B Cells, Memory B Cells and Plasma Cells: B Cell Activation, Development and the B Cell Receptor

The immune system consists of two arms, both of which are essential in protecting the body against disease. The innate immune system is the first responder and identifies invaders and non-self cells through general microbial associated molecular patterns (MAMPs) and damage associated molecular patterns (DAMPs). In comparison, the adaptive immune system is highly specific and relies on the binding of specific peptide sequences to complementary receptors on T and B cells. In this article, we focus on B cells, and discuss their development, their roles and what happens when they don’t function as they should.

B cells, also known as B lymphocytes, are immune cells that drive the humoral adaptive immune response. They are one of three types of lymphocyte in the adaptive immune response – the others being T cells and natural killer (NK) cells.¹ B cells are distinguished by their ability to produce antibodies (known as immunoglobulins (Ig) when attached to B cells via a transmembrane domain), and the structure of their surface antigen receptor – the B-cell receptor (BCR), which is genetically and structurally similar to antibodies. B cells can also be identified by a small number of cluster of differentiation (CD) surface markers, including CD19 and CD20.²

B cells were first characterized in the 1960s and early 1970s, when Max Cooper and Robert Good showed that surgical removal of the bursa of Fabricius (a primary immune organ in birds) rendered chickens unable to produce Ig.³

Subsequently, mouse models were used to identify the lymphocytes that mediated these Ig responses.⁴ These cells were then further characterized by the discovery that surface expression of Ig was a marker of B lymphocytes.⁵ It was from the original bursa of Fabricius that the name “B” cells was derived. Since then, B cells have been studied extensively, and we now have a deep understanding of their complexity, their development and their role in immune response and regulation.

B cells develop from hematopoietic precursors in the bone marrow (Figure 1). Once fully matured, B cells enter circulation and populate peripheral lymphoid organs, such as lymph nodes. Each naïve B cell has a unique BCR that recognizes a specific antigen, activating the B cell. B cells can be activated in both a T cell-dependent and a T cell-independent manner. Once activated, B cells undergo clonal expansion, proliferating in lymph nodes and producing a large population of B cells with identical BCRs. Thus, the proliferated B cells will all recognize the same antigen, targeting it for destruction. Most proliferating B cells will differentiate into antibody-producing plasma cells, though a small proportion will become long-lasting memory cells.

Figure 1: Diagram showing the differentiation pathway for the development of B cells. Credit: Technology Networks.

BCRs expressed on the B-cell surface allow the activation of the cell by its specific antigen. The BCRs have two functions:

• Internalize and process antigens for presentation to T cells
• Transduce activation signals to the nucleus

BCRs consist of a transmembrane-bound Ig in complex with two transmembrane polypeptides: Igα and Igβ (Figure 2). These two polypeptides (also known as CD79A and CD79B respectively) are linked to immunoreceptor tyrosine-based activation motifs (ITAMs), which carry the activation signal across the cell membrane. This initiates a signaling cascade that results in the expression of genes coding for proliferation, survival and differentiation into plasma or memory cells.⁶

Figure 2: The B-cell receptor. Credit: Technology Networks.

The BCR Ig is comprised of four domains: two identical light chains and two identical heavy chains, which are linked together by multiple disulfide bonds. Both the light and heavy chains have constant and variable regions, and it is these variable regions that convey antigen specificity and bind the antigen. The two sets of identical variable regions enable simultaneous binding to two identical antigens, thereby increasing the total strength (avidity) of the interaction. The strength of the interaction between an individual binding site and antigen is known as the affinity.

The adaptive immune system relies on an enormous range of T and B-cell receptors to bind specifically to an equally large number of MAMPs. In order to allow a relatively small number of genes to encode the high numbers of unique receptors required, a gene rearrangement process called V(D)J recombination is employed. BCR genes are made up of multiple DNA segments at three different genetic loci, which are randomly combined in developing lymphocytes. Two random V and J segments are combined to make the variable regions of the light chains (V_L), while variable regions of heavy chains (V_H) are comprised of randomly selected V, J and D segments. The segments are brought together by RNA splicing.

However, this combinational diversity can only account for some of the variation in BCR receptors, there are not enough different segments to account for the number of different BCRs. The remaining variation is produced by junctional diversity, as extra short nucleotide sequences are added or deleted at the junctions between segments during recombination.

