CHAPTER 16: Somatic DNA Recombination and Hypermutation in the Immune System

Edited by Paolo Casali, MD

16.1 The Immune System: Innate and Adaptive Immunity

All somatic cells of a eukaryotic organism have the same genetic information, and their phenotypes are determined by the differential control of expression of the same gene(s). A most important exception to this axiom of genetics occurs in the immune system. In developing B and T lymphocytes, genomic DNA changes in antigen receptor–encoding loci through somatic recombination create functional genes consisting of DNA sequences that are not found in the germline. In B lymphocytes that are activated by antigens to divide and differentiate, additional DNA recombination and hypermutation in the previously recombined Ig loci further diversify the biological effector functions and change the antigen-binding affinity of the produced antibodies.

The immune system of vertebrates mounts a protective response that distinguishes foreign (nonself) soluble or microorganism-associated molecules (antigens) from molecules or cells of the host (self-antigens). Innate immunity provides an immediate (without latency) first line of host defense against invading microbial pathogens by using receptors encoded in the germline, recognizing conserved structural patterns that are present across microbial species. It triggers responses by different effector white blood cells (e.g., macrophages and neutrophils), depending on the nature of the inducing microbial components. The innate response is relatively nonspecific for any given pathogen and generally elicits no immune memory. It can, however, modulate the adaptive immune response elicited by and mounted against a specific microorganism.

In contrast to innate immunity, the adaptive response (i.e., acquired immunity) is elicited by and mounted against a specific antigen. An antigen is in general a protein, a glycoprotein, a lipoprotein, or a glycolipid, such as found on infecting viruses or bacteria. The adaptive immune response triggered by those antigens will eventually destroy the infecting virus or bacterium expressing it. It is effected by B and T lymphocytes, with the assistance of other white blood cells, such as dendritic cells (DCs). B and T lymphocytes are named after the lymphoid organ in which they mature. The “B” in B cells stems from the bursa of Fabricius, which is named after Hieronymus Fabricius, the Italian anatomist who is considered the “Father of Embryology.” He recognized in the 16th century that this hematopoietic organ in birds is the equivalent of mammalian bone marrow, in which B cell development occurs. The “T” in T cells stems from thymus.

Both B and T lymphocytes use DNA rearrangement as the mechanism for production of the proteins that enable them to specifically recognize an antigen in the adaptive immune response. The adaptive immune response is characterized by a latency period—in general a few days—required for the expansion of foreign antigen–specific B cells and/or T cells that survive clonal deletion, a process by which B and T cell clones showing a high reactivity to self-antigens are deleted. The structural basis for foreign antigen–specific responses is provided by the expression of a large number of unique B cell receptors (BCRs) and T cell receptors (TCRs) on B and T lymphocyte clones, respectively. Such a highly diverse BCR and TCR repertoire allows the host to deal with an almost infinite number of foreign molecules. Binding of antigen to the BCR activates B cells and triggers the antibody response; activation of the TCR triggers T helper cell (T_h)– and cytotoxic T cell (CTL)–mediated responses. Antigen-activated B and T cells also differentiate into memory B and T cells, which underpin immunological memory. This provides protective immunity against the same antigen that drove the original response. The immune memory enables the organism to respond rapidly once exposed again to the same pathogen.

All jawed vertebrates (gnathostomes) display innate and adaptive immune responses. In evolution, immunity arose in the earliest multicellular animals and plants by the need to distinguish self cells and molecules from infectious nonself cells and their products. Invertebrates have an innate immune system but no adaptive system. Among vertebrates, jawless vertebrates (agnathans), such as lamprey and hagfish, display an innate immunity as well as a primitive form of adaptive immunity. In agnathans, thymus-like microanatomical structures, thymoids, and lymph node–like structures, typhlosoles, exist in the intestine of larvae; in adults, gills and kidneys provide residence for cells resembling mammalian monocytes, granulocytes, and lymphocytes. Recirculating lymphocyte-like cells in typhlosoles also express genes that are orthologs of genes important for lymphocyte development. Remarkably, agnathan antigen receptors (variable lymphocyte receptors, VLRs) are also generated by a recombination mechanism involving cytosine deaminase 1 (CDA1) or CDA2, which belong to the AID/APOBEC family of cytosine deaminases. T-like cells express CDA1 to assemble their VLRA gene repertoire, whereas B-like cells express CDA2 to assemble their VLRB gene repertoire. By contrast, they do not express orthologs of genes essential for recombination in T and B lymphocytes in jawed vertebrates. Immunization of lamprey with antigens, such as bacteria and synthetic antigens, elicits proliferation of VLRA⁺ and VLRB⁺ cells as well as cytokine- and antibody-like responses, similar to T and B cell responses in jawed vertebrates.

16.2 The Innate Response Utilizes Conserved Recognition Molecules and Signaling Pathways

As the first line of defense against microbial pathogens, innate immunity is activated upon recognition of certain predefined patterns in microorganisms by immune cell–associated pattern recognition receptors (PRRs). Most PRR ligands are conserved among microorganisms and are not found in higher eukaryotes, thereby allowing the immune system to quickly distinguish dangerous nonself from self. These microbe-associated molecular patterns (MAMPs) are synthesized by several sequential microbial enzyme reactions and, therefore, mutate more slowly than protein antigens (TABLE 16.1). Notably, nonpathogenic bacteria, such as commensal bacteria residing in the gut, also display conserved MAMPs.

TABLE 16.1 Innate immunity: A summary of MAMPs and PRRs.

Microorganism	MAMP	Location	PRR
Bacteria	Triacyl lipopeptides (Pam₃CSK₄)	Cell wall	TLR1/2
Bacteria	Muramyl dipeptide	Cell wall	NOD2
Bacteria	Pili	Cell wall	TLG10
Flagellated bacteria	Flagellin	Flagellum	TLR5
Gram^+ve bacteria	Peptidoglycan	Cell wall	TLR2/6
Gram^–ve bacteria	Lipoteichoic acid	Cell wall	TLR2/6
Gram^–ve bacteria	Lipopolysaccharide	Cell wall	TLR4
Bacteria and viruses	ssRNA	Inside cell/capsid	TLR7/8, NALP3, TLR3/RIG-1
RNA viruses	dsRNA	Inside virus	Helicase
Fungi	B-glycans	Cell wall	Dectin-1
Mycoplasma	Diacyl lipopeptides (Pam₂CSK₄)	Cell wall	TLR2/6
DNA-containing microorganisms	Unmethylated CpG DNA	Inside cell/capsid	TLR9
Toxoplasma gondii	Profilia	Inside cell	TLR10

An important type of PRR is the Toll-like receptors (TLRs). TLR4 recognizes Gram-negative bacterial lipopolysaccharide (LPS), a well-known MAMP; TLR1 and TLR2 recognize lipoteichoic acid from Gram-positive bacteria and peptidoglycans; and TLR5 recognizes bacterial flagellin. These TLRs are expressed on the surface of immune cells. TLRs that recognize nucleic acid variants are normally associated with viruses, such as single-stranded RNA (TLR3), double-stranded RNA (TLR7 and TLR8), or certain unmethylated CpG DNA. TLR9 is localized in the cytoplasm. Upon sensing their ligands, TLRs rapidly activate innate immune responses by triggering activation of transcription factors for inflammatory gene expression. Notably, some TLRs also serve as sensors for selective environmental cues. For example, TLR4 recognizes nickel and mediates allergy to this metal.

Retinoic acid–inducible gene 1 (RIG-I) and RIG-I-like receptors (RLRs) are RNA sensors. RIG-I is activated by the 5′-triphosphate (5′-PPP) moiety of uncapped double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA) of relatively short lengths, as typically found in replication intermediates of RNA viruses. This distinguishes viral RNA from usually capped eukaryotic mRNA. The RNA binding is mediated by the central RNA helicase DEAD box motifs and the C-terminal domain of RIG-I. The N-terminal caspase activation and recruitment domain (CARD) mediates the activation of downstream pathways to induce type I interferons for antiviral responses. Among other known members of the RLR family, MDA5 binds to 5′-PPP and triggers antiviral immunity, and LGP2 can only bind RNA but does not activate downstream pathways due to the lack of a CARD domain, thereby playing mainly regulatory roles.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a recently identified sensor for cytosolic DNA, as associated with DNA virus and retrovirus replication. Upon activation by DNA, cGAS mediates the synthesis of cGAMP, a second messenger signaling molecule that, through its 2′–5′ phosphodiester linkage, activates pathways for the induction of antiviral type I interferon responses. Intercellular transmission of cGAMP, through tight junctions or by virus particles that package cGAMP, also allows the spread of the response to bystander immune cells. A homolog of cGAS is the oligoadenylate synthase (OAS) family of proteins, which can sense dsRNA and mediate the synthesis of 2′,5′–linked oligonucleotides to trigger immunity.

Innate response pathways are widely conserved and are found in organisms ranging from flies to humans. As the first identified and most studied PRRs, TLRs are orthologs of the Drosophila protein Toll. Toll, in addition to orchestrating dorsal–ventral organization during development, mediates innate antimicrobial activities. It is triggered by Spatzle, an insect cytokine produced by a proteolytic cascade upon infection by fungi or Gram-positive bacteria to activate Dorsal-related immunity factor (DIF), which is related to the mammalian transcription factor NF-κB. DIF, in turn, promotes expression of genes encoding antifungal peptides, such as drosomycin, which kill their respective target organisms through membrane permeabilization (FIGURE 16.1). The antibacterial response in flies also relies on peptidoglycan recognition proteins (PGRPs), which have high affinities for bacterial peptidoglycans. Such responses lead to production of bactericidal peptides in a manner dependent on DIF or Relish, another NF-κB–related transcription factor, in response to Gram-positive and Gram-negative bacteria, respectively.

The TLR pathway in vertebrates is parallel to the Toll pathway with several equivalent components. About 10 human homologs of the TLRs can activate several immune response genes. Once a TLR is activated by an MAMP (as contrasted to the cytokine Spätzle in insects) it undergoes conformational changes and interacts, through homo- and heterodimerization, with one or more of five known Toll/interleukin 1/resistance (TIR) domain–containing adapters. These include myeloid differentiation primary response gene 88 (MyD88) and TIR domain–containing adapter-inducing interferon-β (TRIF), which, in turn, relay the signal, eventually leading to the induction of transcription factors such as NF-κB, AP-1, and IRFs for specific gene expression (FIGURE 16.2). The downstream pathways of TLRs are more expanded and versatile in mammals, as compared to those in insects. Notably, plants also use proteins with a leucine-rich region (LRR), which is the MAMP-binding site in TLRs, to detect pathogens and activate a mitogen-activated protein kinase (MAPK) cascade for induction of disease-resistance genes.

FIGURE 16.1 One of Drosophila’s innate immunity pathways is closely related to the mammalian pathway for activating NF-κB; the other has components related to those of apoptosis pathways.

FIGURE 16.2 Innate immunity is triggered by MAMPs. In mammals, MAMPs cause the production of peptides that activate Toll-like receptors. The receptors lead to a pathway that activates a transcription factor for the Rel family. Target genes for this factor include bactericidal and antifungal peptides. The peptides act by permeabilizing the membrane of the pathogenic organism.

PRRs, particularly TLRs, are highly expressed in immune cells of the myeloid origin, such as neutrophils, macrophages, and DCs, which are capable of phagocytosing or killing pathogens directly, consistent with their innate immune functions. Several TLRs are also highly expressed in lymphocytes (i.e., B cells and selected T cell subsets).

In general, the innate response contains the first wave of invasion by pathogens, but cannot deal effectively with the later stages of virulent infections, which require the specificity and potency of the adaptive response. Innate and adaptive responses overlap and crosstalk, in that cells activated by the innate response subsequently participate in the adaptive response. This is exemplified by the B cell–intrinsic function of TLR signaling in adaptive immunity and the “innate” function of natural antibodies.

Natural antibodies are produced by B lymphocytes through the same DNA recombination process that generates BCRs and antibodies, in contrast to the aforementioned PRRs, which are encoded by the germline. They are mainly IgM and are polyreactive (i.e., capable of binding multiple antigens). These antigens are often different in nature, such as phospholipids, polysaccharides, proteins, and nucleic acids, and are unlikely to share an identical epitope (which is the binding motif of an antibody). Rather, natural antibodies recognize foreign antigens possessing molecular structures that are different but that can equally fit the same natural antibody binding site—in this sense, natural polyreactive antibodies are also PRRs. This is exemplified by the ability of natural antibodies to bind appropriately spaced phosphate residues in the context of a variety of polynucleotides and phospholipids. Finally, many natural antibodies are “natural autoantibodies,” because they are produced in healthy individuals by B lymphocytes that show a moderate reactivity to a self-antigen and evade clonal deletion. Natural polyreactive antibodies play an important role in early stages of infection, prior to the emergence of class-switched highly antigen–specific antibodies. They can also function as templates for the generation of high-affinity autoantibodies through somatic hypermutation.

16.3 Adaptive Immunity

The defining critical feature of adaptive immunity is the specificity for antigens, such as those expressed by bacteria and viruses. This is made possible by the specificity of the BCRs and TCRs expressed on B and T lymphocytes, respectively. BCRs and TCRs are related in structure and their genes are related in organization. The mechanism underlying the variability is also similar (i.e., gene recombination).

Specific recognition and binding of an antigen by the BCRs expressed on the surface of B cells triggers B cell activation, proliferation, and differentiation, leading to the production of large amounts of antibodies specific for the same antigen. The structure and antigenic specificity (epitope) of the antibody produced by a given B cell are identical to those of the BCRs borne on the same B cell. Antibodies recognize naturally occurring proteins, glycoprotein, carbohydrates, or phospholipids, such as structural components of bacteria and viruses or bacterial toxins (FIGURE 16.3). Binding of antigen by antibody gives rise to an antigen–antibody complex, which, in turn, triggers the activation of soluble mediators and phagocytic cells (mainly macrophages) that eventually lead to the disruption of the antibody-bound bacterium or virus. A major soluble mediator is complement, a multiprotein/enzymatic cascade, whose name reflects its ability to “complement” the action of the antibody itself. Complement consists of a set of more than 20 proteins that function through a proteolytic cascade. If the target antigen is part of a cell—for example, an infecting bacterium—the action of complement culminates in the lysis of the bacterium. The activation of complement also releases proinflammatory soluble mediators and chemotactic mediators; that is, molecules that can attract phagocytic cells, such as macrophages and granulocytes, which scavenge the target cells or their products. Complement is also an important innate immune mediator, integrating the innate and adaptive immune functions when activated by an antibody. Antibody-coated bacteria may also be directly killed by macrophages (scavenger cells) that are recruited by the antigen–antibody complex.

FIGURE 16.3 Free antibodies bind to antigens to form antigen–antibody complexes that are removed from the bloodstream by macrophages or are attacked directly by the activated complement cascade.

T cells are activated upon TCR recognition of peptide fragments derived from a foreign antigen. A crucial feature of TCR recognition is that the antigen must be presented in conjunction with a major histocompatibility complex (MHC) molecule, which is expressed by an antigen-presenting cell (APC). The MHC possesses a groove on its surface that binds a peptide fragment derived from the foreign antigen. The TCR recognizes the combination of a peptide fragment and MHC protein. The requirement that T lymphocytes recognize (foreign) antigen in the context of (self) MHC protein ensures that the cell-mediated response acts only on host cells that have been infected with a foreign antigen. MHC proteins also share some common features with antibodies, as do other lymphocyte-specific proteins; the immune system relies on a series of superfamilies of genes that may have evolved from common ancestors encoding primitive defense elements.

Each individual has a characteristic set of MHC proteins that fall into the general clusters of class I and class II, which restrict the activation of T_h cells and cytotoxic T cells (CTLs), respectively. T_h cells are activated by APCs, such as DCs and B lymphocytes. Cognate interactions of T_h and B cells activated by the same antigen allow the engagement of the CD40 receptor expressed on B cells by the CD40 ligand (also called CD154) expressed on T cells. CD40 ligation, together with the exposure to cytokines produced by T_h cells and other immune cells, induces B cells to undergo optimal proliferation and differentiation. In contrast to T_h cells, CTLs, or killer T cells, mediate responses that kill host cells infected by an intracellular parasite, such as a virus (FIGURE 16.4).

FIGURE 16.4 In cell-mediated immunity, cytotoxic T cells use the T cell receptor (TCR) to recognize a peptide fragment of the antigen that is presented on the surface of the target cell by the MHC molecule.

16.4 Clonal Selection Amplifies Lymphocytes That Respond to a Given Antigen

After an organism has been exposed to an antigen, such as one on an infectious agent, it becomes generally immune to infection by the same agent. Before exposure to a particular antigen, the organism lacks adequate capacity to deal with any toxic effects mediated by or associated with that agent. This ability is acquired through the induction of a specific immune response. After an infection has been defeated, the organism retains the ability to respond rapidly in the event of a reinfection by the same microorganism.

The dynamic distribution of B and T lymphocytes maximizes their chances to encounter their target antigens. Lymphocytes are peripatetic cells. They develop from immature stem cells in the adult bone marrow. They migrate via the bloodstream to the peripheral lymphoid tissues, such as the spleen, lymph nodes, Peyer’s patches, and tonsils. Lymphocytes recirculate between blood and lymph throughout the body, thereby ensuring that an antigen will be exposed to lymphocytes of all possible specificities.

Under appropriate conditions, when a lymphocyte encounters an antigen that binds its BCR or TCR, a specific immune response can be elicited. This is brought about by clonal selection and clonal amplification (FIGURE 16.5). The repertoire of B and T lymphocytes comprises a large variety of BCRs or TCRs. Any individual B lymphocyte expresses one given BCR, which is capable of recognizing specifically only a single antigen; likewise, any individual T lymphocyte expresses only one given TCR. In the lymphocyte repertoire, unstimulated B cells and T cells are morphologically indistinguishable. Upon exposure to antigen, though, a B cell whose BCR is able to bind the antigen, or a T cell whose TCR can recognize it, is activated and induced to divide, by signaling from the surface of the cell through the BCR/TCR and associated signaling molecules. The induced cell then undergoes rigorous proliferation and morphological changes, including an increase in cell size, and differentiation into an antibody-producing cell or effector T cell. The initial expansion of a specific B or T cell upon first exposure to antigen underlies the primary immune response, leading to the production of large numbers of B or T lymphocytes with specificity for the target antigen. Each population represents a clone of the original responding cell. Selected B cells secrete large quantities of antibodies, and they may even come to dominate the antibody response.

FIGURE 16.5 The B cell and T cell repertoires include BCRs and TCRs with a variety of specificities. Encounter with an antigen leads to clonal expansion of the lymphocyte with the BCR or TCR that can recognize the antigen.

After a successful primary immune response has been mounted and the challenging antigen cleared, the organism retains the selected B and T cell clones expressing the BCRs and TCRs that are specific for the antigen that induced the response. These memory cells respond promptly and vigorously with clonal expansion upon encounter with the same antigen that induced their differentiation, leading to a secondary (or memory or anamnestic) immune response. Thus, both memory B and T cells are critical elements in the specific resistance to infections after first exposure to a microbial pathogen or vaccine.

The repertoire of B lymphocytes in a mammal comprises more than 10¹² specificities (i.e., clones). The T cell repertoire is less expansive. Some clones are poorly represented; that is, they consist of a few cells each, as the corresponding antigen had never been encountered before. Others consist of as many as to 10⁶ cells, because clonal selection has selected and expanded the progeny of lymphocyte in response to a specific antigen. Naturally occurring antigens are in general relatively large molecules and efficient immunogens, inducing an effective immune response. Small molecules may identify antigenic determinants and can be recognized by antibodies, although owing to their small size they are not effective in inducing an immune response. They do, however, induce a response when conjugated with a larger carrier molecule, usually a protein, such as ovalbumin (OVA), keyhole limpet hemocyanin (KLH), or chicken gamma globulin (CGG). A small molecule that is not immunogenic per se but that can elicit a specific response upon conjugation with a carrier is defined as a hapten. Haptens conjugated with protein carriers generally induce T-dependent antibody responses. T-independent immunizations can be induced by dextran, Ficoll, lipopolysaccharides, or biodegradable nanoparticles. Only a small part of the surface of a macromolecular antigen is actually recognized by any one antibody. The binding site consists of only five or six amino acids. Any given protein may have more than one such binding site, in which case it induces antibodies with specificities for different sites. The site or region inducing a response is called an antigenic determinant or epitope. In an antigen containing several epitopes, some epitopes may be more effective than others in inducing a specific immune response. In fact, they may be so effective that they dominate the response, in that they are the targets of all specifically elicited antibodies and/or effector T cells.

