Chapter 5.pdf

The Generation of Lymphocyte Antigen Receptors A lymphocyte expresses many exact copies of a single antigen receptor that has a unique antigen-binding site (see Section 1-12). The clonal expression of antigen receptors means that each lymphocyte is unique among the billions of lymphocytes that each...

The Generation of Lymphocyte Antigen Receptors A lymphocyte expresses many exact copies of a single antigen receptor that has a unique antigen-binding site (see Section 1-12). The clonal expression of antigen receptors means that each lymphocyte is unique among the billions of lymphocytes that each person possesses. Chapter 4 described the structural features of immunoglobulins and T-cell receptors, the antigen receptors on B cells and T cells, respectively. We saw that the vast repertoire of antigen receptors results from variations in the amino acid sequence at the antigenbinding site, which is composed of the two variable regions from the two chains of the receptor. In immunoglobulins, these are the heavy-chain variable region (VH) and the light-chain variable region (VL), and in T-cell receptors, the Vα and Vβ regions. The immunoglobulin domains of these regions contain three loops that comprise three hypervariable regions, or complementaritydetermining regions (CDRs) (see Section 4-6) that determine the receptor’s antigen binding site and allow for seemingly limitless diversity in specificity. In the 1960s and 1970s, immunologists recognized that the limited size of the genome (at roughly 3 billion nucleotides) meant that the genome could not directly encode a sufficient number of genes to account for the observed diversity of antigen receptors. For example, encoding each distinct antibody by its own gene could easily fill the genome with nothing but antibody genes. As we will see, variable regions of the receptor chains are not directly encoded as a complete immunoglobulin domain by a single DNA segment. Instead, the variable regions are initially specified by so-called gene segments that encode only a part of the immunoglobulin domain. During the development of each lymphocyte, these gene segments are rearranged by a process of somatic DNA recombination to form a complete and unique variable-region coding sequence. This process is known generally as gene rearrangement. A fully assembled variable region sequence is produced by combining two or three types of gene segments, each of which is present in multiple copies in the germline genome. The final diversity of the receptor repertoire is the result of assembling complete antigen receptors from the many different gene segments of each type during the development of each individual lymphocyte. This process gives each new lymphocyte only one of many possible combinations of antigen receptors, providing the repertoire of diverse antigen specificities of naive B cells and T cells. The first and second parts of this chapter describe the gene rearrangements that generate the primary repertoire of immunoglobulins and T-cell receptors. The mechanism of gene rearrangement is common to both B cells and T cells, and its evolution was probably critical to the evolution of the vertebrate adaptive immune system. The third part of the chapter explains how the transition from production of transmembrane immunoglobulins by activated B cells results in the production of secreted antibodies by plasma cells. Immunoglobulins can be synthesized as either transmembrane receptors or secreted antibodies, unlike T-cell receptors, which exist only as transmembrane receptors. Antibodies can also be produced with different types of constant regions, or isotypes (see Section 4-1). Here, we describe how the ERRNVPHGLFRVRUJ 5 IN THIS CHAPTER Primary immunoglobulin gene rearrangement. T-cell receptor gene rearrangement. Structural variation in immunoglobulin constant regions. Evolution of the adaptive immune response. 173 174 Chapter 5: The Generation of Lymphocyte Antigen Receptors expression of the isotypes IgM and IgD is regulated, but we postpone describing how isotype switching occurs until Chapter 10, since that process and the affinity maturation of antibodies occurs normally in the context of an immune response. The last part of this chapter briefly examines alternative evolutionary forms of gene rearrangements that give rise to different forms of adaptive immunity in other species. Primary immunoglobulin gene rearrangement. Immunoglobulin variable region N HV3 (CDR3) HV1 (CDR1) C HV2 (CDR2) a N C b D E B A G F C C´ C´´ Virtually any substance can be the target of an antibody response, and the response to even a single epitope comprises many different antibody mole­ cules, each with a subtly different specificity for the epitope and a unique affinity, or binding strength. The total number of antibody specificities available to an individual is known as the antibody repertoire or immunoglobulin repertoire, and in humans is at least 1011 and probably several orders of magnitude greater. The number of antibody specificities present at any one time is, however, limited by the total number of B cells in an individual, as well as by each individual’s previous encounters with antigens. Before it was possible to examine the immunoglobulin genes directly, there were two main hypotheses for the origin of this diversity. The germline theory held that there is a separate gene for each different immunoglobulin chain and that the antibody repertoire is largely inherited. In contrast, somatic diversification theories proposed that the observed repertoire is generated from a limited number of inherited V-region sequences that undergo alteration within B cells during the individual’s lifetime. Cloning of the immunoglobulin genes revealed that elements of both theories were correct and that the DNA sequence encoding each variable region is generated by rearrangements of a relatively small group of inherited gene segments. Diversity