B-cell development begins in the bone marrow, where hematopoietic stem cells (HSCs) go through a process called hematopoiesis to become common lymphoid progenitor cells before differentiating into T cells, B cells or natural killer (NK) cells. The earliest form of B cell is known as a pro-B cell (Figure 3). At this stage of development, the cells do not express any immunoglobulins on their surface, but they do express other B-cell markers, such as CD19.

In the pro-B-cell stage, gene rearrangement begins, and the V, D and J gene segments for the heavy chain variable region are recombined. Once this variable section is created, the constant section of the receptor is produced, and the cell differentiates into the pre-B-cell stage, which is an important maturation checkpoint. During the pre-B-cell stage, part of the constant region of the BCR is transiently expressed on the cell surface as a pre-B-cell receptor. Signaling through the pre-B-cell receptor aids finalization of the gene rearrangement at the heavy chain variable region and induces further cell differentiation and initiation of the light chain variable region.⁷ Following rearrangement of all the variable regions, the BCR is completed and expressed on the surface of the cell as IgM, and the cell becomes an immature B cell. Cells that express these surface immunoglobulins receive signals to prevent any further gene rearrangement and ensure that only one particular variable region is expressed on any one cell.

Figure 3: B-cell development. Credit: Technology Networks.

Before immature B cells can leave the bone marrow to finish the final stages of maturation in the spleen, they must undergo selection. Due to the random nature of variable regions, all B cells must be tested to make sure they do not recognize (and are therefore not reactive against), antigens on the body’s own cells. Immature B cells come into contact with a variety of self-antigens. If recognition occurs, these cells are negatively selected, and are rendered harmless by anergy (the cells remain alive, but completely unresponsive), clonal deletion (the cells die by apoptosis) or receptor editing (the BCR is rearranged again to create a different, non-self-reactive combination). Any cells that do not recognize self-antigens are positively selected and migrate to the spleen.

In the spleen, the B cells will begin to express both IgM and IgD on their surface, completing their maturation. They are finally ready to begin fighting pathogens! Large concentrations of B cells are found in follicular areas of secondary lymphoid organs such as lymph nodes, where they are most likely to come into contact with their specific antigens.

Figure 4: B-cell activation. Credit: Technology Networks.

B-cell activation depends on the binding of a specific antigen to the BCR, and a secondary signal from an activated helper T cell – T cell dependent activation (Figure 4). Stimulation of the BCR both activates the B cell, and triggers internalization of the antigen. Once inside the B cell, the antigen is processed and broken down into short polypeptides, which are then trafficked to the B-cell surface on the major histocompatibility complex (MHC) class II. The combination of MHC class II and antigen are presented to complementary helper T cells that recognize the same antigen. The interaction of the presented antigen with the T cell receptor activates the helper T cell, which in turn begins to secrete cytokines that induce differentiation of the B cell into a plasma cell and the mass proliferation of plasma cell clones. This ensures the rapid production of large amounts of antibodies to bind and neutralize the invading pathogen. Cytokines produced by the helper T cells also direct antibody class switching, dictating which classes of antibody are produced, and triggering the production of long-term memory cells.⁶

Very large antigens with repeating motifs, such as bacterial polysaccharides, can activate B cells in a T cell-independent manner, by crosslinking several BCRs simultaneously. As in T cell-dependent activation, the B cells proliferate, and plasma cells are produced, but without cytokine direction from helper T cells, class switching will not occur, and only IgM class antibodies are produced.

In a primary response, i.e., the first time a specific antigen is encountered, the process of B-cell activation and antibody production takes time. A lag of approximately ten days occurs before antibodies are detected in the blood. Antibody levels then continue to increase, peaking around 2–3 weeks after antigen exposure. However, if a re-exposure to the same antigen occurs, a secondary immune response is triggered and the lag time is far shorter. Memory B cells are reactivated, and rapidly proliferate and differentiate into plasma cells. Any plasma cells produced during a secondary immune response produce higher levels of antibody for a longer period of time. This results in a faster, stronger immune response, which either clears the pathogen before it becomes established and induces symptomatic disease, or results in a milder, shorter disease instance than the primary infection.

