B Cell Receptor (Immunoglobulin) Class Switching
Immunoglobulin
Immunoglobulins, also known as antibodies, are glycoprotein molecules produced by plasma cells or white blood cells. They specifically recognize and bind to particular antigens.
Structure of immunoglobulins
Antibody (or immunoglobulin) molecules are glycoproteins composed of one or more units, each containing four polypeptide chains: two identical heavy chains (H) and two identical light chains (L).
The amino terminal ends of the polypeptide chains show considerable variation in amino acid composition and are referred to as the variable (V) regions to distinguish them from the relatively constant (C) regions.
Each L chain consists of one variable domain, VL, and one constant domain, CL. The H chains consist of a variable domain, VH, and three constant domains CH1, CH2 and CH3.
Each heavy chain has about twice the number of amino acids and molecular weight (~50,000) as each light chain (~25,000), resulting in a total immunoglobulin monomer molecular weight of approximately 150,000.
Heavy and light chains are held together by a combination of non-covalent interactions and covalent interchain disulfide bonds, forming a bilaterally symmetric structure. The V regions of H and L chains comprise the antigen-binding sites of the immunoglobulin (Ig) molecules. Each Ig monomer contains two antigen-binding sites and is said to be bivalent.
The hinge region is the area of the H chains between the first and second C region domains and is held together by disulfide bonds. This flexible hinge (found in IgG, IgA, and IgD, but not IgM or IgE) region allows the distance between the two antigen-binding sites to vary.
Classes of immunoglobulins
The five primary classes of immunoglobulins are IgG, IgM, IgA, IgD, and IgE. These are distinguished by the type of heavy chain found in the molecule. IgG molecules have heavy chains known as gamma-chains; IgMs have mu-chains; IgAs have alpha-chains; IgEs have epsilon-chains; and IgDs have delta-chains.
Differences in heavy chain polypeptides allow these immunoglobulins to function in different types of immune responses and at particular stages of the immune response.
The polypeptide protein sequences responsible for these differences are found primarily in the Fc fragment. While there are five different types of heavy chains, there are only two main types of light chains: kappa (κ) and lambda (λ).
Antibody classes differ in valency as a result of different numbers of Y-like units (monomers) that join to form the complete protein.
For example, in humans, functioning IgM antibodies have five Y-shaped units (pentamer) containing a total of ten light chains, ten heavy chains, and ten antigen-binding.
IgG
Properties of IgG:
1. Molecular weight: 150,000 Da
2. H-chain type (MW): gamma (53,000 Da)
3. Serum concentration: 10 to 16 mg/mL
4. Percent of total immunoglobulin: 75%
5. Glycosylation (by weight): 3%
6. Distribution: intra- and extravascular
7. Function: secondary response
IgM
Properties of IgM:
1. Molecular weight: 900,000 Da
2. H-chain type (MW): mu (65,000 Da)
3. Serum concentration: 0.5 to 2 mg/mL
4. Percent of total immunoglobulin: 10%
5. Glycosylation (by weight): 12%
6. Distribution: mostly intravascular
7. Function: primary response
IgA
Properties of IgA:
1. Molecular weight: 320,000 Da (secretory)
2. H-chain type (MW): alpha (55,000 Da)
3. Serum concentration: 1 to 4 mg/mL
4. Percent of total immunoglobulin: 15%
5. Glycosylation (by weight): 10%
6. Distribution: intravascular and secretions
7. Function: protect mucus membranes
IgD
Properties of IgD:
1. Molecular weight: 180,000 Da
2. H-chain type (MW): delta (70,000 Da)
3. Serum concentration: 0 to 0.4 mg/mL
4. Percent of total immunoglobulin: 0.2%
5. Glycosylation (by weight): 13%
6. Distribution: lymphocyte surface
7. Function: unknown
IgE
Properties of IgE:
1. Molecular weight: 200,000 Da
2. H-chain type (MW): epsilon (73,000 Da)
3. Serum concentration: 10 to 400 ng/mL
4. Percent of total immunoglobulin: 0.002%
5. Glycosylation (by weight): 12%
6. Distribution: basophils and mast cells in saliva and nasal secretions
7. Function: protect against parasites
Subclasses of immunoglobulins
In addition to the major immunoglobulin classes, several Ig subclasses exist in all members of a particular animal species.
Antibodies are classified into subclasses based on minor differences in the heavy chain type of each Ig class. In humans there are four subclasses of IgG: IgG1, IgG2, IgG3, and IgG4 (numbered in order of decreasing concentration in serum).
Variance among different subclasses is less than the variance among different classes. For example, IgG1 is more closely related to IgG2, IgG3 and IgG4 than to IgA, IgM, IgD, or IgE.
Consequently, antibody-binding proteins (e.g., Protein A or Protein G) and most secondary antibodies used in immunodetection methods cross-react with multiple subclasses but usually not multiple classes of Ig.