is further enhanced by the process of somatic hypermutation in mature activated B cells. Thus, the somatic diversification theory was essentially correct, although the germline theory concept of the existence of multiple germline genes also proved true. 5-1 Variable exon region A c FR1 HV1 HV2 HV3 B C C´ C´´ D E F G FR2 FR3 FR4 Immunobiology | chapter 5 | 05_100 Fig. 5.1 Three hypervariable regions Murphy et al | Ninth edition are encoded within a single V-region © Garland Science design by blink studio limited exon. Panel a: the variable region is based on the immunoglobulin (Ig) fold that is supported by framework regions (yellow) composed of nine β sheets and contains three hypervariable (HV) regions (red) that determine its antigen specificity. Panel b: the three HV regions exist as loops of amino acids between the β sheets of B and C, between Cʹ and Cʹʹ, and between F and G. Panel c: a complete variable region in a lymphocyte is encoded within a single exon of the full antigen-receptor gene. The three HV regions are interspersed between four framework regions (FRs) made up of the β sheets of the Ig domain. Immunoglobulin genes are rearranged in the progenitors of antibody-producing cells. Figure 5.1 shows the relationships between a light-chain variable region’s antigen-binding site, its domain structure, and the gene that encodes it. The variable regions of immunoglobulin heavy and light chains are based on the immunoglobulin fold, which is composed of nine β sheets. The antibody-binding site is formed by three loops of amino acids known as hypervariable regions HV1, HV2, and HV3, or also CDR1, CDR2, and CDR3 (see Fig. 5.1a). These loops are located between the pairs of β sheets B and C, Cʹ and Cʹʹ, and F and G (see Fig. 5.1b). In a mature B cell, the variable regions for heavy and light chains are encoded by a single exon, but are separated from one another within this coding sequence (see Fig. 5.1c). This exon is the gene’s second exon (exon 2). The first exon of the variable regions encodes the antibody’s leader sequence, which directs the antibody into the endoplasmic reticulum for surface expression or secretion. Unlike most genes, the complete DNA sequence of the variable-region exon is not present in the germline of the individual, but is originally encoded by two separate DNA segments, as illustrated in Fig. 5.2. These two DNA segments are spliced together to form the complete exon 2 as the B cell develops in the bone marrow. The first 95–101 amino acids of the variable region, encoding β sheets A–F and the first two complete hypervariable regions, originate from a ERRNVPHGLFRVRUJ Primary immunoglobulin gene rearrangement. Fig. 5.2 The CDR3 region originates from two or more individual gene segments that are joined during lymphocyte development. Panel a: a complete light-chain variable region encoding the CDR1, CDR2, and CDR3 loops resides in a single exon. Panel b: the complete variable region is derived from distinct germline DNA sequences. A V gene segment encodes the CDR1 and CDR2 loops, and the CDR3 loop is formed by sequences from the end of the V gene segment and the beginning of the J gene segment, and by nucleotides added or lost when these gene segments are joined during lymphocyte development. The exon for the CDR3 loop of the heavy chain is formed by the joining of sequences from V, D, and J gene segments (not shown). variable region CDR1 CDR2 a In nonlymphoid cells, the V-region gene segments remain in their original germline configuration, and are a considerable distance away from the sequence encoding the C region. In mature B lymphocytes, however, the assembled V-region sequence lies much closer to the C region, as a consequence of a splicing event of the gene’s DNA. Rearrangement within the immunoglobulin genes was originally discovered almost 40 years ago, when the techniques of restriction enzyme analysis first made it possible to study the organization of the immunoglobulin genes in both B cells and non­lymphoid cells. Such experiments showed that segments of genomic DNA within the immunoglobulin genes are rearranged in cells of the B-lymphocyte lineage, but not in other cells. This process of rearrangement is known as ‘somatic’ DNA recombination to distinguish it from the meiotic recombination that takes place during the production of gametes. 5-2 Complete genes that encode a variable region are generated by the somatic recombination of separate gene segments. The rearrangements that produce the complete immunoglobulin light-chain and heavy-chain genes are shown in Fig. 5.3. For the light chain, the joining of a VL and a JL gene segment creates an exon that encodes the whole lightchain VL region. In the unrearranged DNA, the VL gene segments are located relatively far away from the exons encoding the constant region of the light chain (CL region). The JL gene segments are located close to the CL region, however, and the joining of a VL gene segment to a JL gene segment also brings the VL gene segment close to a CL-region sequence. The JL gene segment of the rearranged VL region is separated from a CL-region sequence only by a short intron. To make a complete immunoglobulin light-chain messenger RNA, the V-region exon is joined to the C-region sequence by RNA splicing after transcription. For the heavy-chain, there is one additional complication. The heavy-chain V region (VH) is encoded in three gene segments, rather than two. In addition to the V and J gene segments (denoted VH and JH to distinguish them from the light-chain VL and JL), the heavy chain uses a third gene segment called the diversity or DH gene segment, which lies between the VH and JH gene segments. The recombination process that generates a complete heavychain V region is shown in Fig. 5.3 (right panel), and occurs in two separate stages. First, a DH gene segment is joined to a JH gene segment; then a VH gene segment rearranges to DJH to make a