The primary function of plasma cells is to produce enormous amounts of secreted antibodies. Plasma cells tend to live only for a few days, but can produce antibodies at a rate of around 2,000 antibodies per second, providing a rapid response following antigen recognition.⁸ The antibodies produced by these short-lived plasma cells almost exclusively produce the IgM class of antibodies.

Some proliferating B cells will enter germinal centers in the follicles of lymph nodes to undergo further differentiation, a process called “affinity maturation” or “somatic hypermutation”. In this process, the genes encoding the variable regions of the BCR undergo point mutations to produce antibodies with altered binding sites. Follicular dendritic cells present the target antigen to the altered BCRs, in an attempt to find B cells producing optimized and improved antibodies with higher antigen affinity. Only B cells with improved affinity are selected, while those with disadvantageous mutations undergo apoptosis.

Optimized plasma cells have a much longer lifespan than short-lived plasma cells, and can survive for years after antigen exposure.⁹These plasma cells may also undergo antibody class switching, allowing the production of different antibody classes, e.g. IgG, IgA and IgE. Different antibody classes have distinct effector functions, allowing a B-cell response to be tailored to the type of pathogen present. As antibody classes are dictated by the tail region of the antibody, the variable region remains the same, and antigen specificity is unaffected.

Memory B cells are long-lived B cells that arise during the initial adaptive response to a pathogen, in order to generate a rapid and robust response in the event of a repeat exposure to the same antigen. Memory B cells can survive for years, if not decades.¹⁰ To enable rapid antibody responses and prevent a pathogen becoming established, memory B cells must be positioned in areas where the chance of encountering their specific antigen is high. Thus, large reservoirs of memory B cells are found in the spleen and lymph nodes.¹⁰ Memory B cells can be identified by the presence of class-switched Ig (i.e., IgG, IgA or IgE) on the cell surface. In comparison, naïve B cells express only IgD and IgM, while plasma cells express very little surface Ig at all.

In the adaptive immune response, B cells control the antibody-based humoral response, while T cells drive the cellular response. Although both T and B cells are classed as lymphocytes, there are some clear differences between their structure and their roles (Table 1). Both T and B cells begin their development in the bone marrow as hematopoietic stem cells, but while B cells finish their development in the bone marrow, developing T cells leave the bone marrow relatively early, finishing their maturation in the thymus.

Receptors that recognize specific antigens are found on the surface of T and B cells, but T cell receptors (TCRs) have a different structure to the BCR, that does not resemble an antibody. T cells rely on antigen-presenting cells (e.g., macrophages, dendritic cells, neutrophils) to bring antigens into contact with their TCRs in order to activate the cell and the adaptive immune response.

Table 1: The differences between T cells and B cells

Characteristic	B cells	T cells
Site of maturation	Bone marrow	Thymus
Receptor	Immunoglobulins	T cell receptor
Antigen interaction	Responds directly to antigens in the lymph nodes	Interacts with antigens via antigen-presenting cells
Types of active cells	Plasma cells, memory cells	Cytotoxic cells, helper cells, regulatory cells, memory cells
Targets	Extracellular pathogens, e.g., bacteria, parasites	Intracellular pathogens, e.g., viruses, intracellular bacteria
Functions	Produces antibodies that bind to and neutralize pathogens	Recognizes and kills infected cells, aids B cells in antibody production and modulates immune responses

B cells play an important, powerful role in the immune response, but can cause severe issues if not functioning correctly. The immune system can malfunction in several different ways:

• Autoimmunity (a self-directed immune response)
• Hypersensitivity (an immune response against an innocuous antigen
• Immunodeficiency (a reduced or absent immune response)

B cells can also become cancerous and defective, giving rise to B-cell malignancies.

B-cell autoimmune diseases

Autoimmune disorders result from the immune system reacting to self-antigens causing an unwanted immune response. This occurs due to a breakdown in tolerance, when self-reactive immune cells are not removed from circulation during cell development. Self-reactive B cells produce autoantibodies, which target a body’s own cells for destruction.

