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B LYMPHOCYTES


Lymphocyte B-Cell

Introduction

B-cells are a subtype of lymphocyte involved in the adaptive immune system. They were first discovered in the bursa (from which the B in B-cells is derived) of birds. In humans they develop in the bone marrow and migrate to secondary lymphoid organs such as the spleen and lymph nodes for maturation[1]. B cells express B cell receptors which bind to a specific antigen to elicit an immune response. Upon antigen interaction, B cells differentiate into memory B cells and plasma B cells. Plasma B cells secrete antigen-specific antibodies, which play a vital role in humoral immunity. Antibodies opsonise the antigen for phagocytosis, initiate the complement cascade and may directly neutralise toxins[2]. Memory B cells are vital to sustaining an immune response over a longer period of time in the instance of antigen re-exposure[3].

B lymphocytes are a population of cells that express clonally diverse cell surface immunoglobulin (Ig) receptors recognising specific antigenic epitopes. Each B cell produces a single species of antibody, each with a unique antigen-binding site. When a naïve or memory B cell is activated by antigen, it proliferates and differentiates into an antibody-secreting effector cell. Such cells make and secrete large amounts of soluble (rather than membrane bound) antibody, which has the same unique antigen-binding site as the cell surface antibody that served earlier as the antigen receptor. Plasma cells continuously secrete antibodies at the rate of about 2000 molecules per second.

History

Year B cell Related Discovery
1890: Antibodies were shown to mediate humoral immunity
1948: Plasma cells suggested as a source of antibody production
1956 The role of bursa Fabricius in the development of B cell lineage was defined by Glick B et. al. [4]
1960 Proposal of 'clonal selection theory' by Macfarlane Burnet, suggesting that antibodies were cellular receptors (Nobel Prize was awarded for this discovery in 1960)
1965: Two lymphocyte lineage model published by Good, Cooper and Peterson: lead to an understanding that immunity was derived from two separate origins; thymus derived (leading to T- cell production and cell mediated immunity), and bursa derived lymphocytes (leading to B- cell production and humoral immunity) [5]
1966- 1968: B cells and T cells were shown to cooperate in the antibody response, with B cells functioning in the production on antibodies and T cells functioning in the cell- mediated immune response [6]5918139
1970: B cells were shown to switch from IgM to IgG production in birds

Antibody forming B cells were accepted as being distinctly different from Thymus derived T cells [7]

1974: B cells were shown to originate from fetal liver and bone marrow (mice)
1975 Discovery of monoclonal antibodies by Cesar Milstein [8]
1976: Pre- B cells were identified by Martin Raff and Cooper[9]
1987 First x-ray crystallographic image of a major histocompatibility complex produced, providing a greater understanding of its structure [10]
2002: The term 'regulatory B cells' was first published, describing B cells that regulate the immune response by secreting IL-10 [11]
2014: Explanation of the mechanism by which B cells produce antibodies with a high affinity for a pathogen. [12]
2016 Discovery that Follicular T cell (Tfh cells) control the maturation of B cells, and promote the proliferation of those which produce highly selective antibodies [13]

Development

File:B-cell development and B-cell subsets.jpg
B-cell development and B-cell subsets[14]

Production of all B cells begins in the bone marrow, where the microenvironment encourages hematopoietic stem cells to differentiate into progenitor B (pro-B) cells [15]. Pro-B cells differ from hematopoietic stem cells by the expression of B220, CD43, and c-kit on the cell surface [15]. Once in the Pro-B cell stage, gene rearrangement occurs. In order to express a complete IgM surface molecule that is unique to that B cell, the heavy (H) and light (L) chain genes must be rearranged. This is what gives B cells the ability to respond to a vast amount of different antigens. The L and H chains both consist of a constant region and a variable region. It is in the variable region that gene rearrangement happens. In the Pro-B stage, rearrangement of the H chain genes occurs. Once rearrangement occurs, H chain gene is expressed, forming a precursor B cell receptor (pre-BCR). As reviewed in, [16] a cell that expresses a functional pre-BCR on its surface is now a pre-B cell . Signaling from this new Pre-BCR stimulates the cell to proliferate as a large pre-B cell. After 3-5 rounds of division, these cells are stimulated through BCR signalling to differentiate into small non-dividing pre-B cells [17]. The L chain genes are then rearranged. The L chain is expressed, and the immature B cells now have a complete BCR on their surface. These newly formed immature B cells migrate out of the bone marrow and travel to the spleen via the bloodstream as transitional B cells to complete their maturation [15].

Transitional B cells bridge the gap between immature bone marrow B cells, and naïve peripheral B cells. In the transition stage of development, B cells undergo various checkpoints to test for auto-reactivity. Transitional b cells that are not auto-reactive eventually develop into naïve b cells [18]. Transitional cells that are found to be auto reactive are either deleted, undergo receptor editing or become anergic [19]. Successful B cells are now ready for activation.