Isotype Class Switching
Key Terms
Isotype: Antibodies can come in different varieties known as isotypes, which refer to the genetic variations or differences in the constant regions of the heavy and light chains of the antibody.
Class Switch Recombination: A biological mechanism that changes a B cell’s production of antibody from one class to another; for example, from an isotype called IgM to an isotype called IgG.
Immunoglobulin (Ig) [Ab] Class Switching
Antibodies can come in different varieties, known as isotypes or classes. In placental mammals there are five antibody isotypes: IgA, IgD, IgE, IgG and IgM. They are each named with an “Ig” prefix that stands for immunoglobulin (another name for antibody) and differ in their biological properties, functional locations, and ability to deal with different antigens.
The antibody isotype of a B cell changes during cell development and activation. Immature B cells, which have never been exposed to an antigen, are known as naïve B cells and express only the IgM isotype in a cell surface bound form.
B cells begin to express both IgM and IgD when they reach maturity; the co-expression of both of these immunoglobulin isotypes renders the B cell ‘mature’ and ready to respond to an antigen.
B cell activation follows engagement of the cell-bound antibody molecule with an antigen, causing the cell to divide and differentiate into an antibody-producing cell, called a plasma cell.
In this activated form, the B cell starts to produce antibodies in a secreted form rather than a membrane-bound form. If these activated B cells encounter specific signaling molecules via their CD40 and cytokine receptors (both modulated by T helper cells), they undergo antibody class switching to produce IgG, IgA or IgE antibodies (from IgM or IgD) that have defined roles in the immune system.
Immunoglobulin class switching (or isotype switching, or isotypic commutation, or class switch recombination (CSR)) is a biological mechanism that changes a B cell’s production of antibody from one class to another; for example, from an isotype called IgM to an isotype called IgG.
During this process, the constant region portion of the antibody-heavy chain is changed, but the variable region of the heavy chain stays the same (the terms “constant” and “variable” refer to changes or lack thereof between antibodies that target different epitopes).
Since the variable region does not change, class switching does not affect antigen specificity. Instead, the antibody retains affinity for the same antigens, but can interact with different effector molecules. This allows different daughter cells from the same activated B cell to produce antibodies of different isotypes or subtypes (e.g. IgG1, IgG2 etc.).
Class switching occurs by a mechanism called class switch recombination (CSR) binding. Class switch recombination is a biological mechanism that allows the class of antibodies produced by an activated B cell to change during a process known as isotype or class switching.
During CSR, portions of the antibody-heavy chain locus are removed from the chromosome, and the gene segments surrounding the deleted portion are rejoined to retain a functional antibody gene that produces antibodies of a different isotype.
Double-stranded breaks are generated in DNA at conserved nucleotide motifs, called switch (S) regions, which are upstream from gene segments that encode the constant regions of antibody-heavy chains; these occur adjacent to all heavy chain constant region genes with the exception of the δ-chain.
DNA is nicked and broken at two selected S-regions by the activity of a series of enzymes, including Activation-Induced (Cytidine) Deaminase (AID), uracil DNA glycosylase and apyrimidinic/apurinic (AP)-endonucleases. The intervening DNA between the S-regions is subsequently deleted from the chromosome, removing unwanted μ or δ heavy chain constant region exons and allowing substitution of a γ, α or ε constant region gene segment.
The free ends of the DNA are rejoined by a process called non-homologous end joining (NHEJ) to link the variable domain exon to the desired downstream constant domain exon of the antibody-heavy chain.
In the absence of non-homologous end joining, free ends of DNA may be rejoined by an alternative pathway biased toward microhomology joins. With the exception of the μ and δ genes, only one antibody class is expressed by a B cell at any point in time.
Key Points
The antibody isotype of a B cell changes during cell development and activation. Immature B cells have never been exposed to an antigen and are known as naïve B cells. B cells begin to express both IgM and IgD when they reach maturity and renders the B cell ‘mature’ and ready to respond to antigen.
If activated B cells encounter specific signaling molecules via their CD40 and cytokine receptors (both modulated by T helper cells), they undergo antibody class switching to produce IgG, IgA or IgE antibodies that have defined roles in the immune system.
During class switch recombination the constant region portion of the antibody-heavy chain is changed, but the variable region of the heavy chain stays the same; thus, class switching does not affect antigen specificity.
The antibody retains affinity for the same antigens, but can interact with different effector molecules. This allows different daughter cells from the same activated B cell to produce antibodies of different isotypes or subtypes (e.g. IgG1, IgG2 etc. ).
Making Memory B Cells
Memory B cells are a B cell subtype that are formed following a primary infection. In the wake of the first (primary response) infection involving a particular antigen, the responding naïve cells (ones which have never been exposed to the antigen) proliferate to produce a colony of cells.
Most of them differentiate into the plasma cells, also called effector B cells (which produce the antibodies) and clear away with the resolution of infection. The rest persist as the memory cells that can survive for years, or even a lifetime.