One such example is systemic lupus erythematosus (SLE), a systemic autoimmune disease driven by autoantibodies targeting a variety of self-antigens, including chromatin, double stranded DNA and RNA-associated proteins. Due to the ubiquitous nature of the self-antigens targeted, SLE manifests in multiple organs, with clinical signs including neurological damage, inflammation in the lungs, splenomegaly, arthritis and skin rashes. There is no one cause of SLE, but a range of triggers have been associated, including cell damage and viral infections, coupled with susceptible genetics.¹¹ Cell damage causes excess apoptotic debris, which, if coupled with a loss of tolerance, can trigger an immune response and the production of self-antibodies by self-reactive B cells. This immune response can cause further cellular damage and production of more self-antigens, creating a positive feedback cycle and further inflammation. SLE can be difficult to treat given the number of different organs and tissues involved, but treatment regimens consist mainly of immunosuppressive drugs such as corticosteroids.

B cells in hypersensitivities

A hypersensitivity is an inappropriate or over-reaction of the immune system to an innocuous environmental antigen. Hypersensitivities are classified into four different types, of which three (type I, type II and type III) are mediated by antibodies and therefore involve B cells. Type IV hypersensitivities are driven by T cells. Type I hypersensitivities manifest as allergic reactions and are by far the most common of all immune disorders.

A type I hypersensitivity arises when an allergen is taken up by an antigen-presenting cell and presented to a helper T cell. The helper T cell then activates B cells and initiates the production of antibodies – specifically IgE. The IgE is captured by mast cells and basophils, where it remains until subsequent exposure to the same antigen. On re-exposure, the allergen crosslinks IgE on the surface of the mast cells and basophils, leading to rapid activation and degranulation of these innate immune cells and the release of highly inflammatory molecules such as histamines.¹² Type I hypersensitivities can range from mild to highly severe and potentially fatal anaphylactic reactions. However, they can be treated by simple evasion of the allergen, anti-inflammatory and anti-histamine medication for mild to moderate reactions and adrenaline auto-injectors for severe allergies.

In comparison, type II hypersensitivities occur when IgG and IgM are generated in response to cell surface modifications, or cell-matrix associated antigens being regarded as foreign by the immune system. The antibodies then activate the complement system or NK cells, resulting in cell damage. The most common cause of type II hypersensitivities are medications such as penicillins and cephalosporins.¹³

Type III reactions are mediated by the aberrant formation of large antibody–antigen aggregates known as immune complexes. These complexes can be deposited in blood vessels, which activates the complement system and results in tissue damage. Type III reactions can occur as a symptom of SLE, or as a reaction to drugs that contain proteins from other species.¹⁴

B-cell immunodeficiencies

Immunodeficiencies arise when one or more components of the immune system are defective or absent, resulting in increased susceptibility to infection. Genetic immunodeficiencies are rare, but the most common immunodeficiencies tend to involve B cells and result in antibody disorders. Selective IgA deficiency, characterized by decreased or absent IgA in serum, is the most common genetic immunodeficiency, and can be caused by a variety of genetic mutations.¹⁵ The majority of IgA deficient patients are asymptomatic, though some are prone to recurrent respiratory and gastrointestinal infections, as well as increased risk of allergies and autoimmune disorders.

In comparison, X-linked agammaglobulinemia (XLA) is a far more serious B-cell-associated immunodeficiency. XLA is caused by a mutation in the Bruton tyrosine kinase (BTK) enzyme, which is essential in B-cell development and differentiation, and therefore results in a severe to complete lack of circulating B cells and antibodies.¹⁶ Patients suffer recurrent infections and may show other symptoms including abnormal lymph nodes and chronic diarrhea. However, it can be treated with injections of antibodies against a wide range of microorganisms.

B-cell malignancies

The production of abnormal B cells results in malignancies, including lymphomas (originating in lymph nodes) and leukemias (originating in bone marrow). Most B-cell lymphomas are non-Hodgkin, meaning that they can arise in any lymph node in the body. B-cell malignancies are often triggered by chromosomal breaks or translocations in the Ig domains of the genome. Other triggers include mutations in tumor-suppressing genes and viral infection such as Epstein-Barr virus, which is found in nearly all patients with Burkitt’s lymphoma and about 40% of patients with Hodgkin lymphomas.¹⁷ B-cell malignancies result in rapidly proliferating but aberrant or non-functional B cells, which cause symptoms such as rapid weight loss, severe night sweats and unexplained fevers. These cancers are traditionally treated using radio- and chemotherapy but immunotherapies are becoming more common and improving survival rates. For example, chimeric antigen receptor (CAR) T cells are T cells, harvested from a donor or the patient themselves and genetically enhanced to improve targeting of malignant cells.¹⁸