SLO) and then eventually circulation in the blood.

B cells in the BM are derived from hematopoietic stem cells and differentiate into either common lymphoid progenitors or multipotent progenitors (Figure 1). Common lymphoid progenitor cells give rise to either T-lineage cells, natural killer cells, or B-lineage cells. B cell lineage cells undergo positive and negative selection within the specified BM niches.

Select B cells can migrate to SLO including spleen or lymph nodes. B cells migrating from the BM to the blood and spleen are referred to as transitional 1 (T1) B cells. Upon entering the spleen, T1 B cells can mature to transitional 2 (T2) B cells. In the spleen, the T2 B cells will either become follicular (FO) B cells, marginal zone (MZ) B cells, or B1 cells (in humans). In mice, the B1 transition occurs in the BM. The SLO is the site where a B cell binds to an antigen via its B cell receptor (BCR) and undergoes activation. B cells are activated in a T cell–dependent or –independent fashion (Figure 2 and Figure 3). In general, the FO B cells undergo T cell–dependent activation, whereas the B1 and MZ B cells favor T cell–independent activation.

T cell–independent activation of B cells requires that the B cell receptor (BCR) cluster at the surface of the B cell (also referred to as crosslinking), along with a secondary signal provided by Toll-like receptor (TLR) engagement (Figure 2). BCR crosslinking occurs when the BCRs encounter evolutionarily conserved repeat epitopes on the surface of bacteria or viruses [1].

T-cell dependent activation of a B cell requires two signals. The first is the crosslinking of the BCRs by binding to antigen surface molecules on foreign targets or free soluble antigens. The second is signaling through the CD40 receptor on T cells (Figure 3).

Figure 3. T cell–dependent activation of B cells. This type of activation produces memory and plasma B cells. Naïve B cells bind to soluble or membrane bound antigens and internalize the antigen. The B cell presents this antigen on a MHCII and is recognized by a helper T cell (Th). A second signaling mechanism is required through CD40 receptor and ligand binding. Cytokines are secreted by the Th cell and this activates the B cell into differentiating into mature B cells capable of IgG, IgA, or IgE production [9].
 

Follicular (FO) B cells

Follicular (FO) B cells arise from the germinal center of lymphoid follicles and comprise the main B cell subset in both human and mice [1]. They are mostly found circulating through the blood; however, some are also present in the SLO [4]. A small subset arises from BM through T cell–dependent activation, whereas most FO B cells are activated in the spleen in a T cell–independent fashion. Their development is driven by strong BCR signaling and a blockade of Notch2 signaling. These cells generate the majority of high-affinity antibodies during an infection. These cells interact with T follicular helper cells [9].

Marginal zone (MZ) B cells

Marginal zone (MZ) B cells serve as the first line of defense against blood-borne pathogens [5, 6]. Found in the MZ of the spleen and other lymphoid tissue, they are well placed to encounter large amounts of circulating blood and the pathogens that blood may carry. MZ B cells are the only B cells that depend on Notch2 signaling for proliferation. In humans, MZ B cells can be found in circulation, as well as in other parts of the body, whereas in mice, MZ B cells are immobile and reside in the spleen.

Plasma cells

Plasma cells develop high-affinity antibodies towards their targets in germinal centers, providing immunological memory [9, 10, 11, 12, 13]. Initially, B cells in the SLO germinal center are activated due to interaction with T - helper (CD4) cells. During this activation B cells undergo somatic hyper-mutation and affinity maturation, which improves the B cell’s antibody (BCR) affinity for its own specific foreign antigen. The activated B cell can undergo BCR receptor isotype class switching, ie. IgG and IgE and differentiate further to become antibody - producing Plasma cells. Plasma cells produce more antibodies than plasmablasts_. In fact, their sole purpose is to produce hundreds to thousands of antibodies per second per cell. Plasma cells in the BM are the main source of circulating antibodies; however, with this massive antibody production, plasma cells lose their ability to act as antigen-presenting cells and tend to be short lived. In general, it is thought that replenishment of short-lived plasma cells is provided by the activation of memory B cells [14, 15].

Memory B cells

In contrast to the plasma cells, memory B cells have a long lifespan, lying dormant until they reencounter their antigen [1, 2]. Memory B cells can immediately start producing antibodies against their antigen upon re-exposure. With re-exposure to their foreign antigen, memory B cells immediately reactivate and differentiate into antibody secreting plasma cells. IgG isotype antibodies are secreted from these plasma cells, with high affinity for their specific foreign antigen. These IgG antibodies are effective at neutralizing viral and bacterial antigens quickly, as part of a secondary immune response that includes immunological memory (especially from memory B and memory T lymphocytes).

