++
B cells perform two important functions: (1) they differentiate into plasma cells that produce antibodies (also called immunoglobulins) and (2) they can become long-lived memory B cells that can rapidly respond to a reinfection. The immunoglobulin on the B-cell surface is its antigen receptor (B-cell receptor or BCR) and the ability of a B-cell precursor to make this antigen receptor determines whether it is allowed to develop into a mature B cell.
++
B-cell precursors first arise from stem cells in the fetal liver, but by the time of birth, these stem cells migrate to the bone marrow, which is their main location during childhood and adult life. Unlike T cells, B cells do not require the thymus for maturation.
++
The maturation of B cells has two phases: the first is the antigen-independent phase, which consists of stem cells, pre-B cells, and B cells, and it is during this phase that the B cell recombines its immunoglobulin genes to make a unique antigen receptor. For pre-B cells to differentiate into B cells, a functional immunoglobulin must be present on the cell surface. A protein called Bruton’s tyrosine kinase (BTK) detects this immunoglobulin and signals to the cell to continue to divide and differentiate. A mutation in the gene encoding this protein causes X-linked agammaglobulinemia, a condition in which cells cannot progress to the pre-B cell stage and no antibodies are made (see Chapter 68).
++
During the second phase, which is the antigen-dependent phase, mature B cells with functional antigen receptors interact with antigens. This phase will be covered in more detail in Chapter 61.
++
The immunoglobulin (Ig), or BCR, of a mature B cell is an IgM molecule with an additional region at the end of its heavy chain that tethers it to the B-cell surface. Approximately 109 B cells are produced each day, but only a small fraction of these make it from the bone marrow into the circulation, and unless they are activated through their antigen receptors, circulating B cells have a short life span (i.e., days or weeks). In this chapter, we will explore the structure and diversity of BCRs, and in Chapter 61, we will describe how these antigen receptors cause activation of B cells and how the resulting antibodies provide host defense.
++
Antibodies are glycoproteins made up of light (L) and heavy (H) polypeptide chains. The terms light and heavy refer to molecular weight; light chains have a molecular weight of about 25,000, whereas heavy chains have a molecular weight of 50,000 to 70,000. The simplest antibody molecule has a Y shape (Figure 59–2) and consists of four polypeptide chains: two identical H chains and two identical L chains. In other words, even though you received copies of H and L chain genes from each of your parents, each B cell ultimately synthesizes only one of the H chain genes and one of the L chain genes to use to form an antibody, and therefore, all of the subsequent antibodies from that B cell and its progeny use the same H and L chains. Table 59–2 is a summary of the properties of the human lymphocyte antigen receptors.
++
++
++
One end of the Y is composed of two identical pieces that bind the antigen, and therefore, this is called the antigen-binding fragment (or Fab). The Fab includes the variable region of the L chain (VL) and the variable region of the H chain (VH), as well as the constant region of the L chain (CL) and the first constant region of the H chains (CH1). The portions of the L and H chains that actually bind the antigen are only 5 to 10 amino acids long, each composed of three extremely variable (hypervariable) amino acid sequences. Antigen–antibody binding involves electrostatic and van der Waals’ forces and hydrogen and hydrophobic bonds rather than covalent bonds. The remarkable specificity of antibodies is due to these hypervariable regions.
++
The other end of the Y is a single stalk, where the H chains come together, and it is made of the remaining three or four constant regions of each of the H chains (CH2, etc.). This is called the constant or “crystallizable” fragment (or Fc). You might think that the Fab is the most important part of the antibody because it binds the antigen, but the Fc is needed to attach the antibody to host cells (e.g., via Fc receptors) or to complement (at the CH2 domain). The Fc is also the region that is used to fuse IgM and IgA together into larger “multimers.” It is also necessary for transport of IgA across epithelial barriers and transport of IgG from mother to fetus through the placenta.