Location and Activation

B cells are aggregated in secondary lymphoid organs, such as the lymph nodes and spleen[1]. Lymph nodes are divided into three discrete regions. Directly beneath the subcapsular sinus is a macrophage-rich sheet that surrounds the cortex (a B cell rich zone). B cells in this region are organised into follicles. Following exposure to antigen, follicles may also contain specialized structures, known as germinal centres, which consist of rapidly proliferating B cells within a network of follicular dendritic cells[20]. Lymph nodes are strategically positioned at branches throughout the lymphatic system to enable extensive antigenic sampling of lymphatic fluid.

The macrophages in the subcapsular sinus are a distinct population to those in the medulla and have limited phagocytic activity, which enables them to present intact antigen on their cell surface. Following encounter with antigen, the macrophages accumulate various larger antigens and subsequently present these to neighboring follicular B cells. The macrophage–B-cell interactions at the subcapsular sinus allow antigen-specific B cells to acquire and internalize antigen through their BCR before their migration to the B-cell–T-cell boundary, where they may receive specific T-cell help[1].

T-cell Dependent Activation

File:B cell activation.jpeg
T cell dependent/independent B cell activation

Activation of B-cells starts with the binding of antigen to the BCR. Once bound, the antigen is endocytosed and broken down into smaller fragments. These fragments are then presented on the surface of the B cell for T cell recognition. T cells constantly survey, and loosely bind to B cells until a B cell presenting the relevant antigen is located. The T and B cells then bind strongly, and interact with each other. T cells secrete Interleukins and other stimulatory factors to activate the bound B cell. These signals cause the B cell to undergo proliferation and immunoglobulin class switching[21].

T-Cell Independent Activation

B cells may undergo Activation without the help of T cells. This is mostly found in organisms that lack T cells. Activation via this pathway begins the same way as T cell dependent activation. Antigen specific binding to the B cells BCR starts the activation process. Unlike T cell dependent activation, independent T cell activation relies on signals from other cells to fully activate a B cell [22]. One such way of doing this is through receptor clustering on the surface of the B Cell. Clusters of 10-20 antigen bound membrane molecules cross link, causing an intracellular influx of calcium. This influx of calcium recruits transcription factors to induce proliferation and differentiation [23]. While this process increases proliferation and induces differentiation, the intracellular influx of calcium does not initiate antibody secretion.

Toll-Like receptors (TLRs) are key in providing the signal required for B cells to start secreting antibodies in response to a pathogen. TLRs associate with motifs on pathogens, initiating secretion of antibody. TLRs have been found to activate B cells that have been partially activated by multiple receptors (T cell independent activation), but not B cells partially activated by single receptor binding events (T cell dependent activation)[24].

Following antigenic activation, B cells can differentiate along two distinct pathways:

1. Differentiate to form extrafollicular plasmablasts that are essential for rapid antibody production and early protective immune responses[25].

2. Activated B cells can enter germinal centres, where they can differentiate into plasma cells or memory B cells. Some of these B cells remain in the follicles and become membrane Ig-expressing germinal center cells. Others become antibody-secreting plasma cells and move to the medulla, probably on their way to the bone marrow[26].

Types of B- cells

Overview

Traditionally in immunology, immune responses are classified as either adaptive or innate. However, recent research in the field has identified a number of immune cells which do not adhere to either category, including the group of B cells referred to as innate-like B cells (ILBs). ILBs are classified as 'unconventional' B cells, and primarily encompass B1 cells and marginal zone B cells. A review of ILBs by Zhang. X [27] makes it evident that these cells differ from conventional B cells in their regulatory function. The regulatory functions of B cells, including antigen presentation and cytokine production are primarily carried out by these ILBs, while B2 cells produce high affinity, specific antibodies. Innate- like lymphocytes function generally in providing a common ground between the rapidly acting innate cells, and the slow acting T- cell dependent antibody response. An important regulatory cytokine produced by ILBs is IL-10, which acts in down-regulating the inflammatory response [28]

Differentiated B cells

File:Th cell–regulated B cell memory development.gif
Th cell–regulated B cell memory development[29]

Developed B cells migrate from bone marrow to be activated. After reaching the lymph nodes, B cells act as antigen presenting cells by internalising antigens and then presenting them to CD4+ T cells. The binding of CD4+ T cells activates the B cell causing it to differentiate.