16.5 Ig Genes Are Assembled from Discrete DNA Segments in B Lymphocytes

Sophisticated evolutionary mechanisms have evolved to guarantee that the organism is prepared to produce specific antibodies for a broad variety of naturally occurring and manmade components that it has never encountered before. Each antibody is a tetramer consisting of two identical immunoglobulin light (L) chains and two identical immunoglobulin heavy (H) chains (FIGURE 16.6). Humans and mice have two types of L chains (λ and κ) and nine types of H chains. The class is determined by the H chain constant (C) region, which mediates the antibody’s biological effector functions. Different Ig classes have different effector functions. L chains and H chains share the same general type of organization in that each protein chain consists of two principal domains: the N-terminal variable (V) region and the C-terminal constant (C) region. These were defined originally by comparing the amino acid sequences of different Ig chains secreted by monoclonal B cell tumors (plasmacytomas). As the names suggest, the V regions show considerable changes in sequence from one protein to the next, whereas the C regions show substantial homology.

FIGURE 16.6 An antibody (immunoglobulin, or Ig) molecule is a heterodimer consisting of two identical heavy chains and two identical light chains. Schematized here is an IgG1, which comprises an N-terminal variable (V) region and a C-terminal constant (C) region.

Corresponding regions of the L and H chains associate to generate distinct domains in the Ig protein. The V domain is generated by association between a recombined H chain V_HDJ_H segment and a recombined L chain V_λJ_λ or V_κJ_κ segment. The V domain is responsible for recognizing the antigen. Generation of V domains of different specificities creates the ability to respond to diverse antigens. The total number of V region genes for either L or H chain proteins is measured in hundreds. Thus, an antibody displays the maximum versatility in the region responsible for binding the antigen. The C regions in the subunits of the Ig tetramer associate to generate individual C domains. The first domain results from association of the single C region of the L chain (C_L) with the C_H1 domain of the H chain C region (C_H). The two copies of this domain complete the arms of the Y-shaped antibody molecule. Association between the C regions of the H chains generates the remaining C domains, which vary in number (three of four) depending on the type of H chain.

Many genes encode V regions, but only a few genes encode C regions. In this context, “gene” means a sequence of DNA coding for a discrete part of the final Ig polypeptide (H or L chain). Thus, recombined V(D)J genes encode variable regions, and C genes encode constant regions. To construct a unit that can be expressed in the form of a whole L or H chain, a V(D)J gene must be joined physically to a C gene.

The sequences encoding L chains and H chains are assembled in the same way: Any one of several V(D)J gene segments may be joined to any one of a few C gene segments. This somatic DNA recombination occurs in the B lymphocyte in which the BCR/antibody is expressed. The large number of available V(D)J gene segments is responsible for a major part of the diversity of Igs. Not all diversity is encoded in the genome, though; more is generated by changes that occur during the assembly process of a functional gene.

Essentially the same mechanisms underlie the generation of functional genes encoding the protein chains of the TCR. Two types of receptor are found on T cells—one consisting of α and β chains, and the other consisting of γ and δ chains. Like the genes encoding Igs, the genes encoding the individual chains in TCRs consist of separate parts, including recombined V(D)J gene segments and C region genes.

The organism does not possess the functional genes in the germline for producing a particular BCR or TCR. It possesses a large repertoire of V gene segments and a smaller number of C gene segments. The subsequent assembly of a productive gene from these parts allows the BCR/TCR to be expressed on B and T cells so that it is available to react with the antigen. V(D)J DNA rearrangement occurs before exposure to antigen. Productive V(D)J rearrangements are expressed by B cells and T cells as surface BCRs and TCRs, which provide the structural substrate for selection of those clones capable of binding the antigen. The arrangement of V(D)J gene segments and C gene segments is different in the cells expressing BCR or TCR from all other somatic cells or germ cells. The entire process occurs in somatic cells and does not affect the germline; thus, the progeny of the organism does not inherit the specific response to an antigen.

The Ig κ and λ chains and H chain loci reside on different chromosomes, and each locus consists of its own set of both V gene segments and C gene segments. This germline organization is found in the germline and in the somatic cells of all lineages. In a B cell expressing an antibody, though, each chain—one L type (either κ or λ) and one H type—is encoded by a single intact DNA sequence. The recombination event that brings a V(D)J gene segment in proximity to, and to be expressed with, a C gene segment creates a productive gene consisting of exons that correspond precisely with the functional domains of the protein. After transcription of the whole DNA sequence into a primary RNA transcript, the intronic sequences are removed by RNA splicing.

V(D)J recombination occurs in developing B lymphocytes. A B lymphocyte, in general, carries only one productive rearrangement of L chain gene segments (either κ or λ) and one of H chain gene segments. Likewise, a T lymphocyte productively rearranges an α gene and a β gene or a δ gene and a γ gene. The BCR and TCR expressed by any one cell is determined by the particular configuration of V gene segments and C gene segments that have been joined.

The principles by which functional genes are assembled are the same in each family, but there are differences in the details of the organization of both the V and C gene segments, and correspondingly of the recombination reaction between them. In addition to these segments, other short DNA sequences (D segments and J, “joining,” segments) are included in the functional somatic loci.

If any L chain can pair with any H chain, about 10⁶ different L chains and about 10⁶ different H chains can pair to generate more than 10¹² different Igs. Indeed, a mammal has the ability to generate 10¹² or more different antibody specificities.

16.6 L Chains Are Assembled by a Single Recombination Event

A λ chain is assembled from two DNA segments (FIGURE 16.7). The V_λ gene segment consists of the leader exon (L) separated by a single intron from the V segment. The J_λ−C_λ gene segment consists of the J_λ segment separated by a single intron from the C_λ exon.

J is an abbreviation for “joining,” because the J segment identifies the region to which the V_λ segment becomes connected. Thus, the joining reaction does not directly involve V_λ and C_λ gene segments, but occurs via the J_λ segment (V_λJ_λ-C_λ joining). The J_λ segment is short and codes for the last few amino acids of the variable region, as defined by amino acid sequence. In the complete gene generated by recombination, the V_λ-J_λ segment constitutes a single exon coding for the entire variable region.

FIGURE 16.7 The C_λ gene segment is preceded by a J_λ segment, so that V_λ-J_λ recombination generates a productive V_λ-J_λC_λ.

A κ chain is also assembled from two DNA segments (FIGURE 16.8). However, the organization of the C_κ locus differs from that of the C_λ locus. A group of five J_κ segments is spread over a region of 500 to 700 bp, separated by an intron of 2 to 3 kb from the C_κ exon. In the mouse, the central J_κ segment is nonfunctional (φJ3). A V_κ segment (which contains a leader exon, such as V_λ) may be joined to any one of the J_κ segments. Whichever J_κ segment is used, it becomes the terminal part of the intact variable exon. Any J_κ segment upstream of the recombining J_κ segment is lost; any J_κ segment downstream of the recombining J_κ segment is treated as part of the intron between the V and C exons.

FIGURE 16.8 The C_κ gene segment is preceded by multiple J_κ segments in the germline. V_κ-J_κ joining may recognize any one of the J segments, which is then spliced to the C gene segment during RNA processing.

All functional J_L segments possess a signal at their 5′ boundary that makes it possible to recombine with a V segment; they also possess a signal at the 3′ boundary that can be used for splicing to the C exon. Whichever J_L segment is recognized in DNA V-J_L joining, it will use its splicing signal in RNA processing.

16.7 H Chains Are Assembled by Two Sequential Recombination Events

The IgH locus includes an additional set of gene segments, the D segments. Thus, the assembly of a complete H chain entails recombination of V_H, D, and J_H genes. The D segment (for diversity) was discovered by the presence in the H chain peptide sequences of an extra 2 to 13 amino acids between the sequences coded by the V_H and the J_H segments. An array of D segments lies on the chromosome between the cluster of V_H segments and that of J_H segments.

V_HDJ_H joining takes place in two stages (FIGURE 16.9). First, one of the D segments recombines with a J_H segment; second, a V_H segment recombines with the already recombined DJ_H segment. The resulting V_HDJ_H DNA sequence is then expressed with the nearest downstream C_H gene, which consists of a cluster of four exons (the use of different C_H genes is discussed in the section in this chapter titled Class Switch DNA Recombination). The D segments are organized in a tandem array. The human locus comprises about 30 D segments, followed by a cluster of 6 J_H gene segments. The same D segment is involved in the DJ_H recombination and related V_HDJ_H recombination.

FIGURE 16.9 Heavy genes are assembled by sequential recombination events. First a D_H segment is recombined with a J_H segment, and then a V_H gene segment is recombined with the D_H segment.

The structure of recombined V(D)J segments is similar in organization in the H chain and λ and κ chain loci. The first exon codes for the signal sequence, which is involved in membrane attachment, and the second exon codes for the major part of the variable region itself, which is about 100 codons long. The remainder of the variable region is provided by the D segment (in the H chain locus only) and by a J segment (in all three loci).

The structure of the C region differs in different H and L chains. In both κ and λ chains, the C region is encoded by a single exon, which becomes the third exon of the recombined V_κJ_κ-C_κ or V_λJ_λ-C_λ gene. In H chains, the C region is encoded by multiple and discrete exons, separately coding for four regions: C_H1; C_H hinge; C_H2 and C_H3 (IgG, IgA, and IgD); or C_H1, C_H2, C_H3, and C_H4 (IgM and IgE). Each C_H exon consists of about 100 codons, with the hinge exon being shorter; the intronic sequences are about 300 bp each.

16.8 Recombination Generates Extensive Diversity

A census of the available V, D, J, and C gene segments provides a measure of the diversity that can be accommodated by the variety of the coding regions carried in the germline. In both the IgH and L chain loci, many V gene segments are linked to a much smaller number of C gene segments.

The human λ locus (chromosome 22) has seven C_λ genes, each preceded by its own J_λ segment (FIGURE 16.10). The mouse λ locus (chromosome 16) is much less diverse. The main difference is that in a mouse there are only two V_λ gene segments, each of which is linked to two J_λC_λ regions. One of the C_λ segments is a pseudogene (nonfunctional gene). This configuration suggests that the mouse suffered in its evolutionary history a large deletion of most of its germline V_λ gene segments.

FIGURE 16.10 The lambda family consists of V_λ gene segments and a small number of J_λ-C_λ gene segments.

Both the human κ locus (chromosome 2) and the mouse κ locus (chromosome 6) have only one C_κ gene segment, preceded by six J_κ gene segments (one of them being a pseudogene) (FIGURE 16.11). The V_κ gene segments occupy a large cluster on the chromosome, upstream of the C_κ region. The human cluster has two regions. Just upstream of the C_κ gene segment a 600-kb region contains the J_κ segments and 40 V_κ gene segments. A gap of 800 kb separates this region from another cluster of 36 V_κ gene segments.

FIGURE 16.11 The human and mouse Igκ families consist of V_κ gene segments and five functional J_κ segments linked to a single C_κ gene segment. V_κ genes include nonfunctional pseudogenes.

The V_H, V_κ, and V_λ gene segments are segregated into families. A family comprises members that share more than 80% amino acid identity. In humans, the V_H locus comprises six V_H families: V_H1 through V_H6. V_H3 and V_H4 are the largest families, each with more than 10 functional members; V_H6 is the smallest family, consisting of one member only. In mice, the V_κ locus comprises about 18 Vκ families, which vary in size from 2 to 100 members. Like other families of related genes, related V gene segments form subclusters, which were generated by duplication and divergence of individual ancestral members. Many of the V segments are pseudogenes. Although nonfunctional, some of these may function as donors of partial V sequences in secondary rearrangements.

A given lymphocyte expresses either a κ or a λ chain to be paired with a V_HDJ_H-C_H chain. In humans, about 60% of B cells express κ chains and about 40% express λ. In the mouse, 95% of B cells express a κ chain, presumably because of the reduced number of λ gene segments available.

The single IgH chain locus (human chromosome 14) consists of multiple discrete segments (FIGURE 16.12). The furthest 3′ member of the V_H cluster is separated by only 20 kb from the first D segment. The D segments (30) are spread over approximately 50 kb, followed by the cluster of 6 J_H segments. Over the next 220 kb lie all the C_H genes. In addition to the nine functional C_H genes, there are two pseudogenes. The human IgH locus organization suggests that a Cγ gene was duplicated to generate the Cγ-Cγ-Cε-Cα subcluster, after which the entire subcluster was then tandemly duplicated. The mouse IgH locus (chromosome 12) has more V_H gene segments, fewer D and J_H segments, and eight (instead of nine) C_H genes.

FIGURE 16.12 A single gene cluster in humans contains all the information for the IgH chain. Depicted is a schematic map of the human IgH chain locus.

The human IgH locus alone can produce more than 10⁴ different V_HDJ_H sequences by combining 51 V_H genes, 30 D segments, and 6 J_H segments. This degree of diversity is further compounded by the imprecision in the V_HDJ_H joinings, the insertion of unencoded nucleotide (N) additions, and use of multiple D-D segments. By combining any one of more than 50 V_κ gene segments with any 1 of 5 J_κ segments the human κ locus has the potential to produce 300 different V_κJ_κ segments. These, however, are conservative estimates, because more diversity is introduced by insertion of untemplated N nucleotides, albeit at lower frequency than in V_HDJ_H. Further diversity is produced by pairing of the same V_HDJ_H-C chain with different V_κJ_κ-C_κ or V_λJ_λ-C_λ chains. Finally, diversification in individual genes after V_HDJ_H, V_κJ_κ, and V_λJ_λ recombination occurs by somatic hypermutation (SHM) (see the section in this chapter titled Somatic Hypermutation Generates Additional Diversity and Provides the Substrate for Higher-Affinity Submutants).

16.9 V(D)J DNA Recombination Relies on RSS and Occurs by Deletion or Inversion

The recombination of Igκ, Igλ, and IgH chain genes involves the same mechanism, although the number and nature of recombining elements differ. The same consensus sequences are found at the boundaries of all germline segments that participate in the joining reactions. Each consensus sequence consists of a heptamer (7-bp sequence) separated by an either 12- or 23-bp spacer from a nonamer (9-bp sequence). These sequences are referred to as recombination signal sequences (RSSs) (FIGURE 16.13). In the κ locus, each V_κ gene segment is followed by an RSS sequence with a 12-bp spacer. Each J_κ segment is preceded by an RSS with a 23-bp spacer. The V_κ and J_κ RSSs are inverted in orientation. In the λ locus, each V_λ gene segment is followed by an RSS with a 23-bp spacer; each J_λ gene segment is preceded by an RSS with a 12-bp spacer. The rule that governs the joining reaction is that an RSS with one type of spacer can be joined only to an RSS with the other type of spacer. This is referred to as the 12/23 rule.

FIGURE 16.13 RSS sequences are present in inverted orientation at each pair of recombining sites. One member of each pair has a 12-bp spacer between its components; the other has a 23-bp spacer.

In the IgH locus, each V_H gene segment is followed by an RSS with a 23-bp spacer. The D segments are flanked on either side by RSSs with 12-bp spacers, and the J_H segments are preceded by RSSs with 23-bp spacers. The RSSs at V and J segments can lie in either order; thus the different spacers do not impart any directional information, but instead serve to prevent one V or J gene segment from recombining with another of the same. Thus, a V_H segment must recombine with a D segment, and a D segment must recombine with a J_H segment. A V_H gene segment cannot recombine directly with a J_H segment, because both possess the same type of RSS. The spacer between the components of the RSS corresponds to close to one (12 bp) or two turns (23 bp) of the double helix. This may reflect geometric constraints in the recombination reaction. The recombination protein(s) may approach the DNA from one side, in the same way that RNA polymerase and repressors approach recognition elements, such as promoters and operators.

Recombination of the components of Ig genes is accomplished by a physical rearrangement of different DNA segments that involves DNA breakage and ligation. In the H chain locus, two recombination events occur: first DJ_H, then V_HDJ_H. DNA breakage and ligation occur as separate reactions. A DSB is made in each of the heptamers that lie at the ends of the coding units. This releases the DNA between the V and J-C gene segments; the cleaved termini of this fragment are called signal ends. The cleaved termini of the V and J-C loci are called coding ends. The two coding ends are covalently linked to form a coding V-C joint.

Most V_L and J_L-C_L gene segments are organized in the same orientation. As a result, the cleavage at each RSS releases the intervening DNA as a linear fragment, which, when relegated at the signal ends gives rise to a circle (FIGURE 16.14). Deletion to release an excised DNA circle is the predominant mode of recombination at the Ig and TCR loci.

In some cases, the V_λ gene segment in germline configuration is inverted in orientation on the chromosome relative to the J_λ-C_λ DNA, and DNA breakage and ligation invert the intervening DNA instead of deleting it. The outcomes of deletion versus inversion in terms of the coding sequence are the same. Recombination with an inverted V gene segment, however, makes it necessary for the signal ends to be joined or a DSB in the locus is generated. Recombination by inversion occurs also in some cases in the κ locus, the IgH locus, and the TCR locus.

FIGURE 16.14 Breakage and recombination at RSSs generate VJC sequences. A generic V-J rearrangement is shown for simplicity. In most cases, the V and J segments undergoing recombination are arranged in the same transcriptional orientation and rearrangement occurs by deletion of the intervening DNA, as shown. Less commonly, V and J segments undergoing recombination are arranged in opposite transcriptional directions and rearrangement occurs by inversion (not shown).

Data from D. B. Roth, Nat. Rev. Immunol. 3 (2003): 656–666.

16.10 Allelic Exclusion Is Triggered by Productive Rearrangements

Virtually all B cells express a single κ or λ chain and a single type (isotype) of IgH chain, because only a single productive rearrangement of each type occurs in a given lymphocyte in order to express only one L and one H chain. Each event involves the genes of only one of the homologous chromosomes. Thus, the alleles on the other chromosome are not expressed in the same cell. This phenomenon is termed allelic exclusion.

The occurrence of allelic exclusion complicates the analysis of somatic recombination, because both homolog alleles can be recombined: one in a productive (expressed H or κ or λ chain), the other in a nonproductive rearrangement. A DNA probe reacting with a region that has rearranged on one homolog will also detect the allelic sequences on the other homolog. Thus, the V(D)J configuration on both homolog chromosomes must be analyzed in order to understand the natural history of the V(D)J rearrangement of a given B cell.

Two different configurations of Ig locus can exist in B cells:

A DNA probe specific for the expressed V gene may reveal one rearranged copy and one germline copy, indicating that recombination has occurred on one chromosome, whereas the other chromosome has remained unaltered.
A DNA probe specific for the expressed V gene reveals two different rearranged patterns, indicating that both chromosomes underwent independent V(D)J recombination events involving the same gene.

In general, in those cases in which both chromosomes in a B cell underwent recombination, only one of them underwent a productive rearrangement to express a functional IgH or L chain. The other suffered a nonproductive rearrangement. This can occur in different ways, but in each case the gene sequence cannot be expressed as an Ig chain. The rearrangement may be incomplete (e.g., because DJ_H joining has occurred but V_HDJ_H joining has not followed), or it may be aberrant (nonproductive), with the process completed but failing to generate a gene that encodes a functional protein.

The coexistence of productive and nonproductive rearrangements suggests the existence of a feedback mechanism controlling the recombination process (FIGURE 16.15). A B lineage progenitor cell starts with two IgH chain loci in the (unrearranged) germline configuration (Ig⁰). Either locus may recombine V_H, D, and J_H-C_H to generate a productive gene (IgH⁺) or a nonproductive gene (IgH^–) rearrangement. If the first rearrangement is productive, the expression of a functional IgH chain provides an inhibitory signal to the B cell to prevent rearrangement of the other IgH allele. As a result, the configuration of this B cell with respect to the IgH locus will be IgH⁺/Ig⁰. If the first rearrangement is nonproductive, it will result in a configuration Ig⁰/Ig^–. The lack of an expressed IgH chain will not provide an inhibitory (negative) feedback for rearrangement of the remaining germline allele. If this undergoes a productive rearrangement, the B cell will have the configuration Ig⁺/Ig^–. Two successive nonproductive rearrangements will result in an Ig^–/Ig^– configuration. In some cases, a B cell in an Ig^–/Ig^– configuration can attempt an atypical rearrangement utilizing cryptic RSSs embedded in the coding DNA of a V gene. Indeed, certain Ig locus DNA configurations found in B cells can only be explained as having been generated by sequential rearrangements of nonproductively rearranged sequences.