++
There are five classes of antibodies: IgM, IgD, IgG, IgE, and IgA. Each class has structural differences that make it unique. For example, IgG and IgA have three CH domains, whereas IgM and IgE have four. The structural differences between the antibody classes translate into important functional differences. Mature naïve B cells start out making only IgM and IgD but later “switch” to making the other classes. We will discuss the different antibody functions and how the B cells class-switch in Chapter 61.
++
L chains can be of two types, κ (kappa) or λ (lambda), which differ in their constant regions. Either type can pair with H chains in all classes of immunoglobulins (IgG, IgM, etc.), but once a B cell chooses to use κ or λ, it shuts off the other L chain gene, so that all of the immunoglobulin from any one B cell contains only one type of L chain. In humans, the ratio of immunoglobulins containing κ chains to those containing λ chains is approximately 2:1, and a value dramatically different can be a sign of a monoclonal immunoglobulin-producing malignancy such as multiple myeloma.
++
H chains are distinct for each of the five immunoglobulin classes and are designated γ (gamma), α (alpha), μ (mu), ε (epsilon), and δ (delta). The VH hypervariable region of the H chain joins with VL in binding antigen; the opposite regions of the VH chains form the Fc fragment, which determines which class the antibody is and, therefore, what its biologic activities will be (see Chapter 61).
++
As described earlier, each antibody is composed of four immunoglobulin chains (two light chains and two heavy chains). There are two light chain gene clusters, one encoding kappa light chain (κL), on human chromosome 2, and one encoding lambda light chain (λL), on chromosome 22. All heavy chain genes (μH, δH, γH, εH, and αH) are together in a cluster on chromosome 14. The heavy chains and light chains are assembled after recombining gene segments within their respective gene clusters, a process that is directed by recombinase enzymes (RAG1 and RAG2). A schematic diagram of gene recombination is shown in Figure 59–3.
++
++
First, the VH and VL genes are recombined. Each cluster contains dozens of different V gene segments widely separated from the D (diversity, seen only in H chains), J (joining), and C gene segments. The VH region of each heavy chain is encoded by three gene segments (V + D + J). In the synthesis of a heavy chain, one particular V region (out of ~45) is translocated to lie close to one particular D segment (out of ~23), one particular J segment (out of 6), and one C segment.
++
The VH/CH combination is transcribed together on an RNA molecule and spliced to produce an mRNA that codes for the complete heavy chain, encoded by a single V, D, and J segment attached to a C segment. Why are IgM and IgD the first antibodies that are produced? The newly assembled V + D + J gene segments are closest to the Cμ and Cδ genes! In Chapter 61, we will describe how class switching leads to IgG, IgE, and IgA, which are further downstream in the heavy chain locus.
++
The VL region of each L chain is encoded by two gene segments (V + J). In the assembly of an L chain, the same process occurs except that there are slightly fewer possible V segments (~30–35 in kappa and lambda), and neither of the L chains have D segments. Also, the kappa chain gene has a single Cκ, whereas the lambda chain gene has four Cλ segments, one already associated with each J segment. The L chain comes from a similar translocation in which a single V and J are brought close together and then transcribed and translated with the appropriate C segment. Note that the DNA of the unused V, D, and J genes is discarded; once a particular B cell has recombined its light and heavy chains, it is committed to making antibody with only one specificity.
++
The H and L chains are synthesized as separate peptides and then folded and assembled in the cytoplasm by means of disulfide bonds to form H2L2 units. Finally, an oligosaccharide is added to the constant region of the heavy chain, and the BCR molecule is transported to the cell surface.
++
Note that the genetic recombination outlined earlier can lead to an enormous number of possible combinations. There are approximately 1011 possible heavy chain–light chain combinations! Antibody diversity depends on (1) multiple gene segments, (2) their rearrangement into different sequences, (3) the combining of different L and H chains in the assembly of immunoglobulin molecules, and (4) mutations. A fifth mechanism called junctional diversity applies primarily to the antibody heavy chain. Junctional diversity occurs by the addition of new nucleotides at the splice junctions between the V-D and D-J gene segments. The resulting antibodies have the potential to recognize the three-dimensional structure of a wide range of proteins, carbohydrates, nucleic acids, and lipids.