  • Plasmablast: Plasmablasts are immature plasma cells which are generated early on in an infection. Plasmablasts do secrete antibodies but they are low in number and weak in affinity compared to plasma cells[30]. The life of a plasmablast is short as after a few days, it either dies or transcription factors Blimp-1/PRDM1 and IRF4 activate and further differentiates the cell to become a mature plasma cell[31][32].
  • Plasma cell: Plasma cells are generated in later phases of infection and unlike plasmablasts, they have a long lifespan of up to months and can reside in bone marrow as long-lived plasma cells. Plasma cells produce antibodies of higher affinity as they are modeled off the precursor B cell's receptors. These antibodies then bind to their specific antigen to neutralise or signal its destruction[33].
  • Memory cell: Memory B cells do not have any immediate effect on the current infection. Instead, they lay dormant and circulate the body to provide a stronger and more rapid secondary antibody response upon re-infection of the same specific antigen. Memory B cells are activated by signals from follicular T helper cells after recognition and binding of specific antigen[3]. These signals cause the memory B cells to either differentiate into plasmablasts and plasma cells or generate plasma cells and more memory B cells through a germinal center reaction.
Receptor editing as a major mechanism of central tolerance in B cells[34]

B-1 cells

B-1 cells are produced in the fetus and undergo self-renewal in the periphery. They are found predominantly in the peritoneal and pleural cavities as opposed to the spleen and lymph nodes[35]. B-1 cells perform the same duties as other B cells by acting as antigen presenting cells and producing antibodies. There is a difference though in that B-1 cells express greater amounts of IgM antibodies compared to the IgG isotype[36]. B-1 cells also do not form memory cells and therefore are not part of the adaptive immune system. B-1 cell receptors are polyspecific and can bind many different antigens but have a lower affinity.

B-2 cells

The three major B cell compartments in peripheral lymphoid organs[37]

B-2 cells undergo a different developmental pathway from B-1 cells as they are produced after birth and are later replaced in the bone marrow. They are also activated by thymus dependant antigens[38].

  • Follicular B cell: Follicular B cells are found in secondary lymph organs and reside in lymphoid follicles when they are not circulating the body. They constitute the majority of matured B cells from the spleen, with the latter being marginal zone B cells. During an infection, follicular B cells generate high-affinity antibodies with the help of follicular helper T cells. Follicular B cells also differentiate into memory cells[3].
  • Marginal Zone B cell: Marginal zone B cells reside in the marginal zone of lymphoid follicles in the spleen and do not circulate the body. They are involved in the early phases of the adaptive immune response against blood-borne pathogens as they do not require T cell activation, and are positioned on the marginal zone with constant contact with circulating blood[39]. Marginal zone B cells further accelerate the primary antibody response as they have a natural tendency to differentiate into plasma cells.

Regulatory B cells

Regulatory B cells are involved in the suppression of the immune system. They secrete the anti-inflammatory cytokines IL-10 and TGF-β to inhibit T cell mediated inflammatory reactions[40]. Another function of regulatory B cells is to signal cell death by interacting with the Fas ligand and Fas receptor. The generation of regulatory T cells is also promoted by regulatory B cells by skewing T cell differentiation.

Structure

Surface structures in quiescent B cells

As a cell whose functions are based on communication with other cells or antigens, it is the surface of the B lymphocyte that requires structural attention. Up until the advent of monoclonal antibody (mAb) technology, relatively little was known about the constitution of the B cell surface. The secreted immunoglobulins (see Antibody Structure), were well characterised, but structural aspects of the membrane-bound form of immunoglobulins were less well known. In the past 20 years, the use of mAbs has facilitated the discovery of approximately 10 B-cell specific surface molecules [41]. Termed clusters of differentiation (CD), these target molecules have diverse functions (see table for summary), from regulating intracellular signal transduction (CD19), acting as a membrane-embedded Ca2+ channel (CD20), and serving as a critical survival factor for germinal centre B cells (CD40). As has been discussed, these receptors also play a critical role in B cell maturation and development .

Surface molecule Function
CD19 Expressed by all B-cell lineages and regulates intracellular signal transduction.
CD20 Mature B-cell specific and functions as a membrane-embedded Ca2+ channel
CD21 Interacts with CD19 to generate transmembrane signals and informs inflammatory response
CD40 Serves as a critical survival factor for germinal centre B cells
CD179a Regulates Ig gene rearrangements during B-cell differentiation
CD179b Critical in B cell differentiation

The antigen-binding portion of the B-cell receptor (BCR) complex is a cell-surface immunoglobulin that has the same antigen specificity as the secreted antibodies that the B cell will eventually produce. It is identical to the secreted form, except that the C termini of its heavy chains attaches to the membrane. As seen in diagram below, B-cell receptors have a general structure (proceeding in the direction of the C-terminus)[42]:

  • Several negatively charged (polar) residues on the outer surface of the membrane;
  • Followed by a hydrophobic stretch of amino acids within the lipid bilayer;
  • Followed by several positively charged residues on the cytoplasmic side of the membrane.

However, an experiment by “” showed that when cells are transfected with heavy- and light-chain cDNA (derived from a cell expressing surface immunoglobulin), the immunoglobulin that was synthesized remained in the cytosol and did not translocate to the membrane. From these and similar experiments, it was apparent that other molecules must be required in order for the immunoglobulin receptor to be expressed on the cell surface. Two such proteins are Igα and Igβ [43].