FIGURE 16.15 A successful rearrangement to produce an active light (depicted) or heavy chain suppresses further rearrangements of the same type, resulting in allelic exclusion.

Thus, allelic exclusion is caused by the suppression of further rearrangements as soon as a productive IgH or L chain rearrangement is achieved. Allelic exclusion in vivo is exemplified by the creation of transgenic mice in which a rearranged V_HDJ_H-C_H or V_κJ_κ-C_κ or V_λJ_λ-C_λ DNA has been inserted into the Ig locus. Expression of the transgene in B cells suppresses the corresponding rearrangement of endogenous V(D)J genes. Allelic exclusion is independent for the IgH, κ, and λ chain loci. IgH chain genes generally rearrange first. Allelic exclusion for L chains applies equally to both families (cells may express either productive κ or λ chains). In most cases, a B cell rearranges its κ locus first. It then tries to rearrange the λ locus only if both κ rearrangement attempts are unsuccessful.

The same consensus sequences and the same V(D)J recombinase are involved in the recombination reactions at IgH, κ, and λ loci, and yet the three loci rearrange in a sequential order. It is unclear why the IgH chain rearrangement precedes L chain rearrangement and why κ precedes λ chain rearrangements. The DNA in the different loci may become accessible to the enzyme(s) effecting the rearrangement at different times, possibly reflecting each locus transcription status. Transcription starts before rearrangement, although some Ig-locus mRNA, such as germline I_H-C_H transcripts, have no coding function. Transcription events may change the structure of chromatin, making the consensus sequences for recombination available to the enzyme effecting the rearrangement.

16.11 RAG1/RAG2 Catalyze Breakage and Religation of V(D)J Gene Segments

The recombination activating gene (RAG) proteins, RAG1 and RAG2, are necessary and sufficient for DNA cleavage in V(D)J recombination. They are encoded by two genes, separated by less than 10 kb: RAG1 and RAG2. RAG1/RAG2 gene transfection into fibroblasts causes a suitable DNA substrate to undergo the V(D)J recombination. Mice that lack RAG1 or RAG2 are unable to recombine their BCR and TCR, and as a result abort B lymphocyte and T lymphocyte development. RAG1/RAG2 proteins together undertake the catalytic reactions of cleaving and rejoining DNA, and also provide a structural framework within which the whole recombination reaction occurs.

RAG1 recognizes the RSS (heptamer/nonamer signal with the appropriate 12- or 23-bp spacing) and recruits RAG2 to the complex. The nonamer provides the site for initial recognition, and the heptamer directs the site of cleavage. The complex nicks one strand at each junction (FIGURE 16.16). The nick has 3′–OH and 5′–P ends. The free 3′–OH end then attacks the phosphate bond at the corresponding position in the other strand of the duplex. This creates a hairpin at the coding end, in which the 3′ end of one strand is covalently linked to the 5′ end of the other strand, and leaves a blunt DSB at the signal end.

FIGURE 16.16 Processing of coding ends introduces variability at V_κJ_κ, V_λJ_λ, or V_HDJ_H junctions. Depicted is a V_κJ_κ junction.

This second cleavage is a transesterification reaction in which bond energies are conserved. It resembles the topoisomerase-like reactions catalyzed by the resolvase proteins of bacterial transposons (see the section titled Transposition Occurs by Both Replicative and Nonreplicative Mechanisms in the chapter titled Transposable Elements and Retroviruses). The parallel with these reactions is further supported by a homology between RAG1 and bacterial invertase proteins, which invert specific segments of DNA by similar recombination reactions. In fact, the RAG proteins can insert a donor DNA whose free ends consist of the appropriate signal sequences (heptamer-12/23 spacer-nonamer) into an unrelated target DNA in an in vitro transposition reaction, suggesting that somatic recombination of immune genes evolved from an ancestral transposon.

The hairpins at the coding ends provide the substrate for the next stage of reaction. The Ku70/Ku80 heterodimer binds to the DNA ends and a nuclear protein, Artemis, opens the hairpins. The joining reaction that works on the coding end uses the same pathway of nonhomologous end joining (NHEJ) that repairs DSBs in all cells. If a single-strand break is introduced into one strand close to the hairpin, an unpairing reaction at the end generates a single-stranded protrusion. Synthesis of a complement to the exposed single strand then converts the coding end to an extended duplex. This reaction explains the introduction of P nucleotides at coding ends. P nucleotides are a few extra base pairs related to, but reversed in orientation from, the original coding end.

In addition to P nucleotides, some extra bases called N nucleotides can also be inserted between the coding ends in an untemplated and random fashion. Their insertion occurs via the activity of the enzyme terminal deoxynucleotidyl transferase (TdT), which, like RAG1/RAG2, is expressed at the stages of B and T lymphocyte development when V(D)J recombination occurs, at a free 3′ coding end generated during the joining process through NHEJ.

The initial stages of the V(D)J recombination reaction were identified by isolating intermediates from lymphocytes of mice with a severe combined immunodeficiency (SCID) mutation, which results in a much-reduced level of BCR and TCR V(D)J gene recombination. SCID mice accumulate DSBs at Ig V gene segment coding ends and cannot complete the V(D)J joining reaction. This particular SCID mutation displays a defective DNA-dependent protein kinase (DNA-PK). This kinase is recruited to DNA by the Ku70/Ku86 heterodimer, which binds to the broken DNA ends. DNA-PK_cs (DNA-PK catalytic subunit) phosphorylates and thereby activates Artemis, which, in turn, nicks the hairpin ends; Artemis also possesses exonuclease and endonuclease activities that function in the NHEJ pathway. The actual ligation is undertaken by DNA ligase IV and also requires XRCC4. Mutations in Ku proteins, XRCC4, or DNA ligase IV are found in patients with congenital diseases involving deficiencies in DNA repair that result in increased sensitivity to radiation. The free (signal) 5′-phosphorylated blunt ends at the heptamer sequences of the intervening DNA, which are looped out by the V(D)J recombinations, also bind Ku70/Ku86. Without further modification, a complex of DNA ligase IV/XRCC4 joins the two signal ends to form the signal joint.

Thus, changes in DNA sequence during V(D)J recombination are a consequence of the enzymatic mechanisms involved in breaking and rejoining the DNA. In IgH chain V_HDJ_H recombination, base pairs are lost and/or N nucleotides inserted at the V_HD or DJ_H junctions. Deletions also occur in V_κJ_κ and V_λJ_λ joining, but N insertions at these joints are less frequent than in V_HD or DJ_H junctions. The changes in sequence affect the amino acid coded at V_HDJ_H junctions or at V_LJ_L junctions.

The above mechanisms will ensure that most coding joints will display a different sequence from that predicted as a result of direct joining of the coding ends of the V, D, and J segments involved in each recombination. Variations in the sequence of V_LJ_L junctions make it possible for different amino acid residues to be encoded here, generating diverse structures at this site that contacts antigen. The amino acid at position 96 is created by V_κJ_κ and V_λJ_λ recombination. It forms part of the antigen-binding site and also is involved in making contacts between the L chains and the H chains. Thus, maximum diversity is generated at the site that contacts the target antigen.

Changes in the number of base pairs at coding joints affect the reading frame. V_LJ_L recombination appears to be random with regard to reading frame, so that only one-third of the joined sequences retain the proper reading frame through the junctions. If a V_κJ_κ or V_λJ_λ recombination occurs so that the J_L segment is out of frame, translation is terminated prematurely by a nonsense codon in the incorrect frame. This may be the price a B cell pays for being able to generate maximal diversity of the expressed V_κJ_κ and V_λJ_λ sequences. Even greater diversity is generated by recombinations that involve the V_H, D, and J_H gene segments of the Ig H chain, mainly due to random and variable “chopping off” of D and J_H DNA, as well as random and variable N nucleotide insertions. Nonproductive recombinations are generated by a joining that places V_H out of frame with the rearranged D-J_H gene segment.

Germline (unrearranged) V gene segments about to undergo recombination are transcribed, albeit at a moderate level. Once V(D)J gene segments are productively recombined, the resulting sequence is transcribed at a higher rate. The sequence upstream of a V gene segment is not altered by the joining reaction, though, and as a result the promoter is conserved in unrearranged, nonproductively rearranged, and productively rearranged V genes. The V promoter lies upstream of every V gene segment but is only moderately active when in germline configuration. Its activation is significantly enhanced by its downstream relocation closer to the C region after V(D)J rearrangement, suggesting that the V promoter activation depends on downstream cis-elements (FIGURE 16.17). Indeed, an enhancer element located within or downstream of the V, D, and J gene clusters significantly enhances the activation of V promoter. This enhancer is referred to as intronic enhancer (iEμ in the H chain and iEκ in the κ chain). It is tissue specific, being active only in B cells.

FIGURE 16.17 A V gene promoter is inactive until recombination brings it into the proximity (and therefore under the influence) of the iEμ enhancer that lies downstream of the Sμ region and upstream of the Cμ exon cluster. The enhancer is active only in B lymphocytes.

16.12 B Cell Development in the Bone Marrow: From Common Lymphoid Progenitor to Mature B Cell

B cells differentiate from hematopoietic stem cells (HSCs) in the bone marrow. In the first step, an IgH D segment is recombined with a J_H segment. Cells at this stage (recombined DJ_H) are referred to as pro-B cells. DJ_H recombination is followed by V_HDJ_H recombination, which generates an IgH μ chain; these cells are now pre-B cells. Several recombination events involving a succession of nonproductive and productive rearrangements may occur, as discussed previously. As a pro-B cell differentiates to a pre-B cell, it expresses on the surface a productively recombined IgH V_HDJ_H-Cμ paired with a surrogate L chain (λ-Vpre-B, a protein resembling a λ chain) to give rise to pre-BCR, a monomeric IgM molecule (L₂μ₂), which consists of the Cμm version of the constant region (FIGURE 16.18). The pre-BCR is similar in function and structure to a BCR, but signals in a different way upon engagement. The pre-BCR signaling drives the pre-B cell through five or six divisions (large pre-B cells) until the pre-B cell stops dividing and reverts back to a small size, thereby signaling the rearrangement of a V gene segment with a J gene segment in the κ or λ locus. After V_κ or V_λ rearrangement, the B cell, now referred to as an immature B cell, will express a BCR consisting of two identical V_HDJ_H-Cμ chains paired with two identical V_κJ_κ-C_κ or V_λJ_λ-C_λ chains, thereby forming a functioning BCR. Thus, the whole process that eventually gives rise to mature B cells depends upon successful Ig V(D)J gene rearrangement. If V(D)J rearrangement is blocked, B cell development is aborted.

FIGURE 16.18 B cell development proceeds through sequential stages of H chain and L chain V(D)J gene rearrangement.

A B cell emerges from the bone marrow as an immature B cell. This expresses a full-fledged BCR consisting of two identical V_HDJ_H-Cμ chains paired with two identical V_κJ_κ-C_κ or V_λJ_λ-C_λ chains, as a membrane-bound monomeric form of IgM (mIgμ; “m” indicates that IgM is located in the membrane). An immature B cell expresses the same BCR, also in an Igδ (mIgδ) context, V_HDJ_H-Cδ, but at a lower density than the corresponding V_HDJ_H-Cμm chains. As the immature B cell transitions to a mature B cell in the periphery, it will increase the expression of surface BCR with IgH δ chains, eventually resulting in a high surface Igδ:Igμ chain ratio. The intracytoplasmic tails of the two IgH chains are associated with transmembrane proteins called Igα and Igβ. These proteins provide the structures that trigger the intracellular signaling pathways in response to BCR engagement by antigen (FIGURE 16.19).

FIGURE 16.19 The BCR consists of an immunoglobulin tetramer (H₂L₂) linked to two copies of the signal-transducing heterodimer (IgαIgβ).

The Cμm-encoding mRNA transcripts have six exons, among which the first four exons (C_H1 through C_H4) code for the four domains of the C_H region and the last two exons, M1 and M2, code for the 41-residue hydrophobic C_H-terminal region and contain the 3′ nontranslated region. This hydrophobic sequence anchors Igμ to the plasma membrane. An alternative splicing event of the same gene transcript gives rise to mRNA that encodes the Cμs (secreted) version of the C_H region—that is, IgM—which exists in general as a pentamer IgM₅J. J (unrelated to the J region gene) is a joining polypeptide that forms disulfide linkages with μ chains. During the alternative splicing, the 5′ splicing donor site at the end of the C_H4 exon is bypassed, resulting in the extension of transcription beyond C_H4 for an additional 20 codons (FIGURE 16.20). These encode a shorter hydrophilic sequence that replaces the 41-residue hydrophobic sequence in Cμm, thereby allowing the Igμ chain to be secreted. A similar transition from membrane to secreted forms occurs for the other Ig isotypes.

FIGURE 16.20 The 3′ end of each C_H (C_μ, C_γ, C_α, or C_δ) gene cluster controls the use of splicing junctions so that alternative forms (membrane or secretory) of the heavy gene are expressed.

16.13 Class Switch DNA Recombination

Class switch recombination (CSR) and somatic hypermutation (SHM) are the two central processes that underlie the antigen-driven differentiation of mature B cells in high-affinity, class-switched, antibody-producing cells and memory B cells. This differentiation process recruits mature naïve B cells and generally occurs in peripheral lymphoid organs, including the spleen, lymph nodes, and Peyer’s patches, in either a T-dependent or T-independent fashion.

B lymphocytes start their “productive” life as naïve B cells expressing IgM and IgD on their surfaces. After encountering antigen, a B cell undergoes activation, proliferation, and differentiation from an IgM- to an IgG-, IgA-, or IgE-producing cell. This process occurs in peripheral lymphoid organs, such as the lymph nodes and spleen, and is referred to as class switching. Class switching is induced either in a T-dependent fashion through engagement of surface B cell CD40 by CD154 expressed on the surface of T_h cells and exposure to T cell–derived cytokines, such as IL-4 (IgG and IgE) and TGF-β (IgA), or in a T-independent fashion through, for instance, engagement of TLRs on B cells by conserved molecules on bacteria or viruses (MAMPs), such as bacterial lipolysaccharides or CpG or viral dsRNA. After undergoing class switching from IgM, a B lymphocyte expresses only a single class of Ig at any one time.

IgM is the first Ig to be produced by a differentiating B cell and activates complement efficiently. IgD is subsequently expressed when the mature B cell exits the bone marrow. The class of Ig is defined by the type of C_H region. The remaining three C_H classes—IgG, IgA, and IgE (TABLE 16.2)—are exposed on a B cell after undergoing class switching. IgG comprises four subclasses—IgG1, IgG2, IgG3, and IgG4 in humans and IgG1, IgG2a, IgG2b, and IgG3 in mice—and is the most abundant Ig in the circulation. Unlike IgM, which is confined to circulation, IgG passes into the extravascular spaces. IgA is abundant on mucosal surfaces and on secretions in the respiratory tract and the intestine. IgE is associated with the allergic response and with defense against parasites. It is secreted on mucosal surfaces of the respiratory tract.

TABLE 16.2 Immunoglobulin type and functions are determined by the H chain. J is a joining protein in IgM, unrelated to J (joining) gene segments. IgM exists mainly as a pentamer (i.e., 5 IgM μ₂L₂ tetramers) and IgA as a dimer. IgD, IgG, and IgE exist as single H₂L₂ tetramers.

Type	IgM	IgD	IgG	IgA	IgE
C_H chain	μ	δ	γ	α	ε
Structure	(μ₂L₂)₅J	δ₂L₂	γ₂L₂	(α₂L₂)_2J	ε₂L₂
Proportion in circulating blood	5%	1%	80%	14%	< 1%
Effector function	Activates complement Effectively clears bacteria in circulation; does not pass into the extravascular fluid	Development of tolerance (?) Activates basophils and mast cells to produce antimicrobial factors	Activates complement Provides the majority of antibody-based immunity against invading pathogens	Found in secretions Prevents colonization of muscle by pathogens	Allergic responses clear intestinal parasites

Class switching involves only C_H genes; the V_HDJ_H segment originally expressed as part of an IgM and IgD (naïve B cell) continues to be expressed in a new context (IgG, IgA, or IgE). A given recombined V_HDJ_H segment can be expressed sequentially in combination with more than one C_H gene region. The same V_κJ_κ-C_κ or V_λJ_λ−C_λ chain continues to be expressed throughout the lineage of the cell. CSR, therefore, allows the type of biological effector response (mediated by the C_H region) to change while maintaining the same specificity of antigen recognition (mediated by the combination of V_HDJ_H and V_κJ_κ or V_HDJ_H and V_λJ_λ regions).

CSR involves a mechanism different from that effecting V(D)J recombination and is active later in B cell differentiation, generally in peripheral lymphoid organs. B cells that undergo CSR show deletions of the DNA encompassing Cμ and all the other Cμ gene segments preceding the expressed C_H gene. CSR entails a recombination that brings a (new) downstream C_H gene segment into juxtaposition with the expressed V_HDJ_H unit. The sequences of switched V_HDJ_H-C_H units show that the sites of switching (i.e., DSBs) lie upstream of each C_H gene. The switching sites segregate within specialized DNA sequences, the switch (S) regions. The S regions lie within the introns that precede the C_H coding regions—all C_H gene regions have S regions upstream of the coding sequences. As a result, CSR does not alter the translational IgH reading frame. In a first CSR event, such as from Cμ to Cγ1, expression of Cμ is succeeded by expression of Cγ1. The Cγ1 gene segment is brought into its new functional location by recombination between Sμ and Sγ1. The Sμ site lies between V_HDJ_H and the Cμ gene segment. The Sγ1 site lies upstream of the Cγ1 gene. The DNA sequence between the two S region DSBs is excised as circular DNA (S circle) that is transiently transcribed as circle transcripts (FIGURE 16.21). This deletion event imposes a restriction on the IgH locus: Once a CSR event has occurred, a B cell cannot express any C_H gene segment that used to lie between the first C_H and the new C_H gene segment. For instance, human B cells expressing Cγ1 cannot give rise to cells expressing Cγ3, because the Cγ3 exon cluster was deleted in the first CSR event. They can, however, undergo CSR to any C_H gene segment downstream of the expressed Cγ1 gene, such as Cα or Cε. This is accomplished by recombination between the Sμ and Sγ1 DNA (juxtaposed by the original CSR event) and Sα or Sε to give rise to a new Sμ/Sα or Sμ/Sε DNA junction (FIGURE 16.22). Multiple sequential CSR events can occur, but they are not obligatory means to proceed to later C_H gene segments, because IgM can switch directly to any other Ig class.

FIGURE 16.21 Class switching of C_H genes occurs by recombination between switch (S) regions and deletion of the intervening DNA between the recombining S sites as switch circles. Circles are transiently transcribed in the switching cell. Sequential recombinations can occur. The mouse IgH locus is depicted.

FIGURE 16.22 Class switching occurs through sequential and discrete stages. The I_H promoters initiate transcription of sterile transcripts. The S regions are cleaved and recombination occurs at the cleaved regions. Depicted is class switch DNA recombination from Sμ to Sε.

16.14 CSR Involves AID and Elements of the NHEJ Pathway

CSR initiates with transcription from the I_H promoters of the C_H regions that will be involved in the DNA recombination event. An I_H promoter lies immediately upstream of each S region. I_H promoters are activated upon binding of transcription factors induced by CD40 signaling, TLR signaling, occupancy of receptors by cytokines (such as IL-4, IFN-γ, or TGF-β), or BCR crosslinking by antigen. The I_H promoters that lie upstream of the S regions that will be involved in the CSR event are activated to induce germline I_H-C_H transcripts, which are then spliced at the I_H region to join with the corresponding C_H region (FIGURE 16.23).

FIGURE 16.23 When transcription separates the strands of DNA, one strand forms a single-stranded loop if 5′-AGCT-3′ motifs in the same strand are juxtaposed.

S regions vary in length, as defined by the limits of the sites involved in recombination, from 1 to 10 kb. They contain clusters of repeating units that vary from 20 to 80 nucleotides in length, with the major component being 5′-AGCT-3′ repeats. The CSR process continues with the introduction of DSBs in S regions followed by rejoining of the cleaved ends. The DSBs do not occur at obligatory sites within S regions, because different B cells expressing the same Ig class have broken the upstream and downstream S regions at different points, yielding different recombined S-S sequences.