The quintessential characteristic of a B cell is the B cell receptor (BCR) and this contributes to adaptive immunity. The BCR is an antibody that is anchored on the B cell membrane and functions to stimulate activation of the B cell by intracellular signaling. This transmembrane receptor consists of two heavy chains and two light chains that are made by recombination of the Variable (V), Diversity (D), Joining (J), and Constant (C) regions of the gene (Figure 4) [16]. Each mature B cell only produces one type of BCR with a single specificity, and therefore produces only antibodies with the same single specificity.

Figure 4. B cell receptor Ig Heavy chain. The Variable (V), Diversity (D), Joining (J), and Constant (C) regions of the BCR recombine to make Igs. Complementarity-determining regions (CDRs) are part of the variable chains that bind to a specific antigen. Framework Regions (FR) are conserved sites within the variable (V) region on the BCR.
 

Isolated B cells will not replicate without stimulus. B cells exposed to exogenous signals to activate and replicate will divide under culture conditions [19, 20]. One signal is provided by the BCR and this signal can be mimicked using anti-IgM or IgD antibodies. The second signal is achieved through engagement of co-stimulatory molecules such as CD40 and cytokine signaling such as IL-4. Alternatively, components of bacterial cell walls, such as lipopolysaccharide (LPS), and antigens with highly repetitious molecules may signal B cell activation directly [21].

For example, if culturing mature, naïve or memory B-cells, media with CD40L expressing stromal feeder cells with a cocktail of specific cytokines including IL-2, IL-4, IL-21, and BAFF can be used [22]. The activated B cells can then differentiate further in co-culture with Th cells. The Th cells bind and stimulate the co-cultured B-cells. Activated B cells will incorporate CFSE or BrdU and exhibit cell proliferation.

Espstein-Barr virus (EBV) infected transformed lines transform resting B cells into continuously proliferating lymphoblastoid cell lines (LCL).There are multiple transformed cell lines available derived from cancer samples or those infected with Epstein-Barr virus (EBV) including Akata, Mutu I, and Mutu III cells. These cell lines can be grown in RPMI medium supplemented with 10% fetal bovine serum (FBS) [18].

Several optimized multicolor immunofluorescence panels (OMIPs) have been published that can be used to design flow cytometry panels for phenotyping B cell populations (Table 1).

Table 1. Published OMIPs for phenotyping B cells.

When analyzing B cells by flow cytometry, gating is relatively straight forward for B cells. Most mouse and human B cell panels should include cell surface markers CD19 or CD20. B220 can be used as a marker for mouse B cells and not a pan B cell marker in humans. Figure 5 illustrates an example gating strategy for phenotyping B cells using CD19 and IgG expression.

Figure 5. Example gating strategy for phenotyping B cells. SLO tissues (spleen, lymph node, tonsil) and blood can be isolated and put into single cell suspension in FACS buffer for antibody staining. Once stained with an antibody cocktail, the cells may be acquired by a flow cytometry analyzer. Cells should be gated in the following order: Single, live cells should be gated first. Dump channel or a combination of markers with the same fluorochrome that mark other cell lineages including T, NK, monocytes, macrophages, and dendritic cells will ensure a purer population of cells. Live subpopulation should then be positive for CD19 and IgG expression. FSC-A: Forward Scatter Area in linear scale, this measures cells size; FSC-W: Forward Scatter Width in linear scale, this is used to discriminate doublet cells by size; SSC-W: Side Scatter Width in linear scale, this is used to discriminate doublet cells by granularity.
 

A variety of cytokines impact the growth, survival and isotype class switching in the development of B cells. IFN- alpha and beta are involved in generation and selection of B cell in the bone marrow whereas IL-4 and IL-6 influence the preferential secretion of certain classes of antibodies by B cells. IL-7 is an important cytokine that mainly act on developing B cells and acts as a survival factor.

Table 3: Key cytokines involved in B Cell differentiation, proliferation, survival, recruitment, isotype class switching, and secretion

Naïve B cells do not secrete many cytokines but activated B cells produce several pro and anti-inflammatory cytokines which influence multiple aspects of immunity including the development of effector and memory CD4+ T cell responses. Based on cytokine production B cells can be subdivided into anti-inflammatory cytokine producing “regulatory” and pro-inflammatory cytokine producing “effector” B subsets. In addition to cytokine, activated B cell also secrete a variety of chemokines such as CCL22 and CCL17 which are involved in the recruitment of Th2 cells. The Invitrogen Cytokine & Chemokine 34-pex human panel and the 36-plex mouse cytokine panel provide a cost-effective way to measure B cell associated cytokine production in plasma, serum, or cell cultures. In addition to detection of cytokines and chemokines, multiplexed isotyping panels provides a convenient way to determine the class (e.g., IgG vs. IgM) and subclass (e.g., IgG1 vs. IgG2a) of a monoclonal antibody which is a critical and beneficial tool in hybridoma development.