These two proteins not only facilitate binding to the membrane; they also generate the signal upon ligand binding to the extracellular recognition site. The immunoglobulin spans the membrane, but it has only a very short cytoplasmic tail, which is insufficient to transmit an intracellular signal. The Igα and Igβ proteins extend further into the cytoplasm and their cytoplasmic tails each have immunoreceptor tyrosine-based activation motifs (ITAMs). The tyrosine in these motifs becomes phosphorylated, and this brings about a cascade of protein tyrosine kinase activity (e.g. Syk or Lyn activation)[44]. This enables the signal to be transmitted inside the cell. It is currently unknown whether BCRs form oligomers or monomers, since evidence has been found in support of both theories[44].

Schematic of B cell receptor. Note that it is similar to the secreted immunoglobulin form, but there are two associated intracytoplasmic proteins

B cell co-receptors are expressed on mature B cells. These are a complex of the cell-surface molecules CD19, CD21, and CD81. When antigen binds to the primary receptor, there is cross-linking with CD21 and subsequent phosphorylation of CD19. This cross-linking augments signaling through the B-cell receptor.

Dynamic structural processes: the immunological synapse and the role of the cytoskeleton

The immunological synapse is an important dynamic structure that forms upon B cell recognition of antigen. High resolution imaging of B-cell interactions with antigen-presenting cells (in vitro) has found that B cells form synapses shortly after initial contact, in a similar way to T-cells[45]. In basic terms, formation of an immunological synapse follows a sequence of structural changes, as reviewed in[46]:

  1. BCR encounters antigen→ stimulates signaling
  2. Intracellular signaling recruits more BCRs to the site via actin flow→ BCR microcluster formation
  3. Microclusters are pulled in by actomyosin contraction→ invagination of B cell membrane
  4. Further actin polymerization and formation of clathrin-coated pits→ endocytosis of BCR microclusters

The cytoskeleton plays a crucial structural role in this immunological synapse and the subsequent activation of B cells. Recent studies have examined cytoskeleton interactions with BCRs in resting and activated states of the B cell.[47] [48]. An experiment by Harwood [49] utilised total internal reflection microscopy (TIRFM) to track single BCR particles in the resting membrane. They found that the BCRs do not diffuse freely in the plane of the membrane in a resting B cell (contrary to Singer and Nicholson’s Fluid Mosaic Model). This was explained by their finding of co-incident regions of high-density actin filament networks. Actin filaments tether the BCR to a confined region of the membrane before its activation by antigen. As reviewed in [50], pharmacological agents that broke down the actin network resulted in intracellular signalling similar to that observed upon antigen stimulation. These findings have led researchers to the conclusion that the cytoskeleton plays a structural role in regulating distribution and dynamics of the BCR in the resting cell. Further studies by Batista et al. (2008) [51] found that B cells reorganize their actomyosin cytoskeleton, initially spreading over the surface of the antigen-presenting cell and then contracting to pull in the membrane and endocytose the antigen. B These findings confirm the structural importance of cytoskeletal elements in B cells and their role in the immunological synapse.

Cytoskeletal rearrangements following BCR activation. Note the recruitment of BCRs via actin flow leading to microcluster formation. The actomyosin cytoskeleton then spreads and contracts to form a mature immunological synapse. The remaining details are beyond the scope of this project[52]

Change in structure in activated B cells- endosomes and lysosomes

As discussed in function, B-cells can function as professional antigen-presenting cells (APCs), presenting specific antigen to CD4+ T-cells. Each B cell captures a single antigen and presents peptides from this antigen to specific T-cells. Antigen-processing compartments are crucial to this process. Following BCR internalization at the immunological synapse (described above), the antigen is taken up by late endosomal or lysosomal structures (collectively called multivesicular bodies, or MVBs)[53]. These structures contain the proteolytic enzymes that can break down the internalized antigen. They remain near the membrane and store peptide fragments of the antigen. These MVBs can pinch off in tubulovesicular endosomes and can be inserted into the plasma membrane for recognition by CD4+ T-cells. Thus, endosomes and lysosomes are important structures which link antigen recognition and antigen presenting functions in B cells.[53]

Villous surface architecture

Scanning electron microscopy (SEM) has revealed some of differences in cell surface architecture of B and T-cells. B-cells are characteristically covered in microvilli (~200 over the surface of a single B cell), while T-cells have a comparatively smooth surface with stubbier digitations (as seen in the electron micrographs)[54]. Importantly, these morphological differences can be used in combination with immunological staining to determine whether a disease such as chronic lymphocytic leukaemia has a B-cell or T-cell origin. The microvilli in B cells may have several functions, including motility, adhesion and signal transduction. For example, a study by Greicius et al.(2004)[54] found that microvilli represent a membrane domain rich in major histocompatability class II and CD86. This suggests that the microvilli function in adhesion and antigen presentation. In vitro imaging shows the adjacent B-cells interacting with each other via their microvilli.