Ku70/Ku80 and DNA-PKcs, which are required for the joining phase of V(D)J recombination and for NHEJ in general, are also required for CSR, indicating that the CSR joining reaction uses the NHEJ pathway. CSR can occur, though, albeit at a lower efficiency, in the absence of XRCC4 or DNA ligase IV, suggesting that an alternative end joining (A-EJ) pathway can be used in the ligation of S region DSB ends.

A-EJ in CSR entails inclusion of nucleotide microhomologies at S–S junctions, a signature of microhomology-mediated end-joining (MMEJ). The microhomology-mediated A-EJ in CSR is mediated by HR factor Rad52, a DNA-binding element that promotes annealing of complementary DSB single-strand ends. Rad52 competes with Ku70/Ku80 for binding to S region DSB free ends. There, it facilitates a DSB synaptic process which favors intra-S region recombination. It also mediates, particularly in the absence of a functional NHEJ pathway, inter-S–S region recombinations.

The key insight into the mechanism of CSR has been the discovery of the requirement for the enzyme activation-induced (cytidine) deaminase (AID). In the absence of AID, CSR aborts before the DNA nicking or breaking stage. SHM is also abrogated, revealing an important connection between these two processes, which are central to the maturation of the antibody response and the generation of high-affinity antibodies (see the section in this chapter titled SHM Is Mediated by AID, Ung, Elements of the Mismatch DNA Repair Machinery, and Translesion DNA Synthesis Polymerases).

AID is expressed late in the natural history of a B lymphocyte, after the B cell encounters the antigen and differentiates in germinal centers of peripheral lymphoid organs, restricting the processes of CSR and SHM to this stage. AID deaminates deoxycytidines in DNA and possesses structural similarities to the members of APOBEC proteins that act on RNA to deaminate a deoxycytidine to a deoxyuridine (see the section RNA Editing Occurs at Individual Bases in the chapter titled Catalytic RNA). The expression and activity of AID are tightly regulated at multiple levels. Transcription of the AID gene (Aicda) is modulated by multiple transcription factors, such as the homeodomain protein HoxC4 and NF-κB. HoxC4 expression is upregulated by estrogen receptors, resulting in upregulation of AID and potentiation of CSR and SHM in antibody and autoantibody responses.

Ung is another enzyme that is required for both CSR and SHM. Ung, a uracil-DNA glycosylase, deglycosylates the deoxyuridines generated by the AID-mediated deamination of deoxycytidines to give rise to abasic sites. B cells that are deficient in Ung have a 10-fold reduction in CSR, suggesting that the sequential intervention of AID and Ung creates abasic sites that are critical for the generation of DSBs. Different events follow in the CSR and SHM processes.

AID more efficiently deaminates deoxycytidine in DNA that is being transcribed and that, therefore, exists as a functionally single-strand DNA, such as in germline I_H-C_H transcription, in which the S region nontemplate strand of DNA is displaced when the bottom strand is used as a template for RNA synthesis (FIGURE 16.24). Although this has been proposed as an operational model for DNA deamination by AID, it would not explain how AID deaminates both DNA strands, which it does. The abasic site emerging after sequential AID-mediated deamination of deoxycytidine and Ung-mediated deglycosylation of deoxyuridine is attacked by an apyridinic/apurinic endonuclease (APE) or MRE11/RAD50, which creates a nick in the DNA strands. Generation of nicks in a nearby location on opposite DNA strands would give rise to DSBs in S regions. The DSB free ends in upstream and downstream S regions are joined by NHEJ (see the section Nonhomologous End-Joining Also Repairs Double-Strand Breaks in the Repair Systems chapter). Aberrant repair of the DSBs would lead to chromosomal translocations. How the CSR machinery specifically targets S regions, and what determines the targeting of the upstream and downstream S regions recruited into the recombination process, is just starting to be understood. 14-3-3 adaptor proteins are involved in recruiting/stabilizing AID to S regions by targeting 5′-AGCT-3′ repeats in S regions. 5′-AGCT-3′ repeats account for more than 40% of the “core” of S regions and constitute the primary sites of DSBs. Accessibility of S regions by 14-3-3, AID, and other elements of the CSR machinery is dependent on germline I_H-C_H transcription and chromatin modifications, including histone posttranslational modifications (PTMs). In certain pathological conditions, such as cancer and autoimmunity, AID off-targeting (i.e., targeting of DNA by AID outside the Ig loci) occurs in the genome at large, leading to widespread DNA lesions, such as DSBs, aberrant chromosomal recombinations, and accumulation of mutations in genes that are not physiologically targets of SHM.

FIGURE 16.24 Somatic mutation occurs in the region surrounding the V segment and extends over the recombined V(D)J segment.

16.15 Somatic Hypermutation Generates Additional Diversity and Provides the Substrate for Higher-Affinity Submutants

The sequences of rearranged and expressed Ig V(D)J genes in B cells, which underwent proliferation and differentiation in the periphery after encountering antigen, are changed at several locations compared with the corresponding germline V, D, and J gene segment templates. Some of these changes result from sequence changes at the VJ or V(D)J junctions that occurred during the recombination process. Other changes are superimposed on these and accumulate within the coding sequences of the recombined V(D)J DNA sequence, as a result of different mechanisms in different species. In mice and humans, the mechanism is SHM. In chickens, rabbits, and pigs, a different mechanism, gene conversion, is at work, in addition to SHM. Gene conversion substitutes a rearranged and expressed V gene segment with a sequence from a different germline V gene.

SHM inserts mostly point mutations in the expressed V(D)J sequence. The process is referred to as hypermutation, because it introduces mutations at a rate that is 10⁶-fold higher (10^–3 change/base/cell division) than that of the spontaneous mutation rate in the genome at large (10^–9 change/base/cell division). An oligonucleotide probe synthesized according to the sequence of an expressed unmutated V gene segment can be used to identify the possible corresponding template segment(s) in the germline. Any expressed V gene whose sequence is different from any germline V gene in the same organism must have been generated by somatic changes. Until a few years ago, not every potential germline V gene segment template had actually been identified. This was not a limitation, however, in the mouse λ chain system, because this is a relatively simple locus. A census of several myelomas producing λ1 chains showed that the same germline gene segment encoded many expressed V genes. Others, however, expressed new sequences that must have been generated by mutation of the germline gene segment. The current availability of mouse and human genomic DNA maps, including the complete IgH, Igκ, and Igλ loci, has made it possible to readily identify germline Ig V gene templates.

To analyze the intrinsic frequency and nature of somatic mutations accumulating during an ongoing immune response, one can analyze the intronic region between J_H and iEμ that is targeted by SHM but does not undergo negative or positive selection of point mutations. To analyze the nature of antigen-selected mutations, one approach is to characterize the Ig V(D)J sequences of a cohort of B cells, all of which respond to a given antigen or, even better, an antigenic determinant. Haptens are used for this purpose. Unlike a large protein, whose different parts induce different antibodies, haptens are small molecules whose discrete structure induces a consistently restricted antibody response. A hapten is not immunogenic per se, in that it does not induce an immune response if injected as such. It does, however, induce an immune response after conjugation with a “carrier” protein to form an antigen. A hapten–carrier conjugate is then used to immunize mice of a single strain. After induction of a strong antibody response, B lymphocytes (usually from the spleen) are obtained and fused with non-Ig–expressing myeloma fusion partner (immortal tumor) cells to generate a monoclonal hybridoma that indefinitely secretes the antibody expressed by the primary B cell used for the fusion. In one example, 10 out of 19 different B cell lines producing monoclonal antibodies directed against the hapten phosphorylcholine utilized the same V_H sequence. This sequence was that of the V_H gene segment T15, one of four related V_H genes. The other nine expressed gene segments, which differed from each other and from all four germline members of the family. They were more closely related to the T15 germline sequence than to any of the others, and their flanking sequences were the same as those around T15. This suggested that they arose from the T15 member through SHM.

The sequence changes (mutations) were concentrated in the V_HDJ_H DNA, which encodes the IgH chain antigen-binding site, but tapered off throughout a region downstream of the V_H gene promoter for approximately 1.5 kb (Figure 16.24). The mutations consisted in all cases of substitutions of individual nucleotide pairs. Most sequences bore 3 to 15 substitutions, corresponding to fewer than 10 amino acid changes in the protein. Only some mutations were replacement mutations, because they affected the amino acid sequence; others were silent mutations, because they were in third-base coding positions or in nontranslated regions. The large proportion of silent mutations suggests that SHM randomly targets the expressed V(D)J DNA sequence and extends beyond it. A tendency exists for some mutations to recur on multiple occasions in the same residue(s). These are referred to as mutational “hotspots,” as a result of some intrinsic preference by the SHM machinery. The best-characterized hotspot is 5′-RGYW-3′, where R is a purine (dA or dG), G is dG, Y is a pyrimidine (dC or dT), and W is dA or dT. Interestingly, the 5′-AGCT-3′ iteration of 5′-RGYW-3′ is the major target of SHM and the preferential site of DSBs in S regions. Like CSR, which requires germline I_H-C_H transcription of the target S_H-C_H sequences, SHM requires transcription of the target V_HDJ_H, V_κJ_κ, and V_λJ_λ sequences. This is emphasized by the requirement for the so-called intronic enhancer that activates transcription at each Ig locus, namely, iEμ in the IgH locus and iEκ in the Igκ locus.

Upon exposure to antigen of a polyclonal B cell population, such as the human B cell repertoire, selected B cell submutants expressing a BCR with high intrinsic affinity for that antigen are selected, activated, and induced to proliferate. SHM occurs during B proliferation or clonal expansion. It randomly inserts one point mutation in the V(D)J sequence of approximately half of the progeny cells; as a result, B cells expressing mutated antibodies become a high fraction of the clone within a few divisions. Random replacement mutations have unpredictable effects on protein function; some decrease the affinity of the BCR for the antigen driving the response, whereas others increase BCR intrinsic affinity for the same antigen. The B cell clone(s) expressing a BCR with the highest affinity for antigen is positively selected and acquires a growth advantage over all other clones; the other clones are gradually counterselected (selected against) for survival and proliferation. Further positive selection of the clone(s) that accumulated mutations conferring the highest affinity for antigen will result in narrowing clonal restriction and accumulation of clones with a very high affinity for antigen.

16.16 SHM Is Mediated by AID, Ung, Elements of the Mismatch DNA Repair Machinery, and Translesion DNA Synthesis Polymerases

The deamination or removal of a deoxycytosine base leads to insertion of somatic mutation(s) in different ways (FIGURE 16.25). When AID deaminates a deoxycytosine, it gives rise to deoxyuridine. This is not germane to DNA and can be dealt with by the B cell in different ways. The deoxyuridine can be “replicated over”; it will pair with deoxyadenine during replication. The emerging mutation is an obligatory dC → dT transition and dG → dA transition on the complementary strand. The net result is the replacement of the original dC-dG pair with a dT-dA pair in half of the progeny cells. Alternatively, the deoxyuridine can be removed from DNA by Ung to give rise to an abasic site. Indeed, the key event in generating a random spectrum of mutations is the creation of an abasic site. This can be replicated over by an error-prone TLS DNA polymerase, such as polymerase ζ, polymerase η, or polymerase θ, which can insert all three possible mismatches (mutations) across the abasic site (see the section Error-Prone Repair in the Repair Systems chapter). In another mechanism, the dU-dG mispair recruits the MMR machinery, starting with Msh2/Msh6, to excise the stretch of DNA containing the damage, thereby creating a gap that needs to be filled in by resynthesis of the missing DNA strand (see the section Controlling the Direction of Mismatch Repair in the Repair Systems chapter). This resynthesis is carried out by an error-prone TLS polymerase, which will introduce mutations. What restricts the activity of the SHM machinery to only target V(D)J regions is still unknown. Ung can be blocked by introducing into cells the bacteriophage PSB-2 gene encoding the uracil-DNA glycosylase inhibitor (UGI) protein. When the UGI gene is expressed in a lymphocyte cell line or Ung is knocked out, the pattern of mutations changes dramatically, with almost all mutations from dC-dG pairs comprising the predicted transition from dC-dG to dA-dT.

FIGURE 16.25 Deamination of C by AID gives rise to a U-G mispair. U can be replicated over, resulting in C-G to A-T transitions in 50% of progeny B cells. When the action of cytidine deaminase (top) is followed by that of uracil-DNA glycosylase, an abasic site is created. Replication past this site should insert all four bases at random into the daughter strand (center). If the uracil is not removed from the DNA, its replication gives rise to a C-G to T-A transition. Alternatively, the U-G mispair is recognized by the MMR machinery, which excises DNA containing the mismatch and then fills in the resulting gap using an error-prone DNA polymerase. This will lead to insertion of further mismatches (mutations).

The main difference between CSR and SHM is the nature of DNA lesions underpinning the two processes. DSBs are introduced as obligatory intermediates in CSR, whereas individual point mutations are introduced as events of single-strand cleavages in SHM. AID and/or DNA repair factor(s) also function as scaffolds to assemble different protein complexes in CSR and SHM. Thus, AID and DNA repair factors contribute to these processes through both enzymatic and nonenzymatic functions, possibly in different ways. AID plays a central role in both CSR and SHM. However, whereas Ung intervention is a central event in CSR, it is not necessarily in SHM, and TLS polymerases play a greater role in SHM than CSR.

16.17 Igs Expressed in Avians Are Assembled from Pseudogenes

The chicken Ig locus is the paradigm for the Ig somatic diversification mechanism utilized by rabbits, cows, and pigs; that is, gene conversion. A similar mechanism is used by both the single (λ-like) L chain locus and the H chain loci. The chicken λ locus comprises only one functional V gene segment, one J_λ segment, and one C_λ gene segment (FIGURE 16.26). Upstream of the functional V_λ1 gene segment lie 25 V_λ pseudogenes, organized in either orientation. In the pseudogenes, either the coding segment is deleted at one or both ends or proper RSSs are missing, or both. This is emphasized by the fact that only the V_λ1 gene segment recombines with the J_λ-C_λ gene segment.

FIGURE 16.26 The chicken λ light chain locus has 25 V pseudogenes upstream of the single functional V_λ-J_λ-C region. Sequences derived from the pseudogenes, however, are found in active rearranged VJC genes.

Nevertheless, sequences of rearranged V_λJ_λ-C_λ gene segments show considerable diversity. A rearranged gene has one or more positions at which a cluster of changes occurred in its sequence. A sequence identical to the new sequence can almost always be found in one of the pseudogenes. The sequences that are not found in a pseudogene always represent changes at the junction between the original sequence and the altered sequence. The unmodified V_λ1 sequence is not expressed, even at early times during the immune response. Sequences from the pseudogenes, between 10 and 120 bp in length, are integrated into the active V_λ1 region by gene conversion. A successful conversion event probably occurs every 10 to 20 cell divisions to every rearranged V_λ1 sequence. At the end of the immune maturation period, a rearranged V_λ1 sequence has four to six converted segments spanning its entire length, which are derived from different donor pseudogenes. If all pseudogenes can participate in this gene conversion process, more than 2.5 × 10⁸ possible combinations are allowed.

The enzymatic basis for copying pseudogene sequences into the recombined Ig V gene depends on AID and enzymes involved in homologous recombination, and is related to the mechanism of human and mouse SHM (see the section Eukaryotic Genes Involved in Homologous Recombination in the Homologous and Site-Specific Recombination chapter). For example, gene conversion is prevented by deletion of RAD54. Deletion of other homologous recombination genes, such as XRCC2, XRCC3, and RAD51B, has another interesting effect: Somatic mutations occur in the V gene of the expressed locus. The frequency of the somatic mutations is 10-fold greater than the rate of gene conversion.

Thus, the absence of SHM in chicken is not due to a deficiency in the enzymatic machinery that is responsible for SHM in humans and mice. The most likely explanation for a connection between (lack of) recombination and SHM is that unrepaired DSBs in the recombined Ig V(D)J segments trigger the induction of mutations. The reason why SHM occurs in mice and humans but not in chickens may, therefore, lie with the nature of the repair system that operates on DSBs in the Ig locus. It would be more efficient in chickens, so that DSBs in the Ig locus are repaired through gene conversion before mutations can be induced.

16.18 Chromatin Architecture Dynamics of the IgH Locus in V(D)J Recombination, CSR, and SHM

During B and T cell development, the coding elements for BCR and TCR are assembled from widely dispersed gene segments. Antigen receptor loci contain multiple V, D, and/or J and C coding elements, and the assembly of these antigen receptors is controlled at multiple levels, including chromatin architecture, nuclear location, and epigenetic marking. This will bring into close proximity elements that are separated by about 2.5 Mb for their recombination (FIGURE 16.27). The Ig H and L chain loci and TCR loci are not simple linear chromosomal structures but possess a three-dimensional configuration, which orchestrates DNA recombination at these loci. Indeed, the IgH chain locus tends to fold into a comprehensive pattern of loop arrangements that shorten the distances between gene segments and allow long-range genomic interactions to occur at relatively high frequencies to facilitate V(D)J recombination.

FIGURE 16.27 Chromatin architecture of Ig locus facilitates V(D)J recombination and CSR. CTCF, which is important for implementing chromatin conformation, modulates V(D)J recombination by regulating enhancer-promoter interaction and locus compaction. Iem:3’Ea interactions create long-range chromatin interactions directed by the Ih promoters and Igh enhancers, which create spatial proximity between Sm and downstream S region loci and facilitates recombination between the broken S regions and creates a matrix of chromatin contacts.

Left panel is modified from Figure 5 of Ong and Corces (2014) Nat. Rev. Gent. 15:234–246.

The DNA-binding zinc finger nuclear protein CCCTC-binding factor (CTCF) mediates long-range chromatin looping and is important for implementing chromatin conformation. CTCF may modulate V(D)J recombination by regulating locus compaction and promoter–enhancer interactions, thereby influencing the spatial conformation of the IgH locus and antisense transcription. This generates noncoding RNAs that can further shape the chromatin architecture. The Ig, and possibly TCR, alleles are sequestered at the transcriptionally repressive nuclear lamina in lymphoid progenitor cells. Before the pro-B cell stage, the IgH locus is released from the lamina to associate with the transcription and/or recombination machineries. Committed pro-B cells undergo broad chromatin conformational changes, in which chromatin looping of CTCF-binding sites at the IgH locus occurs independently of the iEμ enhancer and contributes to the compaction of the locus. Two CTCF-binding sites within the intergenic control region 1 (IGCR1), located between the V_H and D_H clusters, mediate ordered and lineage-specific V_H-DJ_H recombination and bias distal over proximal V_H rearrangements. IGCR1 suppresses the transcriptional activity and the rearrangement of proximal V_H segments by forming a CTCF-mediated loop that presumably isolates the proximal V_H promoter from the influence of the downstream iEμ enhancer. Likewise, before pro-B cell stages, CTCF promotes distal over proximal V_κ rearrangement by blocking the communication between specific enhancer and promoter elements in the Igκ locus

The formation of the S-S synapsis, which is essential for CSR, is mediated by long-range intrachromosomal interactions between distantly located IgH transcriptional elements. This three-dimensional chromatin architecture simultaneously brings I_H promoters into close proximity with iEμ and 3′Eα enhancers to facilitate transcription. Transcription across S-region DNA leads to RNA polymerase II accumulation that promotes the introduction of activating chromatin modifications and hyperaccessible chromatin to ensure AID activity. In mature resting B cells, the iEμ and 3′Eα enhancers are in close spatial proximity by forming a chromatin loop. B cell activation leads to cytokine-dependent enrollment of the I_H promoters to the iEμ–3′Eα complex and allows transcription of S regions targeted for CSR, likely facilitated by a three-dimensional structure adopted by the IgH locus.

Although AID specifically targets the Ig locus, it also acts with much lower efficiency on a limited number of non-Ig genes (off-targets), leading to mutations and translocations that contribute to B cell tumorigenesis. AID targets, however, are not randomly distributed across the genome, but rather predominantly associated with topologically complex and highly transcribed super-enhancers and regulatory clusters. These include multiple interconnected transcriptional regulatory elements and strong convergent transcription, in which normal-sense transcription of the gene overlaps with super-enhancer–derived antisense enhancer RNA (eRNA) transcription. AID deaminates active promoters and eRNA⁺ enhancers that are interconnected in some instances over megabases of linear chromatin. This would provide a critical step toward recombination of widely spread V(D)J regions.