++
Despite the enormous potential diversity, the actual specificities represented among the pool of circulating B cells that we each have is somewhat smaller (about 106). Each immunologically responsive B cell bears copies of a single BCR on its surface (initially composed of its VDJ + Cμ or Cδ, paired with a VJ + Cκ or VJ + Cλ chain) that can react with one antigen (or closely related group of antigens). Even after that B cell divides, all of its progenies, or clones, will continue to make antibodies with the same antigen specificity.
++
There are two steps by which B-cell precursors are “auditioned” and selected to be available to become activated antibody-producing plasma cells. (This process is similar to that of T-cell clonal selection, described later in this chapter and in Figure 59–6, but selection of B cells occurs in the bone marrow rather than in the thymus.) The first step of B-cell clonal selection is called positive selection. Pre-B cells lack surface BCR. If a B-cell precursor fails to rearrange its immunoglobulin gene segments and generate a functional BCR, it dies before it reaches the mature B-cell stage. This is called positive selection because only those cells that do generate a BCR are allowed to survive and mature. For example, mutations in the genes encoding the recombinase enzymes (see above) result in a failure to generate antigen receptors and therefore a deficiency of lymphocytes (severe combined immunodeficiency).
++
Similarly, a mutation in the gene on the X chromosome that encodes BTK, which is important for transmitting the BCR signal from the cell surface, results in the disease X-linked agammaglobulinemia, in which B cells and antibodies are absent. These patients are more susceptible to bacterial infections in their sinuses, lungs, and gastrointestinal tract because they lack the antibodies that usually protect these barrier surfaces (see Chapter 68).
++
Pre-B cells that do successfully generate surface IgM pass through positive selection and progress to become B cells. At this stage, their IgM BCRs immediately encounter self-antigens. Remember that, whereas TCRs can only bind peptides complexed with major histocompatibility complex (MHC) proteins, the BCR can potentially bind to any circulating proteins, lipids, carbohydrates, or nucleic acids. However, because this phase of development occurs in the bone marrow rather than in the peripheral tissues or secondary lymphoid organs, all of the antigens that the B cell could encounter at this stage are self-antigens. During this phase, called negative selection, if the BCR strongly binds a self-antigen, this indicates high potential for autoreactivity.
++
This cell will be removed from the pool of mature B-cell clones, although it has one chance to escape this fate by a process called receptor editing. In this process, an alternate VL combination using an unused light chain allele can replace the previous allele, creating a new IgM receptor. But if this receptor is also autoreactive, the B cells are either killed by apoptosis or rendered “anergic” (their production of surface IgM is turned off and they become insensitive to activation). It is estimated that 25% to 50% of circulating B cells have undergone receptor editing. This phase is called negative selection because it ensures that only B cells that do not strongly bind self-antigens are allowed to leave the bone marrow and, therefore, will be self-tolerant.
++
Initially, all B cells that exit the bone marrow carry IgM specific for antigen. At this stage, they may be considered mature, because they have a functional BCR, but naïve, because they have not yet encountered their cognate antigen. Later, in a process called class switching, further gene rearrangement enables new antibodies that use the same VH but different CH chains. (In Chapter 61, we will describe how activation of B cells causes this class switching and the function of the different Ig classes.) A B cell that has class switched from IgM can never go back.
++
A single B cell has one maternal and one paternal copy of the L chain genes (both κ and λ) and the H chain gene. As described earlier, B cells that recognize self-antigens during clonal selection can attempt “receptor editing,” swapping in the alternate allele, to escape apoptosis or anergy. But once they have succeeded in exiting the bone marrow as a mature B cell, the alleles that gave them the successful BCR are fixed and the others are silenced. This is called allelic exclusion. We all have a diverse mixture of B-cell clones expressing different combinations of paternal and maternal genes. The precise mechanism of how the alternate alleles are turned off is unknown.