Electron micrograph of a single B cell. Note the villous surface architecture, which is distinct from smooth surface of T cells and is functionally important in B cell interactions[54]

How cell surface markers vary across stages of B-cell differentiation

B-cells vary the expression of surface molecules throughout their process of development. Specific arrays of surface molecules are characteristic of each stage of development, as reviewed by Greaves et al. (1974) [55]:

  • Persistent: Pan-B reagents are present on cells from an early stage of B-commitment in primary tissue and persist throughout antigen-independent and antigen-dependent differentiation until they differentiate to form plasma cells. Examples are CD19 and CD20.
  • Stem-cell to pre-B cell: Most of the studies of B-cell phenotypes in this stage derive from studies of leukaemic cells in patients with acute lymphoblastic leukaemia. The earliest identifiable lymphoid precursor cell contains the nuclear enzyme terminal deoxynucleotidyl transferase (TdT), as well as the CD22. CD10 is also present, but disappears after the pre-B stage.
  • Pre-B to early B-cell: the synthesis of immunoglobulin u-chains and their appearance in the cytoplasm is the hallmark of the pre-B stage. Igm is produced and appears first inside the cell and then at low density on the surface (marker for early B-cell). Surface structures asspcoated with immunological function begin to appear (e.g. Fc receptors).
  • Early B-cell to immunoglobulin-secreting cell: the B cells produced daily in large numbers in bone marrow leave the marrow and enter the peripheral lymphoid tissue whre most of them die. Most of the B-lymphocytes in primary follicles have IgM and IgD on their surface

Plasma cells (effector B-cells) have structure distinct from B cells

  • Since they synthesise immunoglobulins, their cytoarchitecture is geared for these processes. Their cytoplasm is plentiful and they have a round, eccentric nucleus. They have lost most of their cell-surface receptors (MHC II and immunoglobulins). Chromatin is arranged in dense-staining ‘stokes’ that give their nuclei a ‘clock-face’ appearance. There is pronounced basophilia due to large concentrations of cytoplasmic RNA. Electron microscopy shows abundant endoplasmic reticulum and polyribosomal units, as well as extensive Golgi apparatus and centrioles[56].

Structure of antibodies

IgG Structure and features

An antibody is composed of two heavy (50kDa each) and two light chains (25kDa each). It is a Y-shaped molecule, with two identical antigen-binding sites at the end of each arm of the Y. The amino acid sequence at the ends of the Y is highly variable (V region), giving the antibody specificity for binding antigen[57]. It is the tail region that gives the antibody its different functional properties, by interacting with effector cells and molecules. The constant (C) region determines the mechanism used to destroy antigen[58]. Based on the C region amino acid sequence, antibodies are characterised into four major classes: IgM, IgG, Iga, IgD and IgE (see table). Because they are bivalent, they can cross-link antigens. If an antigen has two antigenic determinants, the antibody will cross link to form small cyclic complex or linear chains; if the antigen has three antigenic determinants, large, complex three-dimensional lattices will form and may precipitate readily out of solution[59].

Function

Antibody Production

B-lymphocytes, or B cells are a specialised type of white blood cell that comprise an important part of the adaptive immune response. Their primary role is secreting antibodies, small, Y shaped molecules, which target antigens on the surface of foreign molecules in the body. Each B cell secretes a unique species of antibody, which binds to its specific antigen at an antigen- binding site on its surface. When the antigen of a specific B cell enters the body, the naive B cell becomes activated, and differentiates into its antibody secreting, mature form - a plasma cell. B lymphocyte activation is initiated after recognition of a specific antigen by the B-cell receptor.

Fleire et al. (2006) [60] found that the early response is a two-phase response that is both signalling and actin-dependent. Using fluorescence and interference reflection microscopy, they determined that the B-cells first spread over the antigen-bearing membrane and then contract, collecting bound antigen into a central defined cluster. At the same time, there is reorganisation of membrane-bound proteins on the cell surface and formation of an immunological synapse. B cells require T cell help in order to produce specific antibody. It was first thought that this interaction took place via an antigen ‘bridge’. However, a study by Lanzavecchia (1985) [61] uncovered a different mechanism. The antigen is first internalised and processed by specific B cells and is then presented to T cells in a major histocompatability complex (MHC) restricted manner.

Control of Antibody Production

IgM structure and features

A recent study by Pedros et al [13] has found that follicular helper T cells are an essential activating factor in the maturation of B cells. They selectively enhance the proliferation of B cells which produce useful, pathogen fighting antibodies, while inhibiting those B cells which produce potentially harmful antibodies. The study showed that depletion of the TBK1 kinase pathway in T cells, which leads to the differentiation of the germinal centre T follicular helper cells, disrupted the B cell differentiation process, which in tern, lead to impaired antibody signalling. The interaction between B and T lymphocytes is imperative to an effective immune response.