16.19 Epigenetics of V(D)J Recombination, CSR, and SHM

DNA recombination and/or mutagenesis in Ig and TCR loci are stringently orchestrated at multiple levels, including regulation of chromatin structure and transcriptional elongation. Both DNA and its associated histones in Ig and TCR loci chromatin are epigenetically marked during B and T cell development and differentiation.

Epigenetic modifications are changes in the cell progeny that are independent from the genomic DNA sequence. They include histone posttranslational modifications, DNA methylation, and alteration of gene expression by noncoding RNAs, including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) (discussed in the chapters Chromatin, Epigenetics I, Epigenetics II, and Regulatory RNA). Epigenetic modifications act in concert with transcription factors and play critical roles in B and T cell development and differentiation. Upon antigen encounter by mature B cells in the periphery, alterations of the epigenetic landscape in these lymphocytes are induced by the same stimuli that drive the antibody response. Such alterations instruct B cells to undergo CSR and SHM, as well as differentiation to memory B cells or long-lived plasma cells. Inducible histone modifications, together with DNA methylation and miRNAs, modulate the transcriptome, particularly the expression of AID. These inducible B cell–intrinsic epigenetic marks guide the maturation of antibody responses.

For the V(D)J recombination, CSR, and SHM machineries to access their respective DNA targets in the antigen receptor loci, the targeted regions need to be in an open chromatin state, which is associated with transcription and specific patterns of epigenetic modifications. The transcription is mediated by cis-activating elements, such as V_H and I_H promoters as well as iEμ and 3′Eα enhancers, and transcription factors specifically recruited by these elements. During transcription elongation, chromatin remodeling generates nucleosome-free regions by repositioning or evicting nucleosomes or acts more subtly by transiently lifting a loop of DNA off of the nucleosome surface. Transcription elongation results in nucleosome disassembly or disassociation from DNA. DNA freed from repressive associations with nucleosomes is, therefore, amenable to react with factors of the V(D)J recombination, CSR, or SHM machinery. Accordingly, RNA polymerase II is detected at a high density in S regions that will undergo CSR, suggesting that this molecule facilitates recruitment or targeting of CSR factors.

lncRNAs generated by noncoding transcription in the IgH loci have been shown to play an important role in the targeting of the V(D)J recombination and CSR machineries. lncRNAs are evolutionarily conserved noncoding RNA molecules that are longer than 200 nucleotides and located within the intergenic stretches or overlapping antisense transcripts of coding genes (see the Regulatory RNA chapter). Production of lncRNA transcripts from V(D)J region DNA in Ig or TCR loci can trigger changes in chromatin structure and modulate recombination. In addition, lncRNA transcription targets AID to divergently transcribed loci in B cells. In B cells undergoing CSR, the RNA exosome, a cellular RNA-processing/degradation complex, associates with AID, accumulates on S regions in an AID-dependent fashion, and is required for optimal CSR. RNA exosome-regulated, antisense-transcribed regions of the B cell genome recruit AID and accumulate single-strand DNA structures containing RNA–DNA hybrids. The RNA exosome regulates transcription of lncRNAs that are engaged in long-range DNA interactions to regulate the function of IgH 3′ regulatory region super-enhancer and modulate CSR. In addition, an lncRNA generated by S region transcription followed by lariat debranching can fold into G-quadruplex structures, which can be directly bound by AID and mediate targeting of AID to S region DNA. A critical role of chromatin accessibility in antibody diversification is emphasized by the fact that though all S regions contain 5′-AGCT-3′ repeats and can, therefore, potentially be targeted by 14-3-3 adaptors for the recruitment of AID to unfold CSR, only the S regions that undergo germline I_H-S-C_H transcription and enrichment of activating histone modification can be targeted by the CSR machinery, including 14-3-3 and AID.

As a potent mutator, AID is tightly regulated to avoid damages, such as chromosomal translocations, resulting from its dysregulation in both B cells and non-B cells. The expression of Aicda is modulated by changes of Aicda epigenetic status. Repression of Aicda expression in naïve B cells is mediated by promoter DNA hypermethylation. Upon B cell activation, Aicda DNA is demethylated and the locus becomes enriched in H3K9ac/K14ac and H3K4me3. These epigenetic changes, together with induction of Homeobox protein HoxC4, NF-κB, and other transcription factors, activate gene transcription. Transcription elongation depends on induction of H3K36me3, an intragenic mark of gene activation. miRNAs provide an additional and more important mechanism of modulation of AID expression. miR-155, miR-181b, and miR-361 modulate AID expression by binding to the evolutionarily conserved target sites in the 3′ UTR of AICDA/Aicda mRNA, thereby reducing both AICDA/Aicda mRNA and AID protein levels. These miRNAs likely repress AID in naïve B cells and in B cells that completed SHM and CSR. Histone deacetylase inhibitors (HDIs) can upregulate these miRNAs by increasing histone acetylation, and therefore expression of their host genes, and lead to downregulation of AID expression.

AID targets are predominantly associated within super-enhancers and regulatory clusters, which are enriched in chromatin modifications associated with active enhancers (such as H3K27Ac). They are also associated with marks of active transcription (such as H3K36me3), indicating that these features are universal mediators of AID recruitment. In both human and mouse B cells, a strong overlap exists between hypermutated genes and super-enhancer domains. Chromatin in the target region(s) of V(D)J recombination, CSR, and SHM is also marked by multiple activating histone modifications. One of the most important activating histone modifications, trimethylation of the Lys4 residue of H3 (H3K4me3), is a specific mark of open chromatin in the genome and is highly enriched in V(D)J gene segments and S regions that will undergo V(D)J recombination and CSR, respectively. Concomitant with enrichment of activating histone modifications in those regions, repressive histone modifications, such as H3K9me3 and H3K27me3, are decreased.

The change from a repressive to a permissive chromatin state in targeted Ig loci regions is controlled by the stage of lymphoid differentiation, tissue specificity, and allelic exclusion in a fashion virtually identical to how V(D)J recombination, CSR, and SHM per se are regulated. Transcription and change of combinatorial patterns of histone modifications in those regions are coregulated by cis-activating elements and transcription factors activated by environmental cues, such as cytokines critical for B cell development or specification of Ig isotypes. In addition, the transcription process itself plays a role in the induction (“writing”) of selective histone modifications, as suggested by profoundly decreased H3K4me3 in the TCRα locus downstream of an artificially inserted transcription termination sequence.

According to the histone code hypothesis, combinatorial patterns of histone modifications not only encrypt information on the specification of distinct chromatin states but also increase the complexity of chromatin-interacting effectors (histone code “reading”), thereby determining specific biological information outputs. In V(D)J recombination, RAG2 is a specific reader of H3K4me3, which is enriched in the recombination center, a small region containing J gene segments (and the D gene segments in some cases). This, together with strong RAG1 binding to RSSs, ensures targeting of the RAG1/RAG2 complex to the recombination center. In CSR, a combinatorial histone modification H3K9acS10ph (acetylation of Lys9 and phosphorylation of Ser10 of the same H3 tail) is read by 14-3-3 adaptors, thereby stabilizing 5′-AGCT-3′-bound 14-3-3 on the S regions that will undergo recombination.

Some histone code readers, such as RAG2, can directly mediate enzymatic reactions upon reading histone modifications. Others do not possess intrinsic enzymatic activities and, by virtue of their scaffold functions, instead transduce epigenetic information to downstream enzymatic factors. For instance, 14-3-3 adaptors read H3K9acS10ph (as well as binding to 5′-AGCT-3′ repeats) and, in turn, recruit AID to S-region DNA. Together with elements of the CSR and SHM machinery, such as Rev1 in Ung, these histone code transducers nucleate the assembly of multicomponent complexes through simultaneous interaction with multiple protein and/or nucleic acid ligands via different domains or subunits.

Another potential mechanism of accessibility control is DNA methylation, which occurs mainly at dCs of CpG sites (see the chapter Epigenetics I). CpG methylation has an important function in regulating transcription and chromatin structure. It represses gene expression directly by impeding the binding of transacting factors, and indirectly by the recruitment of HDACs through methyl CpG-binding–domain (MBD) family proteins. Differences in methylation status are also correlated with antigen–receptor gene rearrangement and expression. In addition, DNA methylation around the RSS may also regulate V(D)J recombination by directly inhibiting the cleavage activity of the RAG1/RAG2 complex. Although the density of CpG sites is much lower than overall genome-wide CpG level, increased DNA methylation at these CpG sites results in significantly reduced germline transcription and CSR. The role of DNA hypomethylation in SHM has also been suggested by the finding that only the hypomethylated allele is hypermutated in B cells carrying two nearly identical pre-rearranged transgenic Igκ alleles, despite comparable transcription of both alleles. DNA demethylation probably facilitates SHM targeting by promoting H3K9ac/K14ac, H4K8ac, and H3K4me3 histone modifications that are associated with an open chromatin state and are enriched in the V(D)J region.

16.20 B Cell Differentiation Results in Maturation of the Antibody Response and Generation of Long-lived Plasma Cells and Memory B Cells

A primary antibody response is induced by activation of the mature naïve B cell through antigen-mediated BCR cross-linking. This leads to clonal expansion, but only to a limited extent. Vigorous proliferation of antigen-specific B cells requires engagement of other immune receptors. In particular, engagement of TLRs by MAMP molecules on microbial pathogens plays an important role in the early stage of the antibody response before specific T cell help is available. Early B cell response is accompanied by the differentiation of B cells into plasmablasts, which produce mostly unmutated IgM with a moderate intrinsic affinity, but high avidity, for antigen. These antibodies are identical to the BCR expressed by the B cell progenitor, the only difference being the C_H instead of the C_μ terminal of the constant region. TLR engagement can also induce CSR and likely SHM as well as prime B cells for the cognate B-T engagement.

Engagement of CD40 expressed on B cell surface by CD40 ligand (CD154) expressed on T_h cells takes place at a later stage of the primary response. It induces high levels of CSR and SHM for the eventual generation of more specific IgG, IgA, and/or IgE antibodies. These are produced by plasma cells, which are terminal differentiation elements from B cells, and home into bone marrow niches to become long-lived, thereby contributing to the long-term immune memory. Alternatively, activated B cells can differentiate into memory B cells. These cells comprise a minor proportion of the B cells generated at the end of the primary response. They express mutated V(D)J gene segments coding for BCRs that display increased affinity for antigen and have generally undergone CSR. Memory B cells are typically “frozen” with respect to their V(D)J somatic mutations and IgH chain class. They are in a resting state, but are rapidly activated when they re-encounter the same antigen that induced their generation for a secondary antibody response. Upon re-exposure to the same antigen, they can mount a secondary response, rapidly and with vigorous clonal expansion. Activated memory B cells can differentiate into plasma cells producing large amounts of antibodies, thereby mediating a vigorous high-affinity memory or anamnestic response.

Virtually all B cells recruited in an antigen-specific antibody response to undergo CSR and SHM (FIGURE 16.28) are “conventional” B cells, or B-2 cells. In addition to these cells, a separate set of B cells exists, referred to as B-1 cells. B-1 cells also undergo the V(D)J gene rearrangement and apparently are selected for expression of a particular repertoire of antibody specificities. They may be involved in natural immunity; that is, they may possess the intrinsic ability to respond in a T-independent fashion to many naturally occurring antigens, particularly bacterial components, such as polysaccharides and lipopolysaccharides. B-1 cells are the main source of natural antibodies. Natural antibodies are mainly IgM that bind a variety of microbial components and products as well as self-antigens. They are important components of the first line of defense against bacterial and viral infections and may provide the templates for high-affinity antiself autoantibodies that mediate autoimmune pathology.

FIGURE 16.28 B cell differentiation is responsible for acquired immunity. Initial exposure of mature B cells to antigen results in a primary response and generation of memory cells. Subsequent exposure to antigen induces a secondary response through activation of the memory cells.

16.21 The T Cell Receptor Antigen Is Related to the BCR

T cells use evolutionary conserved mechanisms to express significant diversity in TCR-variable regions that are similar to those of B cells (BCR). The TCR consists of two different protein chains. In adult mice, more than 95% of T cells express a TCR consisting of α and β chains (TCRαβ), whereas less than 5% of T cells express TCR consisting of γ and δ chains (TCRγδ). TCRαβ and TCRγδ are expressed at different times during T cell development (Figure 16.29). TCRγδ is synthesized at an early stage of T cell development. It is the only TCR expressed during the first 15 days of gestation, but is virtually lost by birth, at day 20. TCRαβ is synthesized later in T cell development than TCRγδ, being first expressed at days 15 to 17 of gestation. At birth, TCRαβ is the predominant TCR. TCRαβ is synthesized by a separate lineage of cells from those expressing TCRγδ and involves independent rearrangement events.

FIGURE 16.29 The TCRγδ receptor is synthesized early in T cell development. TCRαβ is synthesized later and is responsible for cell-mediated immunity, in which antigen and host MHC are recognized together.

Like the BCR, the TCR must recognize a foreign antigen of virtually any possible structure. The TCR resembles the BCR in structure. The V sequences have the same general internal organization in both the TCR and the BCR. The TCR constant region is related to the Ig constant regions, but has a single C domain followed by transmembrane and cytoplasmic portions. The exon–intron structure reflects the protein function. The organization and configuration of the TCR genes are highly similar to those of the BCR/Ig genes. Each TCR locus (α, β, γ, and δ) is organized in a fashion similar to that of the Ig locus, with separate gene segments that are brought together by a recombination reaction specific to the lymphocyte. The components are similar to those found in the three Ig loci: IgH, Igκ, and Igλ. The TCRα and TCRγ chains are generated by VJ recombination, whereas TCRβ and TCRγ chains are generated by V(D)J recombination.

The TCRα locus resembles the Igκ locus, with V_α gene segments separated from a cluster of J_α segments that precedes a single C_α gene segment (FIGURE 16.30). The organization of the TCRα locus is similar in both humans and mice, with some differences only in the number of V_α gene segments and J_α segments. In addition to the α segments, this locus also contains embedded δ segments. The organization of the TCRβ locus resembles that of the IgH locus, although the large cluster of V_β gene segments lies upstream of two clusters, each containing a D segment, several J_β segments, and a C_β gene segment (FIGURE 16.31). Again, the only differences between humans and mice are in the numbers of V_β and J_β genes.

FIGURE 16.30 The human TCRα locus contains interspersed α and δ segments. A V_δ segment is located within the V_α cluster. The D-J-C_δ segments lie between the V gene segments and the J-C_α segments. The mouse locus is similar, but includes more V_δ segments.

FIGURE 16.31 The TCRβ locus contains many V gene segments spread over approximately 500 kb that lie ~280 kb upstream of the two D-J-C clusters.

Diversity in the TCR is generated by the same mechanisms as in the BCR. Germline encoded (intrinsic) diversity results from the combination of a variety of V, D, and J segments; some additional diversity results from the introduction of new sequences at the junctions between these components, in the form of P and/or N nucleotides. The recombination of TCR gene segments occurs in the thymus through mechanisms highly similar to those of the BCRs in B cells. Appropriate nonamer-spacer-heptamer RSSs direct it. These RSSs are identical to those used in Ig genes and are handled by the same enzymes. As in the BCR/Ig loci, most rearrangements in the TCR loci occur by deletion. Rearrangements of TCR gene segments, like those of BCR genes, may be productive or nonproductive. Like the Ig locus in B cells, the transcription factors that control and mediate the rearrangement of the TCR locus in T cells are just beginning to be appreciated.

The organization of the TCRγlocus resembles that of the Igλ locus, with V_γ gene segments separated from a series of J_γ-C_γsegments (FIGURE 16.32). The TCRγ locus displays relatively little diversity, with about eight functional V_γsegments. The organization is different in humans and mice. The mouse TCRγ locus has three functional J_γ-C_γ segments. The human TCRγ locus has multiple J_γsegments for each C_γ gene segment.

FIGURE 16.32 The TCRγ locus contains a small number of functional V gene segments (and also some pseudogenes not shown) that lie upstream of the J-C loci.

The cluster of genes encoding the TCRδ chain lies entirely embedded in the TCRα locus, between the V_α and C_α genes (see Figure 16.30). The V_δ gene segments are interspersed within the V_α gene segments. Overall, the number of TCR V_γand V_δ gene segments is much lower than that of V_α and V_β gene segments. Nevertheless, great diversity is generated at the TCRδ locus, as DD rearrangements occur frequently, each of them entailing N nucleotide additions. The embedding of the TCRδ cluster of D_δ and J_δ genes and the C_δ gene in the TCRα locus implies that expression of TCRαβ and TCRγδ is mutually exclusive at any one allele, because all the D_δ, J_δ, and C_δ gene segments are lost once a V_α-J_α rearrangement occurs.

DD rearrangements also occur at the TCRβ locus, resulting from DD joinings. The TCRβ locus shows allelic exclusion in much the same way as the Ig locus; rearrangement is suppressed once a productive allele has been rearranged. The TCRα locus may be different; several cases of continued rearrangements suggest the possibility that substitution of V_α sequences may continue after a productive allele has been generated. Unlike the IgH, Igκ, and Igλ loci, none of the TCR loci undergo SHM or a process resembling CSR.

16.22 The TCR Functions in Conjunction with the MHC

T cells expressing TCRαβ comprise subtypes that have a variety of functions related to interactions with other cells of the immune system. CTLs possess the ability to lyse a target cell. T_h cells help the activation/generation of CTLs or aid in the differentiation of B cells into antibody-producing cells.

The BCR/antibody and the TCR differ in their modalities of interaction with their ligands. A BCR/antibody recognizes a small area (epitope) within the antigen, which can be composed of a linear sequence (six to eight amino acids) identifying a linear determinant or a cluster of amino acids brought together by the three-dimensional structure of the antigen (conformation determinant). A TCR binds a peptide derived from the antigen upon processing by an APC. The peptide is generated when the proteasome degrades the antigen protein within the APC. It is “presented” to the T cell by the APC in the context of an MHC protein, in a groove on the surface of the MHC. Thus, the T cell simultaneously recognizes the peptide and an MHC protein carried by the APC. Both T_h cells and CTLs recognize the antigen in this fashion, but with different requirements; that is, they recognize peptides of different sizes and as presented in conjunction with different types of MHC proteins (see the section in this chapter The MHC Locus Comprises a Cohort of Genes Involved in Immune Recognition). T_h cells recognize peptide antigens, 13 to 20 amino acids long, presented by MHC class II proteins, whereas CTLs recognize peptide antigens, 8 to 10 amino acids long, presented by MHC class I proteins. The TCRαβ provides the structural correlate for the helper T_h cell function and for the CTL function. In both cases, TCRαβ recognizes both the antigenic peptide and the self-MHC protein. A given TCR has specificity for a particular MHC, as well as for the associated antigen peptide. The basis for this dual recognition capacity is one of the most interesting structural features of the TCRαβ.

Recombination to generate functional TCR chains is linked to the development of the T lymphocyte (FIGURE 16.33). The first stage consists in rearrangement to form an active TCRβ chain. This binds a nonrearranging surrogate TCRα chain, which is called pre-TCRα. At this stage, the lymphocyte has not yet expressed either CD4 or CD8 on the surface. The pre-TCR heterodimer then associates with the CD3 signaling complex. Signaling from the complex triggers several rounds of cell division, during which TCRα chains are rearranged, and the CD4 and CD8 genes are turned on so that the lymphocyte transitions from CD4^–CD8^–, or double-negative (DN), thymocyte to CD4⁺CD8⁺, or double-positive (DP), thymocyte. TCRα chain rearrangement continues in the DP thymocytes. The maturation process continues through both positive selection (for mature TCR complexes able to bind a self-ligand with moderate affinity) and negative selection (against complexes that interact with self-ligands at high affinity). Both positive and negative selection involve interaction with MHC proteins. DP thymocytes either die within 3 to 4 days or become mature lymphocytes as the result of the selection process. The surface TCRαβ heterodimer becomes cross-linked on the surface during positive selection, which rescues the thymocyte from apoptosis (nonnecrotic cell death). If thymocytes survive the subsequent negative selection, they give rise to the separate T lymphocyte subsets, CD4⁺CD8^– and CD4^–CD8⁺cells.

FIGURE 16.33 T cell development proceeds through sequential stages.

The TCR is associated with the CD3 complex of proteins, which are involved in transmitting a signal from the surface of the cell to the nucleus when the TCR is activated by binding of antigen (FIGURE 16.34). The interaction of the TCR variable regions with antigen causes the ζ chain of the CD3 complex to signal T cell activation, in a fashion comparable to the BCR Igα and Igβ complex signaling B cell activation.