To understand the events taking place, it is important to appreciate that the antibody molecules present on a clone (a group of genetically identical cells) of B cells have a unique paratope (the sequence of amino acids that binds to the epitope on an antigen).
Each time these cells are induced to proliferate due to an infection, the genetic region coding for the paratope undergoes spontaneous mutations with a frequency of about 1 in every 1600 cell divisions. This may not seem high, but because the cells divide so often, it ends up resulting in many mutations. The frequency of mutations in other cells is around 1 in 106, which is much lower.
All these events occur in the highly “eventful” germinal centers of lymphoid follicles, within the lymph nodes.
Some of the resulting paratopes (and the cells elaborating them) have a better affinity for the antigen (actually, the epitope) and are more likely to proliferate than the others.
Moreover, the number of different clones responding to the same antigen increases (polyclonal response) with each such exposure to the antigen and a greater number of memory cells persist. Thus, a stronger (basically, larger number of antibody molecules) and more specific antibody production is the hallmark of secondary antibody response.
The fact that all the cells of a single clone elaborate one (and only one) paratope, and that the memory cells survive for long periods, is what imparts a memory to the immune response. This is the principle behind vaccination and administration of boosters.
The paratope is the part of an antibody which recognizes an antigen, the antigen-binding site of an antibody. It is a small region (15–22 amino acids) of the antibody’s Fc region and contains parts of the antibody’s heavy and light chains. The part of the antigen to which the paratope binds is called an epitope.
Primary and Secondary Antibody Responses
The immune system is a system of biological structures and processes within an organism that protects against disease. To function properly, an immune system must detect a wide variety of agents, from viruses to parasitic worms, and distinguish them from the organism’s own healthy tissue.
Pathogens can rapidly evolve and adapt to avoid detection and neutralization by the immune system. As a result, multiple defense mechanisms have also evolved to recognize and neutralize pathogens. Even simple unicellular organisms such as bacteria possess a rudimentary immune system, in the form of enzymes that protect against bacteriophage infections.
Other basic immune mechanisms evolved in ancient eukaryotes and remain in their modern descendants, such as plants and insects. These mechanisms include phagocytosis, antimicrobial peptides called defensins, and the complement system.
Jawed vertebrates, including humans, have even more sophisticated defense mechanisms, including the ability to adapt over time to recognize specific pathogens more efficiently.
Adaptive (or acquired) immunity creates immunological memory after an initial response to a specific pathogen, leading to an enhanced response to subsequent encounters with that same pathogen. This process of acquired immunity is the basis of vaccination.
Disorders of the immune system can result in autoimmune diseases, inflammatory diseases and cancer. Immunodeficiency occurs when the immune system is less active than normal, resulting in recurring and life-threatening infections.
In humans, immunodeficiency can either be the result of a genetic disease such as severe combined immunodeficiency, acquired conditions such as HIV/AIDS, or the use of immunosuppressive medication.
In contrast, autoimmunity results from a hyperactive immune system attacking normal tissues as if they were foreign organisms. Common autoimmune diseases include Hashimoto’s thyroiditis, rheumatoid arthritis, diabetes mellitus type 1, and systemic lupus erythematosus. Immunology covers the study of all aspects of the immune system.
The immune system protects organisms from infection with layered defenses of increasing specificity. In simple terms, physical barriers prevent pathogens such as bacteria and viruses from entering the organism.
If a pathogen breaches these barriers, the innate immune system provides an immediate, but non-specific response. Innate immune systems are found in all plants and animals. If pathogens successfully evade the innate response, vertebrates possess a second layer of protection, the adaptive immune system, which is activated by the innate response.
Here, the immune system adapts its response during an infection to improve its recognition of the pathogen. This improved response is then retained after the pathogen has been eliminated, in the form of an immunological memory, and allows the adaptive immune system to mount faster and stronger attacks each time this pathogen is encountered.
Both innate and adaptive immunity depend on the ability of the immune system to distinguish between self and non- self molecules. In immunology, self molecules are those components of an organism’s body that can be distinguished from foreign substances by the immune system.
Conversely, non-self molecules are those recognized as foreign molecules. One class of non-self molecules are called antigens (short for antibody generators) and are defined as substances that bind to specific immune receptors and elicit an immune response.
When B cells and T cells are first activated by a pathogen, memory B-cells and T- cells develop. Throughout the lifetime of an animal these memory cells will “remember” each specific pathogen encountered, and are able to mount a strong response if the pathogen is detected again.
This type of immunity is both active and adaptive because the body’s immune system prepares itself for future challenges. Active immunity often involves both the cell-mediated and humoral aspects of immunity as well as input from the innate immune system.
The innate system is present from birth and protects an individual from pathogens regardless of experiences, whereas adaptive immunity arises only after an infection or immunization and hence is “acquired” during life.