Antibody Isotypes

IgM: The IgM antibody isotype is prominent in the primary immune response. The exist as monomers on naive B cells but form pentamers when secreted. Their binding sites have low affinity but can still bind stably to antigen and initiate a response due to a high avidity from their 10 total binding sites[62]. Their main functions are to agglutinate antigen and activate the complement system, an innate immune response[2].
IgG: The IgG antibody isotype is prominent in the secondary immune response. They are abundant in serum and body fluids and have a half life of 30 days after being secreted by plasma B cells[63]. There are four IgG subclasses 1,2,3 and 4 which are dominant in different stages of infection[64]. IgG also activate the complement system and are involved in newborn immunity. Maternal IgG1 crosses the placenta and provides protection to the foetus after 3 months of gestation[65]. After birth, this maternal level of IgG1 drops and protects the newborn for a month, where it can produce its own antibodies.
IgA: The IgA antibody isotype is abundant in mucosal surfaces and secretions. They are secreted by plasma B cells and form dimers which are held together by a protein called a J chain. IgA also has subclasses 1 and 2 with IgA2 being more resistant to proteolytic enzymes produced by bacteria, hence its abundance in the gut[66]. Their main functions are to neutralise viruses, bacteria and toxins and also to prevent the activation of the complement system. Deficiencies in IgA lead to an increase of respiratory and gut infections, autoimmune diseases and allergies[67].
IgD: The IgD antibody isotype only makes up 0.25% of antibodies in serum. They are found on naive B cells and have a half-life of 2.8 days. The main function of IgD is to signal B cell activation[68], but have also been found to bind to basophils and mast cells to activate them. The activated basophils and mast cells contribute to the respiratory immune system by producing antimicrobial factors[69].
IgE: The IgE antibody isotype, like IgD, also has a low serum concentration. They are produced through B cell class switching in response to antigen. Their main function is to defend against parasites, protozoa [70]but are also active in allergic disease. Type 1 hypersensitivity is an IgE-mediated response, where IgE bind mast cells, releasing cytokines to cause bronchoconstriction and nasal vasodilation[71].

Class Switching

Class switching is a cytokine mediated B cell function that allows it to change production of one antibody isotype to another. Naive B cells express both IgM and IgD antibodies but can produce IgG, IgE and IgA through class switching[72]. Only the constant region of the heavy chain of antibodies are changed in class switching, allowing for interaction with different effector molecules[73]. The specificity of the antibody is preserved as the variable chain is not modified.

Initiation of T- cell Immune Response

B cells are essential in initiating the T cell immune response. A study by Bouaziz et. al. has shown that in B cell depleted mice, CD4 T cell activation is depleted, and hence B cells are essential for optimal T cell functioning[74]. An in vivo study by Bouaziz et al. (2007) used CD20 mAb treatment in mice to deplete B cell numbers and found that this significantly inhibited CD4+ T cell responses to antigens. This suggests that B cells cooperate with dendritic cells in functioning as antigen-presenting cells (APCs). A study by Garside et al. (1998)[75] tracked B and T cells in the lymph nodes. They found that antigen-specific B cells migrate from B cell rich follicles to the follicular border following immunisation. Here, they interacted with T cells for an extended period of time, which supports the concept of cognate T-B cell interactions during the immune response.

Maintenance of the Immune System

As well as their contribution to initiating the immune response, B cells also contribute to the maintenance of the immune system through cytokine secretion, making them an important regulator of inflammatory disease. Cytokine producing B cells can be divided into two subsets - regulatory, or effector B cells, defined by the specific cytokines they secrete. Regulatory B cells are known to secrete IL-10, while effector B cells secrete cytokines such as TNF-α, IL-2 and IL-4

File:B cell functions.jpg
Functions of a B cell

Role in Disease

B-cells in autoimmune diseases

B cells contribute to autoimmune pathogenesis through autoantibody production[74]. Normally, B-cells traverse tightly regulated pathways throughout their development. Surviving B cells are screened and may be positively or negatively selected via the concerted action of cytokines and transcription factors. Self-reactive B cells are mostly eradicated in the bone marrow through negative selection via apoptosis, and those that do survive to reach peripheral lymph tissue are either modified by receptor editing, or silenced. When these selection processes fail- due to a) disruption in B-cell signaling, activation or proliferation or b) dysregulated apoptotic genes- there is a greater likelihood that autoreactive B cells will survive and produce autoantibodies[74]. These autoantibodies recognise the body's proteins as antigen, inducing a continuous immune inflammatory response (either systemic or organ-targeted).