FIGURE 16.34 The two chains of the T cell receptor (TCR) associate with the polypeptides of the CD3 complex. The variable regions of the TCR are exposed on the cell surface. The cytoplasmic domains of the ζ chains of CD3 provide the effector function.

Considerable diversity is required in both recognition of a foreign antigen, which requires the ability to respond to novel structures, and recognition of the MHC protein, which is restricted to one of the many different MHC proteins encoded in the genome. T_h cells and CTLs rely upon different classes of MHC proteins; however, they use the same pool of TCRα and TCRβ or TCRγ and TCRδ gene segments to assemble their TCRs. Even allowing for the introduction of additional variation during the TCR recombination process, the number of different TCRs generated is relatively limited, but nevertheless sufficient to satisfy the diversity demands imposed by the variety of TCR ligands. This is made possible by the relatively low binding affinity requirements by the TCR-peptide/MHC interaction, which allows for one TCR to interact with multiple different ligands sharing some similarities.

16.23 The MHC Locus Comprises a Cohort of Genes Involved in Immune Recognition

MHC molecules have evolved to maximize the efficacy and flexibility of their function: to bind peptides derived from microbial pathogens and present them to T cells. In response to a strong evolutionary pressure to eliminate a large variety of microorganisms, the MHC genes encoding these proteins have evolved into polygenic (several sets of genes in all individuals) and polymorphic (multiple variants of gene within the population at large) cohorts of genes. In humans, the MHC is also called human leukocyte antigen (HLA). MHC proteins are dimers inserted in the plasma membrane, with a major part of the protein protruding on the extracellular side. Of the three human MHC classes, class I and class II are the most important in immunobiology and the clinical setting. The structures of MHC class I and class II molecules are related, although they are made up of different components (FIGURE 16.35).

FIGURE 16.35 Class I and class II MHC molecules have a related structure. Class I antigens consist of a single polypeptide (α) with three external domains (α1, α2, and α3) that interacts with β₂-microglobulin (β2M). Class II antigens consist of two polypeptides (α and β), each with two domains (α1 and α2 and β1 and β2) with a similar overall structure.

MHC class I molecules consist of a heterodimer of the class I chain (α) itself and the β2-microglobulin (β2M protein). The class I chain is a 45-kD transmembrane component that has three external domains (each approximately 90 amino acids long), one of which interacts with β2-microglobulin, a transmembrane domain (approximately 40 residues), and a short cytoplasmic domain (approximately 30 residues). MHC class II molecules consist of two chains, α and β, whose combination generates an overall structure in which there are two extracellular domains. Humans have three classified (or major) class Iα-chain genes: HLA-A, HLA-B, and HLA-C. The β2-microglobulin is a secreted protein of 12 kD. It is needed for the class I chain to be transported to the cell surface. Mice lacking the β2-microglobulin gene express no MHC class I antigens on the cell surface. Humans have three major pairs of class IIα- and β-chain genes: HLA-DR, HLA-DP, and HLA-DQ.

The MHC locus occupies a small region of a single chromosome in mice (histocompatibility 2 or H2 locus on chromosome 17) and in humans (human leukocyte antigen or HLA locus on chromosome 6). These regions contain multiple genes. Also located in these regions are genes encoding proteins found on lymphocytes and macrophages that have a related structure and are important in the function of cells of the immune system.

The genes of the MHC locus are grouped into three clusters according to the structures and immunological properties of the respective products. The MHC region was originally defined by genetics in the mouse, where the classical H2 region occupies 0.3 map units. Together with the adjacent region, where mutations affecting immune function are also found, this corresponds to an approximately 2,000-kb region. The MHC region is generally conserved in mammals, as well as in some birds and fish. The genomic regions where the class I and class II genes are located mark the original boundaries of the locus, from telomere to centromere (FIGURE 16.36: right to left). The genes in the class III region, which separate class I from class II genes, encode many proteins with a variety of functions. Defining the ends of the locus varies with the species; the area beyond the class I genes on the telomeric side is called the extended class I region. Likewise, the region beyond the class II gene cluster on the centromeric side is referred to as extended class II region. The major difference between mice and humans is that the extended class II region contains some class I (H2-K) genes in mice.

FIGURE 16.36 The MHC region extends for more than 2 Mb. MHC proteins of classes I and II are encoded by two separate regions. The class III region is defined as the segment between them. The extended regions describe segments that are syntenic on either end of the cluster. The major difference between mouse and human is the presence of H2 class I genes in the extended region on the left. The murine locus is located on chromosome 17, and the human locus is located on chromosome 6.

The organization of class I genes is based on the structure of their products (Figure 16.37). The first exon encodes a signal sequence, cleaved from the protein during membrane passage. The next three exons encode each of the external domains. The fifth exon encodes the transmembrane domain. The last three rather small exons together encode the cytoplasmic domain. The only difference in the genes for human transplantation antigens is that their cytoplasmic domain is coded by only two exons. The exon encoding the third external domain of the class I genes is highly conserved relative to the other exons. The conserved domain probably represents the region that interacts with β2-microglobulin, which explains the need for constancy of structure. This domain also exhibits homologies with the constant region domains of Igs. Most of the sequence variation between class I alleles occurs in the first and second external domains, sometimes taking the form of a cluster of base substitutions in a small region.

FIGURE 16.37 Each class of MHC genes has a characteristic organization, in which exons represent individual protein domains.

The gene for β2-microglobulin is located on a separate chromosome. It has four exons, the first encoding a signal sequence, the second encoding the bulk of the protein (from amino acids 3 to 95), the third encoding the last four amino acids and some of the nontranslated UTR, and the last encoding the rest of the UTR. The length of β2-microglobulin is similar to that of an Ig V gene; there are certain similarities in amino acid constitution, and there are some (limited) homologies of nucleotide sequence between β2-microglobulin and Ig constant domains or type I gene third external domains.

MHC class I genes encode transplantation antigens. They are present on every mammalian cell. As their name suggests, these proteins are responsible for the rejection of foreign tissue, which is recognized as such by virtue of its particular array of transplantation antigens. In the immune system, their presence on target cells is required for cell-mediated responses. The types of class I proteins are defined serologically by their antigenic properties. The murine class I genes encode the H2-K and H2-D/L proteins. Each mouse strain has one of several possible alleles for each of these proteins. The human class I genes encode the classical transplantation antigens: HLA-A, HLA-B, and HLA-C. Some HLA class I–like genes lie outside the MHC locus. Notable among these genes are those of the small CD1 family. CD1 genes encode proteins expressed on DCs and monocytes. CD1 proteins can bind glycolipids and present them to T cells, which are neither CD4 nor CD8.

MHC class II genes encode the MHC class II proteins. These are expressed on the surfaces of both B and activated T lymphocytes, as well as on macrophages and dendritic cells. MHC class II molecules are critically involved in antigen presentation and communications between cells that are necessary to induce a specific immune response. In particular, they are required for T_h cell function. The murine class II genes were originally identified as immune response (Ir) genes; that is, genes whose expression made it possible for an immune response to a given antigen to be triggered (hence, the I-A and I-E terminology). The human class II region (also called HLA-D) is arranged into HLA-DR, HLA-DP, and HLA-DQ subregions. This region also includes several genes that are related to the initiation of antigen-specific response, namely, antigen presentation. These genes include those encoding TAP and LMP, as well as those encoding the DM and DO molecules, which regulate peptide loading onto classical class II molecules. Expression of nonclassical MHC class II is induced by IFN-γ through CIITA, the MHC class II transcriptional activator.

MHC class III genes occupy the “transitional” region between class I and class II regions. The class III region includes genes encoding complement components, including C2, C4, and factor B. The role of complement factors is to interact with antibody–antigen complexes and mediate activation of the complement cascade, eventually lysing cells, bacteria, or viruses. Other genes lying in this transitional region include those encoding tumor necrosis factor-α (TNF-α) and lymphotoxin-α (LTA) and lymphotoxin-β (LTB).

The MHC regions of mammals have several hundred genes, but it is possible for MHC functions to be provided by far fewer genes, as in the case of chickens, where the MHC region is 92 kb and comprises only 19 genes. In comparison to other gene families, the exact numbers of genes devoted to each function differs. The MHC locus shows extensive variation between individuals, and a number of genes may be different in different individuals. As a general rule, however, a mouse genome has fewer active H2 genes than a human genome. The class II genes are unique to mammals (except for one subgroup); birds and fish have different genes in their place. Humans have approximately 8 functional class I genes; mice have approximately 30. The class I region also includes many other genes. The class III regions are very similar in humans and mice. MHC class I and class II genes are highly polymorphic, with the exception of human HLA-DRα and the mouse homologue H2-Eα, and likely arose as a result of extensive gene duplications. Further divergence arose through mutations and gene conversion.

Summary

Virtually all the genes discussed in this chapter likely descended from a common ancestor gene that encoded a primitive protein domain. Such a gene would have encoded a protein that mediated nonspecific defense against a variety of microbial pathogens. It is possibly the ancestor of the conserved genes coding for the more than 20 antifungal, antibacterial, and antiviral peptides in Drosophila. Further duplication and evolution of these genes likely gave rise to the diverse repertoire of Ig V(D)J and C genes in the Ig and TCR loci, as well as the genes in the MHC locus.

The immune system has evolved to respond to an enormous variety of microbial pathogens, such as bacteria, viruses, and other infectious agents. This is accomplished by triggering a virtually immediate response that recognizes common structures or MAMPs shared by many pathogens using PRRs. The diversity of these receptors is limited and encoded in the germline. The PRRs involved are typically members of the Toll-like class of receptors, and the related signaling pathways resemble the pathway triggered by Toll receptors during embryonic development. The pathway culminates in activation of transcription factors that cause genes to be expressed, and whose products inactivate the infective agent, typically by permeabilizing its membrane.

The innate immune response is triggered in different ways, and to different degrees, depending on the nature of the foreign microbial antigen inducing it. It contains (to some degree) the invading microorganism during the early stages of infection, but fails in general to limit the spreading of the infection in later stages or to eradicate the invading microbial pathogen. The innate immune response is nonspecific and does not generate immunological memory. Nevertheless, through differential modulation of the innate effector cells and molecules, the nature of the antigen determines the nature and magnitude of the adaptive response eventually mounted against that antigen.

The adaptive immune response relies on BCRs and TCRs, which play analogous recognition functions on B cells and T cells, respectively. The BCR or TCR components are generated by rearrangement of DNA in a single lymphocyte. Many different rearrangements occur early in the development of B and T cells, thereby creating a large repertoire of immune cells of different specificities. Exposure to an antigen recognized by the BCR or TCR leads to clonal expansion to give rise to many progeny cells that possess the same specificity as the original (parental) cell. The very large number of BCRs and TCRs available in the primary B and T cell repertoire provides the structural basis for this selection process.

Each Ig protein is a tetramer containing two identical H chains and two identical L chains (either κ or λ). Like an Ig molecule, a TCR is a dimer containing two different chains. Like IgH, TCRβ and TCRδ are expressed from a gene created by recombining one of many V gene segments with D segments and J segments, as linked to one of a few C segments. Like IgL, TCRα and TCRγ chains resemble IgL (κ and λ) chains.

V(D)J gene segments and their organization are different for each type of chain, but the principle and mechanism of recombination appear to be the same. The same nonamer-spacer-heptamer RSSs are involved in each recombination; the reaction always involves joining of an RSS with 23-bp spacing to an RSS with 12-bp spacing. The RAG1/RAG2 proteins catalyze the cleavage reaction, and the joining reaction is catalyzed by the same elements of the general NHEJ pathway that repairs DSBs. The mechanism of action of the RAG proteins is related to the action of site-specific recombination catalyzed by resolvases. Recombining different V(D)J segments generates considerable diversity; however, additional variations are introduced in the form of truncations and/or additions of N nucleotides at the junctions between V(D)J DNA segments during the recombination process. A productive rearrangement inhibits the occurrence of further rearrangements (allelic exclusion). Allelic exclusion ensures that a given lymphocyte synthesizes only a single BCR or TCR.

Mature B cells express surface IgM and IgD BCR. After encounter of antigen and activation, these B cells start secreting the corresponding IgM antibodies using a mechanism of differential or alternative splicing. This underlies the expression of a membrane-bound version of a BCR and its corresponding secreted version (antibody). BCRs and TCRs that recognize the body’s own proteins are screened out early in the process. B and T cell clones are expanded and further selected in response to antigen during the primary immune response. Activation of the BCR on B cells triggers the pathways of the humoral response; activation of the TCR on T cells triggers the pathways of the cell-mediated response. The primary immune (adaptive) response is characterized by a latency period—in general a few days—required for the clonal selection and proliferation of the B cells and/or T cells specific for the antigen, be it on a bacterium or a virus or other microorganism, driving the response. Clonal selection of B or T cells relies on binding of antigen to BCR and TCR on selected B and T cells (clones). These clones are significantly expanded in size and undergo SHM and CSR in the late stages of the primary response. Re-exposure to the same antigen induces a secondary response, which has virtually no latency period and is much bigger in magnitude and more specific than the primary response.

SHM and CSR continue to occur in the secondary response, upon re-exposure to the same antigen. SHM inserts point-mutation changes in Ig V(D)J gene sequences. It requires the actions of the AID cytidine deaminase and the Ung glycosylase. Mutations induced by AID lead in most cases to removal of deoxyuridine by Ung, and bypassing of abasic sites by TLS polymerases and/or recruitment of elements of the MMR machinery. The use of the V region is fixed by the first productive rearrangement, but B cells undergo CSR, thereby switching use of C_H genes from the initial C_μ chain to one of the C_H chains lying farther downstream. This process involves a different type of recombination in which the DNA intervening between the V_HDJ_H region and the new C_H gene is deleted and rejoined as a switch circle. More than one CSR event can occur in a B cell. CSR requires the same AID and Ung that are required for SHM. It also uses elements of the NHEJ pathway of DNA repair. Differential or alternative splicing also underlies the expression of membrane and secreted forms of all switched isotypes: IgG, IgA, and IgE.

SHM and CSR occur in peripheral lymphoid organs and are critical in the maturation of the antibody response and the generation of immunological memory. Immunological memory provides protective immunity against the same antigen that drove the original response. Thus, the organism retains a memory of the specific B and/or T cell response. The principles of adaptive immunity are similar, albeit somewhat different in details, throughout the vertebrates. Such memory enables the organism to respond more rapidly and vigorously once exposed again to the same pathogen, and provides the cellular and molecular basis for design and use of vaccines.

Acknowledgments: Dr. Casali would like to thank Dr. Hong Zan for his help with the editing of some sections of this chapter.

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Sen, R., and Baltimore, D. (1986). Inducibility of κ immunoglobulin enhancer-binding protein NF-κB by a posttranslational mechanism. Cell 47, 921–928.
Wesche, H., Henzel, W. J., Shillinglaw, W., Li, S., and Cao, Z. (1997). MyD88: an adapter that recruits IRAK to the IL-1 receptor complex. Immunity 7, 837–847.

16.3 Adaptive Immunity

Reviews

Chang, J. T., Wherry, E. J., and Goldrath, A. W. (2014). Molecular regulation of effector and memory T cell differentiation. Nat. Immunol. 15, 1104–1115.
Goodnow, C. C., Vinuesa, C. G., Randall, K. L., Mackay, F., and Brink, R. (2010). Control systems and decision making for antibody production. Nat. Immunol. 8, 681–688.
Jiang, H., and Chess, L. (2009). How the immune system achieves self-nonself discrimination during adaptive immunity. Adv. Immunol. 102, 95–133.
Koonin, E. V., and Krupovic, M. (2014). Evolution of adaptive immunity from transposable elements combined with innate immune systems. Nat. Rev. Genet. 16, 184–192.
Kurosaki, T., Kometani, K., and Ise W. (2015). Memory B cells. Nat. Immunol. 15, 149–159.
Litman, G. W., Rast, J. P., and Fugmann, S. D. (2010). The origins of vertebrate adaptive immunity. Nat. Rev. Immunol. 10, 543–553.

16.4 Clonal Selection Amplifies Lymphocytes That Respond to a Given Antigen

Reviews

Hodgkin, P. D., Heath, W. R., and Baxter, A. G. (2007). The clonal selection theory: 50 years since the revolution. Nat. Immunol. 8, 1019–1012.
Neuberger, M. S. (2008). Antibody diversification by somatic mutation: from Burnet onwards. Immunol. Cell Biol. 86, 124–132.
Reiner, S. L., and Adams, W. C. (2014). Lymphocyte fate specification as a deterministic but highly plastic process. Nat. Rev. Immunol. 14, 699–704.

Research

Gitlin, A. D., Shulman, Z., and Nussenzweig, M. C. (2014). Clonal selection in the germinal centre by regulated proliferation and hypermutation. Nature 509, 637–640.
Takada, K., Van Laethem, F., Xing, Y., Akane, K, Suzuki, H., Murata, S., Tanaka, K., Jameson, S.C., Singer, A., and Takahama, Y. (2015). TCR affinity for thymoproteasome-dependent positively selecting peptides conditions antigen responsiveness in CD8⁺ T cells. Nat Immunol. 16, 1069–1076.

16.5 Ig Genes Are Assembled from Discrete DNA Segments in B Lymphocytes

Reviews

Cobb, R. M., Oestreich, K. J., Osipovich, O. A., and Oltz, E. M. (2006). Accessibility control of V(D)J recombination. Adv. Immunol. 91, 45–109.
Jung, D., Giallourakis, C., Mostoslavsky, R., and Alt, F. W. (2006). Mechanism and control of V(D)J recombination at the immunoglobulin heavy chain locus. Annu. Rev. Immunol. 24, 541–570.
Kuo, T. C., and Schlissel, M. S. (2009). Mechanisms controlling expression of the RAG locus during lymphocyte development. Curr Opin Immunol. 21, 173–178.
Schatz, D. G., and Swanson, P. C. (2011). V(D)J recombination: mechanisms of initiation. Annu. Rev. Genet. 45, 167–202.

Research

Hozumi, N., and Tonegawa, S. (1976). Evidence for somatic rearrangement of immunoglobulin genes coding for variable and constant regions. Proc. Natl. Acad. Sci. USA 73, 3628–3632.
Schatz, D. G., Oettinger, M. A., and Baltimore, D. (1989). The V(D)J recombination activating gene, RAG-1. Cell 59, 1035–1048.

16.6 L Chains Are Assembled by a Single Recombination Event

Reviews

Langerak, A. W., and van Dongen, J. J. (2006). Recombination in the human Igκ locus. Crit. Rev. Immunol. 26, 23–42.
Schlissel, M. S. (2004). Regulation of activation and recombination of the murine Igκ locus. Immunol. Rev. 200, 215–223.

Research

Johnson, K., Hashimshony, T., Sawai, C. M., Pongubala, J. M., Skok, J. A., Aifantis, I., and Singh, H. (2008). Regulation of immunoglobulin light-chain recombination by the transcription factor IRF-4 and the attenuation of IL-7 signaling. Immunity 28, 335–345.
Lewis, S., Gifford, A., and Baltimore, D. (1985). DNA elements are asymmetrically joined during the site-specific recombination of κ immunoglobulin genes. Science 228, 677–685.

16.7 H Chains Are Assembled by Two Sequential Recombination Events

Reviews

Jung, D., Giallourakis, C., Mostoslavsky, R., and Alt, F. W. (2006). Mechanism and control of V(D)J recombination at the immunoglobulin heavy chain locus. Annu. Rev. Immunol. 24, 541–570.
Schatz, D. G., and Ji, Y. (2011). Recombination centres and the orchestration of V(D)J recombination. Nat. Rev. Immunol. 11, 251–263.

Research

Guo. C., Yoon, H. S., Franklin, A., Jain, S., Ebert, A., Cheng, H. L., Hansen, E., Despo, O., Bossen, C., Vettermann, C., Bates, J. G., Richards, N., Myers, D., Patel, H., Gallagher, M., Schlissel., M. S., Murre, C., Busslinger, M., Giallourakis, C. C., and Alt, F. W. (2011). CTCF-binding elements mediate control of V(D)J recombination. Nature 477, 424–430.

16.8 Recombination Generates Extensive Diversity

Reviews

Bossen, C., Mansson, R., and Murre, C. (2012). Chromatin topology and the regulation of antigen receptor assembly. Annu. Rev. Immunol. 30, 337–356.
Hodgkin, P. D., Heath, W. R., and Baxter, A. G. (2007). The clonal selection theory: 50 years since the revolution. Nat. Immunol. 8, 1019–1012.

Research

Jhunjhunwala, S., van Zelm, M.C., Peak, M. M., Cutchin, S., Riblet, R., van Dongen, J. J., Grosveld, F. G., Knoch, T. A., and Murre, C. (2008). The 3D structure of the immunoglobulin heavy-chain locus: implications for long-range genomic interactions. Cell 133, 265–279.

16.9 V(D)J DNA Recombination Relies on RSS and Occurs by Deletion or Inversion

Reviews

Dadi, S., Le Noir, S., Asnafi, V., Beldjord, K., and Macintyre, E. A. (2009). Normal and pathological V(D)J recombination: contribution to the understanding of human lymphoid malignancies. Adv. Exp. Med. Biol. 650, 180–189.
Liu, Y., Zhang, L., and Desiderio, S. (2009). Temporal and spatial regulation of V(D)J recombination: interactions of extrinsic factors with the RAG complex. Adv. Exp. Med. Biol. 650, 157–165.
Schatz, D. G., and Ji, Y. (2011). Recombination centres and the orchestration of V(D)J recombination. Nat. Rev. Immunol. 11, 251–263.
Swanson, P. C., Kumar, S., and Raval, P. (2009). Early steps of V(D)J rearrangement: insights from biochemical studies of RAG-RSS complexes. Adv. Exp. Med. Biol. 650, 1–15.

Research

Curry, J. D., Geier, J. K., and Schlissel, M. S. (2005). Single-strand recombination signal sequence nicks in vivo: evidence for a capture model of synapsis. Nat. Immunol. 6, 1272–1279.
Du, H., Ishii, H., Pazin, M. J., and Sen, R. (2008). Activation of 12/23-RSS-dependent RAG cleavage by hSWI/SNF complex in the absence of transcription. Mol. Cell 31, 641–649.
Melek, M., and Gellert, M. (2000). RAG1/2-mediated resolution of transposition intermediates: two pathways and possible consequences. Cell 101, 625–633.
Qiu, J. X., Kale, S. B., Yarnell Schultz, H., and Roth, D. B. (2001). Separation-of-function mutants reveal critical roles for RAG2 in both the cleavage and joining steps of V(D)J recombination. Mol. Cell 7, 77–87.
Seitan, V. C., Hao, B., Tachibana-Konwalski, K., Lavagnolli, T., Mira-Bontenbal, H., Brown, K. E., Teng, G., Carroll, T., Terry, A., Horan. K., Marks, H., Adams, D. J., Schatz, D. G., Aragon, L., Fisher, A. G., Krangel, M. S., Nasmyth, K., and Merkenschlager, M. (2011). A role for cohesin in T-cell-receptor rearrangement and thymocyte differentiation. Nature 476, 467–471.

16.10 Allelic Exclusion Is Triggered by Productive Rearrangements

Reviews

Brady, B. L., Steinel, N. C., and Bassing, C. H. (2010). Antigen receptor allelic exclusion: an update and reappraisal. J. Immunol. 185, 3801–3808.
Cedar, H., and Bergman, Y. (2008). Choreography of Ig allelic exclusion. Curr. Opin. Immunol. 20, 308–317.
Levin-Klein, R., and Bergman, Y. (2014). Epigenetic regulation of monoallelic rearrangement (allelic exclusion) of antigen receptor genes. Front. Immunol. 5, 625.
Perlot, T., and Alt, F. W. (2008). Cis-regulatory elements and epigenetic changes control genomic rearrangements of the IgH locus. Adv. Immunol. 99, 1–32.

Research

Hewitt, S. L., Farmer, D., Marszalek, K., Cadera, E., Liang, H. E., Xu, Y., Schlissel, M. S., and Skok, J. A. (2008). Association between the Igk and Igh immunoglobulin loci mediated by the 3′ Igκ enhancer induces “decontraction” of the IgH locus in pre-B cells. Nat. Immunol. 9, 396–404.
Liang, H. E., Hsu, L. Y., Cado, D., and Schlissel, M. S. (2004). Variegated transcriptional activation of the immunoglobulin κ locus in pre-B cells contributes to the allelic exclusion of light-chain expression. Cell 118, 19–29.

16.11 RAG1/RAG2 Catalyze Breakage and Religation of V(D)J Gene Segments

Reviews

Bergeron, S., Anderson, D. K., and Swanson, P. C. (2006). RAG and HMGB1 proteins: purification and biochemical analysis of recombination signal complexes. Methods Enzymol. 408, 511–528.
Schatz, D. G., and Ji, Y. (2011). Recombination centres and the orchestration of V(D)J recombination. Nat. Rev. Immunol. 11, 251–263.

Research

Deriano, L., Stracker, T. H., Baker, A., Petrini, J. H., and Roth, D. B. (2009). Roles for NBS1 in alternative nonhomologous end-joining of V(D)J recombination intermediates. Mol. Cell 34, 13–25.
Difilippantonio, S., Gapud, E., Wong, N., Huang, C. Y., Mahowald, G., Chen, H. T., Kruhlak, M. J., Callen, E., Livak, F., Nussenzweig, M. C., Sleckman, B. P., and Nussenzweig, A. (2008). 53BP1 facilitates long-range DNA end-joining during V(D)J recombination. Nature 456, 529–533.
Ji, Y., Resch., W., Corbett, E., Yamane, A., Casellas, R., and Schatz, D. G. (2010). The in vivo pattern of binding of RAG1 and RAG2 to antigen receptor loci. Cell 141, 419–431.
Lu, C. P., Sandoval, H., Brandt, V. L., Rice, P. A., and Roth, D. B. (2006). Amino acid residues in Rag1 crucial for DNA hairpin formation. Nat. Struct. Mol. Biol. 13, 1010–1015.
Ma, Y., Pannicke, U., Schwarz, K., and Lieber, M. R. (2002). Hairpin opening and overhang processing by an Artemis/DNA-dependent protein kinase complex in nonhomologous end joining and V(D)J recombination. Cell 108, 781–794.
Ru, H., Chambers, M. G., Fu, T.-M., Tong, A. B., Liao, M., and Wu, H. (2015). Molecular mechanism of V(D)J recombination from synaptic RAG1-RAG2 complex structures. Cell 163, 1138–1152.
Tsai, C. L., Drejer, A. H., and Schatz, D. G. (2002). Evidence of a critical architectural function for the RAG proteins in end processing, protection, and joining in V(D)J recombination. Genes. Dev. 16, 1934–1949.
Yarnell Schultz, H., Landree, M. A., Qiu, J. X., Kale, S. B., and Roth, D. B. (2001). Joining-deficient RAG1 mutants block V(D)J recombination in vivo and hairpin opening in vitro. Mol. Cell 7, 65–75.

16.12 B Cell Development in the Bone Marrow: From Common Lymphoid Progenitor to Mature B Cell

Reviews

Bryder, D., and Sigvardsson, M. (2012). Shaping up a lineage-lessons from B lymphopoesis. Curr. Opin. Immunol. 22, 148–153.
Kurosaki, T., Shinohara, H., and Baba, Y. (2010). B cell signaling and fate decision. Annu. Rev. Immunol. 28, 21–55.
Parra, M. (2009). Epigenetic events during B lymphocyte development. Epigenetics. 4, 462–468.

Research

Decker, T., Pasca di Magliano, M., McManus, S., Sun, Q., Bonifer, C., Tagoh, H., and Busslinger, M. (2009). Stepwise activation of enhancer and promoter regions of the B cell commitment gene Pax5 in early lymphopoiesis. Immunity 30, 508–520.
Nechanitzky, R., Akbas, D., Scherer, S., Györy, I., Hoyler, T., Ramamoorthy, S., Diefenbach, A., and Grosschedl, R. (2013). Transcription factor EBF1 is essential for the maintenance of B cell identity and prevention of alternative fates in committed cells. Nat. Immunol. 14, 867–875.

16.13 Class Switch DNA Recombination

Reviews

Robbiani, D. F., and Nussenzweig, M. C. (2013). Chromosome translocation, B cell lymphoma, and activation-induced cytidine deaminase. Annu. Rev. Pathol. 8, 79–103.
Stavnezer, J., and Schrader, C. E. (2014). IgH chain class switch recombination: mechanism and regulation. J. Immunol. 193, 5370–5378.
Xu, Z., Pone, E. J., Al-Qahtani, A., Park, S, R., Zan, H., and Casali, P. (2007). Regulation of Aicda expression and AID activity: relevance to somatic hypermutation and class switch DNA recombination. Crit. Rev. Immunol. 27, 367–397.
Xu, Z., Zan, H., Pone, E. J., Mai, T., and Casali, P. (2012). Immunoglobulin class switch DNA recombination: induction, targeting and beyond. Nature Rev. Immunol., 17, 2595–2615.
Yang, S. Y., and Schatz, D. G. (2007). Targeting of AID-mediated sequence diversification by cis-acting determinants. Adv. Immunol. 94, 109–125.
Zan, H., and Casali, P. (2013). Regulation of Aicda expression and AID activity. Autoimmunity 46, 83–101.

Research

Basu, U., Chaudhuri, J., Alpert, C., Dutt, S., Ranganath, S., Li, G., Schrum, J. P., Manis, J. P., and Alt, F. W. (2005). The AID antibody diversification enzyme is regulated by protein kinase A phosphorylation. Nature 438, 508–511.
Basu, U., Meng, F.-L., Keim, C., Grinstein, V., Pefanis, E., Eccleston, J., Zhang, T., Myers, D., Wasserman, C. R., Wesemann, D. R., Januszyk, K., Gregory, R. I., Deng, H., Lima, C. D., and Alt, F. W. (2011). The RNA exosome targets the AID cytidine deaminase to both strands of transcribed duplex DNA substrates. Cell 144, 353–363.
Geisberger, R., Rada, C., and Neuberger, M. S. (2009). The stability of AID and its function in class-switching are critically sensitive to the identity of its nuclear-export sequence. Proc. Natl. Acad. Sci. USA 106, 6736–6741.
Kinoshita, K., Harigai, M., Fagarasan, S., Muramatsu, M., and Honjo, T. (2001). A hallmark of active class switch recombination: transcripts directed by I promoters on looped-out circular DNAs. Proc. Natl. Acad. Sci. USA 98, 12620–12623.
Mai, T., Zan, H., Zhang, J., Hawkins, J. S., Xu, Z., and Casali, P. (2010). Estrogen receptors bind to and activate the HOXC4/HoxC4 promoter to potentiate HoxC4-mediated activation-induced cytosine deaminase induction, immunoglobulin class switch DNA recombination, and somatic hypermutation. J. Biol. Chem. 285, 37797–37810.
Matsuoka, M., Yoshida, K., Maeda, T., Usuda, S., and Sakano, H. (1990). Switch circular DNA formed in cytokine-treated mouse splenocytes: evidence for intramolecular DNA deletion in immunoglobulin class switching. Cell 62, 135–142.
Muramatsu, M., Kinoshita, K., Fagarasan, S., Yamada, S., Shinkai, Y., and Honjo, T. (2000). Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102, 553–563.
Nagaoka, H., Muramatsu, M., Yamamura, N., Kinoshita, K., and Honjo, T. (2002). Activation-induced deaminase (AID)-directed hypermutation in the immunoglobulin Sμ region: implication of AID involvement in a common step of class switch recombination and somatic hypermutation. J. Exp. Med. 195, 529–534.
Nowak, U., Matthews, A. J., Zheng, S., and Chaudhuri, J. (2011). The splicing regulator PTBP2 interacts with the cytidine deaminase AID and promotes binding of AID to switch-region DNA. Nature Immunol. 12, 160–166.
Okazaki, I. M., Kinoshita, K., Muramatsu, M., Yoshikawa, K., and Honjo, T. (2002). The AID enzyme induces class switch recombination in fibroblasts. Nature 416, 340–345.
Park, S. R., Zan, H., Pal, Z., Zhang, J., Al-Qahtani, A., Pone, E. J., Xu, Z., Mai, T., and Casali, P. (2009). HoxC4 binds to the promoter of the cytidine deaminase AID gene to induce AID expression, class-switch DNA recombination and somatic hypermutation. Nature Immunol. 10, 540–550.
Petersen-Mahrt, S. K., Harris, R. S., and Neuberger, M. S. (2002). AID mutates E. coli suggesting a DNA deamination mechanism for antibody diversification. Nature 418, 99–103.
Pone, E. J., Zhang, J., Mai, T., White, C. A., Li, G., Sakakura, J., Patel, P., Al-Qahtani, A., Zan, H., Xu, Z., and Casali, P. (2012). BCR-signalling signaling synergizes with TLR-signalling to induce AID and immunoglobulin class-switching through the non-canonical NF-κB pathway. Nature Commun. 3, 767.
Rada, C., Williams, G. T., Nilsen, H., Barnes, D. E., Lindahl, T., and Neuberger, M. S. (2002). Immunoglobulin isotype switching is inhibited and somatic hypermutation perturbed in UNG-deficient mice. Curr. Biol. 12, 1748–1755.
Revy, P., Muto, T., Levy, Y., Geissmann, F., Plebani, A., Sanal, O., Catalan, N., Forveille, M., Dufourcq-Labelouse, R., Gennery, A., Tezcan, I., Ersoy, F., Kayserili, H., Ugazio, A.G., Brousse, N., Muramatsu, M., Notarangelo, L. D., Kinoshita, K., Honjo, T., Fischer, A., and Durandy, A. (2000). Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the Hyper-IgM syndrome (HIGM2). Cell 102, 565–575.
Xu, Z., Fulop, Z., Wu, G., Pone, E. J., Zhang, J., Mai, T., Thomas, L. M., Al-Qahtani, A., White, C. A., Park, S. R., Steinacker, P., Li, Z., Yates, J. 3rd, Herron, B., Otto, M., Zan, H., Fu, H., and Casali, P. (2010). 14-3-3 adaptor proteins recruit AID to 5′-AGCT-3′-rich switch regions for class switch recombination. Nature Struct. Mol. Biol. 17, 1124–1135.
Zan, H, White, C. A., Thomas, L. M., Mai, T., Li, G., Xu, Z., Zhang, J., and Casali, P. (2012). Rev1 recruits Ung to switch regions and enhances deglycosylation for immunoglobulin class switch DNA recombination. Cell Rep. 2, 1220–1232.
Zan, H., Zhang, J., Al-Qahtani, A., Pone, E. J., White, C. A., Lee, D., Yel, L., Mai, T., and Casali, P. (2011). Endonuclease G plays a role in immunoglobulin class switch DNA recombination by introducing double-strand breaks in switch regions. Mol. Immunol. 48, 610–622.
Zarrin, A. A., Alt, F. W., Chaudhuri, J., Stokes, N., Kaushal, D., Du Pasquier, L., and Tian, M. (2004). An evolutionarily conserved target motif for immunoglobulin class-switch recombination. Nature Immunol. 5, 1275–1281.

16.14 CSR Involves AID and Elements of the NHEJ Pathway

Reviews

Alt, F. W., Zhang, Y., Meng, F. L., Guo, C., and Schwer, B. (2013). Mechanisms of programmed DNA lesions and genomic instability in the immune system. Cell 152, 417–429.
Gostissa, M., Alt, F. W., and Chiarle, R. (2012). Mechanisms that promote and suppress chromosomal translocations in lymphocytes. Annu Rev Immunol. 29, 319–350.
Lieber, M. R. (2010). The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu. Rev. Biochem. 79, 181–211.

Research

Buerstedde, J. M., Lowndes, N., and Schatz, D. G. (2014). Induction of homologous recombination between sequence repeats by the activation induced cytidine deaminase (AID) protein. Elife 3, e03110.
Chiarle, R., Zhang, Y., Frock, R. L., Lewis, S. M., Molinie, B., Ho, Y. J., Myers, D. R., Choi, V. W., Compagno, M., Malkin, D. J., Neuberg, D., Monti, S., Giallourakis, C. C., Gostissa, M., and Alt, F. W. (2011). Genome-wide translocation sequencing reveals mechanisms of chromosome breaks and rearrangements in B cells. Cell 147, 107–119.
Dong, J., Panchakshari, R.A., Zhang, T., Zhang, Y., Hu, J., Volpi, S. A., Meyers, R. M., Ho, Y. J., Du, Z., Robbiani, D. F., Meng, F., Gostissa, M., Nussenzweig, M. C., Manis, J. P., and Alt, F.W. (2015). Orientation-specific joining of AID-initiated DNA breaks promotes antibody class switching. Nature 525, 134–139.
Yamane, A., Resch, W., Kuo, N., Kuchen, S., Li, Z., Sun, H. W., Robbiani, D. F., McBride, K., Nussenzweig, M. C., and Casellas, R. (2011). Deep-sequencing identification of the genomic targets of the cytidine deaminase AID and its cofactor RPA in B lymphocytes. Nat. Immunol. 12, 62–69.
Yan, C. T., Boboila, C., Souza, E. K., Franco, S., Hickernell, T. R., Murphy, M., Gumaste, S., Geyer, M., Zarrin, A. A., Manis, J. P., Rajewsky, K., and Alt, F. W. (2007). IgH class switching and translocations use a robust non-classical end-joining pathway. Nature 449, 478–482.
Zan, H., Tat, C., Qiu, Z., Taylor, J. R., Guerrero, J. A., Shen, T., and Casali, P. (2017). Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends to modulate antibody class-switch DNA recombination. Nature Commun. 8, 142–144.

16.15 Somatic Hypermutation Generates Additional Diversity and Provides the Substrate for Higher-Affinity Submutants

Reviews

Neuberger, M. S. (2008). Antibody diversification by somatic mutation: from Burnet onwards. Immunol. Cell Biol. 86, 124–132.
Tarlinton, D. M. (2008). Evolution in miniature: selection, survival and distribution of antigen reactive cells in the germinal centre. Immunol. Cell Biol. 86, 133–138.
Teng, G., and Papavasiliou, F. N. (2007). Immunoglobulin somatic hypermutation. Annu. Rev. Genet. 41, 107–120.

Research

Di Noia, J., and Neuberger, M. S. (2002). Altering the pathway of immunoglobulin hypermutation by inhibiting uracil-DNA glycosylase. Nature 419, 43–48.
Gitlin, A. D., Shulman, Z., and Nussenzweig, M. C. (2014). Clonal selection in the germinal centre by regulated proliferation and hypermutation. Nature 509, 637–640.
Muramatsu, M., Kinoshita, K., Fagarasan, S., Yamada, S., Shinkai, Y., and Honjo, T. (2000). Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102, 553–563.
Wei, M., Shinkura, R., Doi, Y., Maruya, M., Fagarasan, S., and Honjo T. (2011). Mice carrying a knock-in mutation of Aicda resulting in a defect in somatic hypermutation have impaired gut homeostasis and compromised mucosal defense. Nat. Immunol. 12, 264–270.

16.16 SHM Is Mediated by AID, Ung, Elements of the Mismatch DNA Repair Machinery, and Translesion DNA Synthesis Polymerases

Reviews

Casali, P., Pal, Z., Xu, Z., and Zan, H. (2006). DNA repair in antibody somatic hypermutation. Trends Immunol. 27, 313–321.
Chandra, V., Bortnick, A., and Murre, C. (2015). AID targeting: old mysteries and new challenges. Trends Immunol. 36, 527–535.
Di Noia, J. M., and Neuberger, M. S. (2007). Molecular mechanisms of antibody somatic hypermutation. Annu. Rev. Biochem. 76, 1–22.
Jiricny, J. (2006). The multifaceted mismatch-repair system. Nat. Rev. Mol. Cell. Biol. 7, 335–346.
Liu, M., and Schatz, D. G. (2009). Balancing AID and DNA repair during somatic hypermutation Trends Immunol. 30, 173–181.
Peled, J. U., Kuang, F. L., Iglesias-Ussel, M. D., Roa, S., Kalis, S. L., Goodman, M. F., and Scharff, M. D. (2008). The biochemistry of somatic hypermutation. Annu. Rev. Immunol. 26, 481–511.
Weill, J. C., and Reynaud, C. A. (2008) DNA polymerases in adaptive immunity. Nat. Rev. Immunol. 8, 302–312.
Xu, Z., Zan, H., Pal, Z., and Casali, P. (2007). DNA replication to aid somatic hypermutation. Adv. Exp. Med. Biol. 596, 111–127.