Systemic Lupus Erythematosus (SLE)

The central feature of SLE is autoantibody production. The pathogenesis is complex, and includes environmental triggers and infectious agents. Genetically, studies have found that SLE is associated with a decrease in proapoptotic genes and the increase in prosurvival gene expression, leading to the proliferation of autoreactive B-cells, as reviewed in [76]. In the active stage of the disease, patients with SLE have elevated serum autoantibody levels. SLE is a heterogeneous disease- that is, its affects patients differently. Its clinical features can be traced to the production of specific autoantibodies. For example anti-double-stranded DNA (dsDNA) antibodies correlates with glomerulonephritis and anti-cardiolipin antibodies correlelates with vascular thrombosis. Interestingly, however, it is unclear whether B-cell autoreactivity in SLE reflects an intrinsic B-cell defect or is secondary to inflammation. Inflammation can perturb B-cell thresholds for signaling, activation and proliferation and thus increase the chance that an autoreactive B-cell will escape negative selection.[77]

Rheumatoid arthritis (RA)

Although RA is classically viewed as primarily T-cell dependent, B-cells play an important role in the pathogenesis. RA is characterized by chronic inflammation of the joint capsule and synovial membrane, eventually leading to joint erosion and deformity[78]. In RA, both the central and peripheral B-cell tolerance checkpoints are faulty. As a result, there is an accumulation of a large number of autoreactive B cells in the mature naive B cell compartment. As reviewed in [78], experiments investigating collagen-induced arthritis of mice found that B-cell depletion delays the onset of the disease and reduces the severity. This suggests that B-cells play a significant regulatory role during the initial stages of the disease. As reviewed in [79], recent studies have focused on the role of regulatory B (Breg) cells in rheumatoid arthritis and other autoimmune diseases. Breg cells produce interleukin-10 and thus function as negative regulators of inflammatory immune responses.

Collagen- induced arthritis is an animal version of rheumatoid arthritis, [80]which has been studied in depth in mice and has contributed significantly to our understanding of the regulatory role of B cells in arthritic patients. CD40 is one of the constituents of the TNF superfamily, and is an important factor in B cell activation, leading to proliferation and isotope switching. Recent research into anti- CD40 treatment has found that stimulation with anti-CD40 mAb and antigen is able to prevent and improve CII/CFA induced arthritis. CD40 stimulated B cells have been able to prevent mice from arthritis. [81] Reduced levels of IL-10 producing B cells have been seen in cases of collagen- induced arthritis. This type of arthritis is thought to be triggered by type of T cell, known as Th17 cells. Current research speculates that B10 cells play an important regulatory role, and and involved in halting the progression of arthritis through suppression of Th17 cell development. [82]

Common Variable Immunodeficiency

Common Variable Immunodeficiency (CVID) arises where B cells are normal in morphology and number,but the process of differentiation into mature plasma cells is impaired. This condition is characterised by reduced levels of IgG, leading to dysfunction in the antibody response to invading cells. [83][84] As a result, patients are increasingly susceptible to infection, and have an increased likelihood of developing autoimmune conditions. A recent study, which identifies Granulomatous-lymphocytic interstitial lung disease (GLILD) as the most common source of morbidity arising from CVID has shown a strong link between low levels of marginal zone and memory B cells and these conditions. [85] Although the genetic basis of CVID is not yet definitively understood,recent research suggests that defects in the gene for inositol 1,4,5- triphosphate 3-kinase β (ITPKB) play a crucial factor in causing this immunodeficiency. This gene functions in controlling the survival and antibody producing functions of B cells. [86]

In a normally functioning immune system, B cells interact with specific T cells, including CD4, CD8, and follicular T cells in one of two ways; through secreted cytokines, which interact with the B cell membrane, or through direct contact between cells. In CVID this interaction is impaired, as the two classes of immune cells are derived from the a common progenitor, and hence are often mutually dysfunctional. [87] Lack of B cell differentiation is present in the majority of CVID patients, while the role of T cell interaction in CVID is less well defined. It is suggested that impaired selective cytokine secretion by CD4+ T cells can be accounted for by the lack of memory B cells, characteristic of of CVID. [84] [88]

There are a number of other B cell related immunodeficiencies, most of which arise as a result of abnormalities in the normal process of B cell maturation. One example is X-linked agammaglobulinemia, which results from a mutation in the BTK gene, and inhibits the maturation of Pro- B cells to pre- B cells. [89]

Chronic Lymphocytic Leukemia

Chronic Lymphocytic Leukemia (CLL) is one of the most common leukemias in adults [90]. It is caused by an over proliferation of B cells and defined by a concentration of >5 G/L of clonal B-lymphocytes in the blood [91]. Several mechanisms contribute to the pathogenesis of the disease, most importantly the deletion of specific micro-RNAs that normally sends B cells down apoptotic pathways. Without these micro-RNAs, B cells that have reached the end of their life cycle are not signaled for apoptosis. This leads to the accumulation of B cells that causes CLL [90]. Patient outcomes varies widely in CLL with some patients living for a long time without the use of therapy, and others requiring immediate treatment for their rapidly developing leukemia [92]

Applications

Monoclonal Antibody Production

Monoclonal antibodies was first discovered in 1975 by Cesar Milstein through the fusion of myeloma cells with B cells. The fusion of the cancer cell with B cells allows the cell to proliferate at a high rate while still performing B cell duties[93]. The antibodies that are produced by these altered immune cells are known as monoclonal antibodies.

Due to monoclonal antibodies being produced by identical immune cells, they possess monovalent affinity, which means that they all bind to the same epitope on an antigen. It is also possible to alter what monoclonal antibodies recognise and bind to and can therefore act as detecting or purifying agents[93]. With the many features of monoclonal antibodies, they have various uses in diagnostic tests as well as therapeutic treatment involving cancer, auto-immune disease and inflammation[94][95].

CD40 activated B cells

Dendritic cells are the most prominent antigen presenting cell. They present antigens to T cells, activating them and inducing an immune response. Despite the immunological capabilities of dendritic cells, there are however limitations to these cells in both research and clinical applications. Dendritic cells are difficult to produce ex vivo not only in technique, but also in cost[96]. Upon being generated, dendritic cells are not homogenous and also lack 1-selectin, a molecule responsible for lymph node homing[97].

CD40-activated B cells (CD40B cells) address the disadvantages of dendritic cells. They are also antigen presenting cells and can induce T cells to generate an immune response. Unlike dendritic cells, CD40B cells can easily be exponentially generated at a high purity without loss of function[98], and can home to secondary lymph organs[99]. CD40B cells also have desired traits to be utilised in clinical applications. They are shown to benefit from or not react to tumour-derived factors which normally inhibit dendritic cells[100]. Additionally, they have displayed no long-term complications such as autoimmunity in vaccinations[101]. Lastly, the effectiveness of CD40B cells have been proven through mice trials, where results indicate delayed tumour growth as well as production of tumour specific antibodies[102]. Due to the advantages of CD40B cells, they have a potential to replace dendritic cells as professional antigen presenting cells in immunotherapy.

Reprogramming B cells to act as Induced Pluripotent Stem Cells

B cells can be transformed in vitro by Epstein-Barr Virus (EBV) to produce lymphoblastoid cell lines (LCLs), a limitless source of proliferating cells. B cells are chosen for this transformation due to them representing 20% of the peripheral blood mononuclear cell population[103]. This high concentration allows for efficiency as only a small amount of blood is needed to generate LCLs and conduct unlimited trials. B cells also possess plasticity to reprogram itself to other cell types. The transdifferentiation of B cells into macrophages and hematopoietic precursor cells by down-regulating Pax5 expression, have been shown in previous studies[104][105].

It is from these traits that inspired induced pluripotent stem cell (iPSC) generation from B cells. This was achieved by utilising oriP/EBNA-1–based vectors on LCLs[106]. The significance of this development is that LCL-iPSCs can be produced in an efficient manner, and can therefore further the understanding of rare diseases and drug development.

Glossary

Immunoglobulin Classes of proteins which act as antibodies in the immune response, and include classes such as IgG, IgA and IgM which each have specific antigen targets.
Antigen Molecules capable of initiating an immune response, stimulating the host to produce specific antibodies against it.
Antibody Large, Y- shaped proteins which target specific antigens on foreign bodies to elicit an immune response through sites on their surface which correspond to sites on the surface of their target antigen
Opsonise/ opsonisation The process of making an antigen more susceptible to engulfment by phagocytes through binding of an opsonin (makes the antigen more 'appetising')
BCR B Cell Receptor - transmembrane receptor proteins located on the surface of a B cell, made up antigen binding chains (heavy and light chains in BCRs)
Heavy and Light chains (H & L) Polypeptide chains that compose an antibody - antibodies are comprised of two heavy chains and two lights chains, joined by disulfide bonds. H & L chains have immunoglobulin domains, which are specific to the antibody they form.
Cytokine Signalling molecules which regulate immunity and inflammation, and are produced by B cells, as well as other immune and non- immune cells. Examples include interleukins and members of the TNF superfamily.
Class switching Also known as isotope switching. It involves changing of a B cells immunoglobulin production from one class to another, e.g. IgM to IgG. Due to the variable region remaining constant, the B cells antigen specificity is not changed, but it can interact with different effector molecules
Germinal centre Sites within secondary lymph nodes where B cells mature, proliferate and undergo class switching
MHC Cell surface proteins which mediate the interactions of leukocytes/ immune cells. They bind peptide fragments of invading pathogens and display them on the cell surface for recognition by immune cells
Hematopoietic The cells that give rise to all blood cells types, prior to differentiation
Monoclonal antibodies Antibodies produced from a single cell line and consist of identical antibody molecules.

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