Research

Aoufouchi, S., Faili, A., Zober, C., D’Orlando, O., Weller, S., Weill, J. C., and Reynaud, C. A. (2008). Proteasomal degradation restricts the nuclear lifespan of AID. J. Exp. Med. 205, 1357–1368.
Di Noia, J., and Neuberger, M. S. (2002). Altering the pathway of immunoglobulin hypermutation by inhibiting uracil-DNA glycosylase. Nature 419, 43–48.
Muramatsu, M., Kinoshita, K., Fagarasan, S., Yamada, S., Shinkai, Y., and Honjo, T. (2000). Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102, 553–563.
Rada, C., Di Noia, J. M., and Neuberger, M. S. (2004). Mismatch recognition and uracil excision provide complementary paths to both Ig switching and the A/T-focused phase of somatic mutation. Mol. Cell 16, 163–171.
Zan, H., Komori, A., Li, Z., Cerutti, A., Schaffer, A., Flajnik, M. F., Diaz, M., and Casali, P. (2001). The translesion DNA polymerase zeta plays a major role in Ig and bcl-6 somatic hypermutation. Immunity, 14, 643–653.
Zan, H., Shima, N., Xu, Z., Al-Qahtani, A., Evinger, A. J., III, Zhong, Y., Schimenti, J. C., and Casali, P. (2005). The translesion DNA polymerase theta plays a dominant role in immunoglobulin gene somatic hypermutation. EMBO J. 24, 3757–3769.
Zan, H., Wu, X., Komori, A., Holloman, W. K., and Casali, P. (2003). AID-dependent generation of resected double-strand DNA breaks and recruitment of Rad52/Rad51 in somatic hypermutation. Immunity 18, 727–738.

16.17 Igs Expressed in Avians Are Assembled from Pseudogenes

Review

Ratcliffe, M. J. (2006). Antibodies, immunoglobulin genes and the bursa of Fabricius in chicken B cell development. Dev. Comp. Immunol. 30, 101–118.

Research

Chatterji, M., Unniraman, S., McBride, K. M., and Schatz, D. G. (2007). Role of activation-induced deaminase protein kinase A phosphorylation sites in Ig gene conversion and somatic hypermutation. J. Immunol. 179, 5274–5280.
Leighton, P. A., Schusser, B., Yi, H., Glanville, J., and Harriman, W. (2015). A diverse repertoire of human immunoglobulin variable genes in a chicken B cell line is generated by both gene conversion and somatic hypermutation. Front. Immunol. 6, 126.
Reynaud, C. A., Anquez, V., Grimal, H., and Weill, J. C. (1987). A hyperconversion mechanism generates the chicken light chain preimmune repertoire. Cell 48, 379–388.
Sale, J. E., Calandrini, D. M., Takata, M., Takeda, S., and Neuberger, M. S. (2001). Ablation of XRCC2/3 transforms immunoglobulin V gene conversion into somatic hypermutation. Nature 412, 921–926.
Yang, S. Y., Fugmann, S., and Schatz, D. G. (2006). Control of gene conversion and somatic hypermutation by immunoglobulin promoter and enhancer sequences. J. Exp. Med. 203, 2919–2928.

16.18 Chromatin Architecture Dynamics of the IgH Locus in V(D)J Recombination, CSR, and SHM

Reviews

Bossen, C., Mansson, R., and Murre, C. (2012). Chromatin topology and the regulation of antigen receptor assembly. Annu. Rev. Immunol. 30, 337–356.
Choi, N. M., and Feeney, A. J. (2014). CTCF and ncRNA regulate the three-dimensional structure of antigen receptor loci to facilitate V(D)J recombination. Front Immunol. 5, 49.
Ong and Corces. (2014). CTCF: an architectural protein bridging genome topology and function. Nat. Rev. Gent. 15, 234–246.
Jhunjhunwala, S., van Zelm, M. C., Peak, M. M., and Murre, C. (2009). Chromatin architecture and the generation of antigen receptor diversity. Cell 138, 435–448.
Shih, H. Y., Krangel, M. S. (2014). Chromatin architecture, CCCTC-binding factor, and V(D)J recombination: managing long-distance relationships at antigen receptor loci. J. Immunol. 190, 4915–4921.

Research

Bonaud, A., Lechouane, F., Le Noir, S., Monestier, O., Cogné, M., and Sirac, C. (2015). Efficient AID targeting of switch regions is not sufficient for optimal class switch recombination. Nat. Commun. 6, 7613.
Guo, C., Yoon, H. S., Franklin, A., Jain, S., Ebert, A., Cheng, H. L., Hansen, E., Despo, O., Bossen, C., Vettermann, C., Bates, J. G., Richards, N., Myers, D., Patel, H., Gallagher, M., Schlissel, M. S., Murre, C., Busslinger, M., Giallourakis, C. C., and Alt, F. W. (2011). CTCF-binding elements mediate control of V(D)J recombination. Nature 477, 424–430.
Hu, J., Zhang, Y., Zhao, L., Frock, R. L., Du, Z., Meyers, R. M., Meng, F. L., Schatz, D. G., and Alt, F. W. (2015). Chromosomal loop domains direct the recombination of antigen receptor genes. Cell 163, 947–959.
Jhunjhunwala, S., van Zelm, M. C., Peak, M. M., Cutchin, S., Riblet, R., van Dongen, J. J., Grosveld, F. G., Knoch, T. A., and Murre, C. (2008). The 3D structure of the immunoglobulin heavy-chain locus: implications for long-range genomic interactions. Cell. 133, 265–279.
Lin, Y. C., Benner, C., Mansson, R., Heinz, S., Miyazaki, K., Miyazaki, M., Chandra, V., Bossen, C., Glass, C. K., and Murre, C. (2012). Global changes in the nuclear positioning of genes and intra- and interdomain genomic interactions that orchestrate B cell fate. Nat. Immunol. 13, 1196–1204.
Meng, F. L., Du, Z., Federation, A., Hu, J., Wang, Q., Kieffer-Kwon, K. R., Meyers, R. M., Amor, C., Wasserman, C. R., Neuberg, D., Casellas, R., Nussenzweig, M. C., Bradner, J. E., Liu, X. S., and Alt, F.W. (2014). Convergent transcription at intragenic super-enhancers targets AID-initiated genomic instability. Cell 159, 1538–1548.
Qian, J., Wang, Q., Dose, M., Pruett, N., Kieffer-Kwon, K. R., Resch, W., Liang, G., Tang, Z., Mathé, E., Benner, C., Dubois, W., Nelson, S., Vian, L., Oliveira, T. Y., Jankovic, M., Hakim, O., Gazumyan, A., Pavri, R., Awasthi, P., Song, B., Liu, G., Chen, L., Zhu, S., Feigenbaum, L., Staudt, L., Murre, C., Ruan, Y., Robbiani, D.F., Pan-Hammarström, Q., Nussenzweig, M. C., and Casellas, R. (2014). B cell super-enhancers and regulatory clusters recruit AID tumorigenic activity. Cell 159, 1524–1537.
Shih H. Y., Verma-Gaur, J., Torkamani, A., Feeney, A. J., Galjart, N., and Krangel, M. S. (2012). Tcra gene recombination is supported by a Tcra enhancer- and CTCF-dependent chromatin hub. Proc. Natl. Acad. Sci. USA 109, E3493–E3502.

16.19 Epigenetics of V(D)J Recombination, CSR, and SHM

Reviews

Chandra, V., Bortnick, A., and Murre, C. (2015). AID targeting: old mysteries and new challenges. Trends Immunol. 36, 527–535.
Li, G., Zan, H., Xu, Z., and Casali, P. (2013). Epigenetics of the antibody response. Trends Immunol. 34, 460–470.
Schatz, D. G., and Ji, Y. (2011). Recombination centres and the orchestration of V(D)J recombination. Nat Rev Immunol. 4, 251–263.
Schatz, D. G., and Swanson, P. C. (2011). V(D)J recombination: mechanisms of initiation. Annu. Rev. Genet. 45, 167–202.
Xu, Z., Zan, H., Pone, E. J., Mai, T., and Casali, P. (2012). Immunoglobulin class switch DNA recombination: induction, targeting and beyond. Nature Rev. Immunol. 12, 517–531.
Zan, H., and Casali, P. (2015). Epigenetics of peripheral B-cell differentiation and the antibody response. Front. Immunol. 6, 631.

Research

Daniel, J. A., Santos, M. A., Wang, Z., Zang, C., Schwab, K. R., Jankovic, M., Filsuf, D., Chen, H. T., Gazumyan, A., Yamane, A., Cho, Y. W., Sun, H. W., Ge, K., Peng, W., Nussenzweig, M. C., Casellas, R., Dressler, G. R., Zhao, K., and Nussenzweig, A. (2010). PTIP promotes chromatin changes critical for immunoglobulin class switch recombination. Science 329, 917–923.
Jeevan-Raj, B. P., Robert, I., Heyer, V., Page, A., Wang, J. H., Cammas, F., Alt, F. W., Losson, R., and Reina-San-Martin, B. (2011). Epigenetic tethering of AID to the donor switch region during immunoglobulin class switch recombination. J. Exp. Med. 208, 1649–1660.
Li, G., White, C. A., Lam, T., Pone, E. J., Tran, D. C., Hayama, K. L., Zan, H., Xu, Z., and Casali, P. (2012). Combinatorial H3K9acS10ph histone modification in IgH locus S regions targets 14-3-3 adaptors and AID to specify antibody class-switch DNA recombination. Cell Rep. 5, 702–714.
Mandal, M., Hamel, K. M., Maienschein-Cline, M., Tanaka, A., Teng, G., Tuteja, J. H., Bunker, J. J., Bahroos, N., Eppig, J. J., Schatz, D. G., and Clark, M. R. (2015). Histone reader BRWD1 targets and restricts recombination to the Igk locus. Nat. Immunol. 16, 1094–1103.
Nowak, U., Matthews, A. J., Zheng, S., and Chaudhuri, J. (2011). The splicing regulator PTBP2 interacts with the cytidine deaminase AID and promotes binding of AID to switch-region DNA. Nat. Immunol. 12, 160–166.
Osipovich, O., Milley, R., Meade, A., Tachibana, M., Shinkai, Y., Krangel, M. S., and Oltz, E. M. (2004). Targeted inhibition of V(D)J recombination by a histone methyltransferase. Nat. Immunol. 5, 309–316.
Pefanis, E., Wang, J., Rothschild, G., Lim, J., Kazadi, D., Sun, J., Federation, A., Chao, J., Elliott, O., Liu, Z.P., Economides, A.N., Bradner, J. E., Rabadan, R., and Basu, U. (2015). RNA exosome-regulated long non-coding RNA transcription controls super-enhancer activity. Cell 161, 774–789.
Ranjit, S., Khair, L., Linehan, E. K., Ucher, A. J., Chakrabarti, M., Schrader, C. E., and Stavnezer, J. (2011). AID binds cooperatively with UNG and Msh2-Msh6 to Ig switch regions dependent upon the AID C terminus. J Immunol. 187, 2464–2475.
Subrahmanyam, R., Du, H., Ivanova, I., Chakraborty, T., Ji, Y., Zhang, Y., Alt, F. W., Schatz, D. G., and Sen, R. (2012). Localized epigenetic changes induced by DH recombination restricts recombinase to DJH junctions. Nat. Immunol. 13, 1205–1212.
Wang, L., Wuerffel, R., Feldman, S., Khamlichi, A. A., and Kenter, A. L. (2009). S region sequence, RNA polymerase II, and histone modifications create chromatin accessibility during class switch recombination. J. Exp. Med. 206, 1817–1830.
Wang, Q., Oliveira, T., Jankovic, M., Silva, I. T., Hakim, O., Yao, K., Gazumyan, A., Mayer, C. T., Pavri, R., Casellas, R., Nussenzweig, M. C., and Robbiani, D. F. (2014). Epigenetic targeting of activation-induced cytidine deaminase. Proc. Natl. Acad. Sci. USA 111, 18667–18672.
White, C. A., Pone, E. J., Lam, T., Tat, C., Hayama, K. L., Li, G., Zan, H., and Casali, P. (2014). Histone deacetylase inhibitors upregulate B cell microRNAs that silence AID and Blimp-1 expression for epigenetic modulation of antibody and autoantibody responses. J. Immunol. 193, 5933–5950.
Zheng, S., Vuong, B. Q., Vaidyanathan, B., Lin, J. Y., Huang, F. T., and Chaudhuri, J. (2015). Non-coding RNA generated following lariat debranching mediates targeting of AID to DNA. Cell 161, 762–773.

16.20 B Cell Differentiation Results in Maturation of the Antibody Response and Generation of Long-lived Plasma Cells and Memory B Cells

Reviews

Igarashi, K., Ochiai, K., and Muto, A. (2007). Architecture and dynamics of the transcription factor network that regulates B-to-plasma cell differentiation. J. Biochem. 141, 783–789.
Kurosaki, T., Kometani, K., and Ise W. (2015). Memory B cells. Nat. Immunol. 15, 149–159.
Nutt, S., L., and Tarlinton, D. M. (2011). Germinal center B and follicular helper T cells: siblings, cousins or just good friends. Nat. Immunol. 12, 472–477.
Pulendran, B., and Ahmed, R. (2006). Translating innate immunity into immunological memory: implications for vaccine development. Cell 124, 849–863.
Sciammas, R., and Davis, M. M. (2005). Blimp-1; immunoglobulin secretion and the switch to plasma cells. Curr. Top. Microbiol. Immunol. 290, 201–224.
Shlomchik, M. J., and Weisel, F. (2012). Germinal center selection and the development of memory B and plasma cells. Immunol. Rev. 247, 52–63.

Research

Martincic, K., Alkan, S. A., Cheatle, A., Borghesi, L,. and Milcarek, C. (2009). Transcription elongation factor ELL2 directs immunoglobulin secretion in plasma cells by stimulating altered RNA processing. Nat. Immunol. 10, 1102–1109.
Pape, K. A., Taylor, J. J., Maul, R. W., Gearhart, P. J., and Jenkins, M. K. (2011). Different B cell populations mediate early and late memory during an endogenous immune response. Science 331, 1203–1207.
Talay, O., Yan, D., Brightbill, H. D., Straney, E. E., Zhou, M., Ladi, E., Lee, W. P., Egen, J. G., Austin, C. D., Xu, M., and Wu, L. C. (2012). IgE(+) memory B cells and plasma cells generated through a germinal-center pathway. Nat Immunol. 13, 396–404.

16.21 The T Cell Receptor Antigen Is Related to the BCR

Reviews

Cobb, R. M., Oestreich, K. J., Osipovich, O. A., and Oltz, E. M. (2006). Accessibility control of V(D)J recombination. Adv. Immunol. 91, 45–109.
Taghon, T., and Rothenberg, E. V. (2008). Molecular mechanisms that control mouse and human TCR-αβ and TCR-γδ T cell development. Semin. Immunopathol. 30, 383–398.

Research

Abarrategui, I., and Krangel, M. S. (2006). Regulation of T cell receptor-alpha gene recombination by transcription. Nat. Immunol. 7, 1109–1115.
Jackson, A. M., and Krangel, M. S. (2006). Turning T-cell receptor beta recombination on and off: more questions than answers. Immunol. Rev. 209, 129–141.
Oestreich, K. J., Cobb, R. M., Pierce, S., Chen, J., Ferrier, P., and Oltz, E. M. (2006). Regulation of TCRβ gene assembly by a promoter/enhancer holocomplex. Immunity 24, 381–391.
Wucherpfennig, K. W. (2005). The structural interactions between T cell receptors and MHC-peptide complexes place physical limits on self-nonself discrimination. Curr. Top. Microbiol. Immunol. 296, 19–37.

16.22 The TCR Functions in Conjunction with the MHC

Reviews

Collins, E. J., and Riddle, D. S. (2008). TCR-MHC docking orientation: natural selection, or thymic selection? Immunol. Res. 41, 267–294.
Garcia, K. C., Adams, J. J., Feng, D., and Ely, L. K. (2009). The molecular basis of TCR germline bias for MHC is surprisingly simple. Nat. Immunol. 10, 143–147.
Godfrey, D. I., Rossjohn, J., and McCluskey, J. (2008). The fidelity, occasional promiscuity, and versatility of T cell receptor recognition. Immunity 28, 304–314.
Jenkins, M. K., Chu, H. H., McLachlan, J. B., and Moon, J. J. (2010). On the composition of the preimmune repertoire T cells specific for peptide-major histocompatibility complex ligands. Annu. Rev. Immunol. 28, 273–294.
Peterson, P., Org, T., and Rebane, A. (2008). Transcriptional regulation by AIRE: molecular mechanisms of central tolerance. Nat. Rev. Immunol. 8, 948–957.
Rudolph, M. G., Stanfield, R. L., and Wilson, I. A. (2006). How TCRs bind MHCs, peptides, and coreceptors. Annu. Rev. Immunol. 24, 419–466.

Research

Borg, N. A., Ely, L. K., Beddoe, T., Macdonald, W. A., Reid, H. H., Clements, C. S., Purcell, A. W., Kjer-Nielsen, L., Miles, J. J., Burrows, S. R., McCluskey, J., and Rossjohn, J. (2005). The CDR3 regions of an immunodominant T cell receptor dictate the “energetic landscape” of peptide-MHC recognition. Nat. Immunol. 6, 171–180.
Feng, D., Bond, C. J., Ely, L. K., Maynard, J., and Garcia, K. C. (2007). Structural evidence for a germline-encoded T cell receptor-major histocompatibility complex interaction “codon”. Nat. Immunol. 8, 975–983.
Gras, S., Burrows, S. R., Kjer-Nielsen, L., Clements, C. S., Liu, Y. C., Sullivan, L. C., Bell, M. J., Brooks, A. G., Purcell, A. W., McCluskey, J., and Rossjohn, J. (2009). The shaping of T cell receptor recognition by self-tolerance. Immunity 30, 193–203.
Kosmrlj, A., Jha, A. K., Huseby, E. S., Kardar, M., and Chakraborty, A. K. (2008). How the thymus designs antigen-specific and self-tolerant T cell receptor sequences. Proc. Natl. Acad. Sci. USA 105, 16671–16676.

16.23 The MHC Locus Comprises a Cohort of Genes Involved in Immune Recognition

Review

Deitiker, P., Atassi, M. Z. (2015). MHC Genes linked to autoimmune disease. Crit. Rev. Immunol. 35, 203–351.
Trowsdale J. (2011). The MHC, disease and selection. Immunol Lett. 30, 1–8.
Rossjohn, J., Stephanie, G., Miles, J. J., Turner, S. J., Godfrey, D. I., and McCluskey, J. (2015). T cell antigen receptor recognition of antigen-presenting molecules. Annu. Rev. Immunol. 33, 169–200.

Research

de Bakker, P. I., McVean, G., Sabeti, P. C., Miretti, M. M., Green, T., Marchini, J., Ke, X., Monsuur, A. J., Whittaker, P., Delgado, M., Morrison, J., Richardson, A., Walsh, E. C., Gao, X., Galver, L., Hart, J., Hafler, D. A., Pericak-Vance, M., Todd, J. A., Daly, M. J., Trowsdale, J., Wijmenga, C., Vyse, T. J., Beck, S., Murray, S. S., Carrington, M., Gregory, S., Deloukas, P., and Rioux, J. D. (2006). A high-resolution HLA and SNP haplotype map for disease association studies in the extended human MHC. Nat. Genet. 38, 1166–1172.
Gregersen, J. W., Kranc, K. R., Ke, X., Svendsen, P., Madsen, L. S., Thomsen, A. R., Cardon, L. R., Bell, J. I., and Fugger, L. (2006). Functional epistasis on a common MHC haplotype associated with multiple sclerosis. Nature 443, 574–577.
Guo, Z., Hood, L., Malkki, M., and Petersdorf, E. W. (2006). Long-range multilocus haplotype phasing of the MHC. Proc. Natl. Acad. Sci. USA 103, 6964–6969.
Nejentsev, S., Howson, J. M., Walker, N. M., Szeszko, J., Field, S. F., Stevens, H. E., Reynolds, P., Hardy, M., King, E., Masters, J., Hulme, J., Maier, L. M., Smyth, D., Bailey, R., Cooper, J. D., Ribas, G., Campbell, R. D., Clayton, D. G., and Todd, J. A. (2007). Localization of type 1 diabetes susceptibility to the MHC class I genes HLA-B and HLA-A. Nature 450, 887–892.