complete VH-region exon. As with the light-chain genes, RNA splicing joins the assembled V-region sequence to the neighboring C-region gene. ERRNVPHGLFRVRUJ A B C C´ C´´ D E F V b variable or V gene segment (see Fig. 5.2). This segment also contributes part of the third hypervariable region. Other parts of the third hypervariable region, and the remainder of the variable region including β sheet G (up to 13 amino acids), originate from a joining or J gene segment. By convention, we will refer to the exon encoding the complete variable region formed by the splicing together of these gene segments as the V-region gene. CDR3 V gene segment Immunobiology | chapter 5 | 05_101 Murphy et al | Ninth edition © Garland Science design by blink studio limited G J J gene segment 175 Chapter 5: The Generation of Lymphocyte Antigen Receptors Light chain L V J Heavy chain C L V D L V DJ C J Germline DNA DNA Somatic recombination D–J rearranged DNA joined Somatic recombination V–J or V–DJ joined rearranged DNA L V J C L V DJ L V J C L V DJ C C Transcription RNA Primary transcript RNA C AAA AAA Splicing mRNA Translation L V J L C C V DJ AAA VL AAA CL C H2 C H3 Polypeptide chain Protein 176 VH Fig. 5.3 V-region are constructed from gene segments. Immunobiology | chaptergenes 5 | 05_001 Murphy et al | Ninth edition genes are constructed from two segments Light-chain V-region Garland Science design blink studio(V) limited © (center panel). A by variable and a joining (J) gene segment in the genomic DNA are joined to form a complete light-chain V-region exon. Immunoglobulin chains are extracellular proteins, and the V gene segment is preceded by an exon encoding a leader peptide (L), which directs the protein into the cell’s secretory pathways and is then cleaved. The light-chain C region is encoded in a separate exon and is joined to the V-region exon by splicing of the light-chain RNA to remove the L-to-V and the J-to-C introns. 5-3 C H1 Heavy‑chain V regions are constructed from three gene segments (right panel). First, the diversity (D) and J gene segments join, and then the V gene segment joins to the combined DJ sequence, forming a complete VH exon. A heavy-chain C-region gene is encoded by several exons. The C-region exons, together with the leader sequence, are spliced to the V-domain sequence during processing of the heavy-chain RNA transcript. The leader sequence is removed after translation, and the disulfide bonds that link the polypeptide chains are formed. The hinge region is shown in purple. Multiple contiguous V gene segments are present at each immunoglobulin locus. For simplicity we have discussed the formation of a complete V-region sequence as though there were only a single copy of each gene segment. In fact, there are multiple copies of the V, D, and J gene segments in germline DNA. It is the random selection of just one gene segment of each type that produces the great diversity of V regions among immunoglobulins. The numbers of functional gene segments of each type in the human genome, as determined by gene cloning and sequencing, are shown in Fig. 5.4. Not all the gene segments discovered are functional, as some have accumulated mutations that prevent them from encoding a functional protein. Such genes are termed ‘pseudogenes.’ Because there are many V, D, and J gene segments in germline DNA, no single gene segment is essential, resulting in a relatively large number of pseudogenes. Since some of these can undergo rearrangement just like a functional gene segment, a significant proportion of rearrangements incorporate a pseudogene and will thus be nonfunctional. ERRNVPHGLFRVRUJ Primary immunoglobulin gene rearrangement. We saw in Section 4-1 that there are three sets of immunoglobulin chains— the heavy chain, and two equivalent types of light chains, the κ and λ chains. The immunoglobulin gene segments that encode these chains are organized into three clusters or genetic loci—the κ, λ, and heavy-chain loci—each of which can assemble a complete V-region sequence. Each locus is on a different chromosome and is organized slightly differently, as shown for the human loci in Fig. 5.5. At the λ light-chain locus, located on human chromosome 22, a cluster of Vλ gene segments is followed by four (or in some individuals five) sets of Jλ gene segments each linked to a single Cλ gene. In the κ light-chain locus, on chromosome 2, the cluster of Vκ gene segments is followed by a cluster of Jκ gene segments, and then by a single Cκ gene. The organization of the heavy-chain locus, on chromosome 14, contains separate clusters of VH, DH, and JH gene segments and of CH genes. The heavy-chain locus differs in one important way: instead of a single C region, it contains a series of C regions arrayed one after the other, each of which corresponds to a different immunoglobulin isotype (see Fig. 5.19). While the Cλ locus contains several distinct C regions, these encode similar proteins, which function similarly, whereas the different heavy-chain isotypes are structurally quite distinct and have different functions. B cells initially express the heavy-chain isotypes μ and δ (see Section 4-1), which is accomplished by alternative mRNA splicing and which leads to the expression of immunoglobulins IgM and IgD, as we shall see in Section 5-14. The expression of other isotypes, such as γ (giving IgG), occurs through DNA rearrangements referred to as class switching, and takes place at a later stage, after a B cell is activated by antigen in an immune response. We describe class switching in Chapter 10. Number of functional gene segments in human immunoglobulin loci Light chains Segment Heavy chain κ λ H Variable (V) 34–38 29–33 38–46 Diversity (D) 0 0 23 Joining (J) 5 4–5 6 Constant (C) 1 4–5 9 Immunobiology chapter 5 | 05_002 Fig. 5.4 The| number of functional gene Murphy et al | Ninth edition segments for the V regions of human heavy and light chains. The numbers shown are derived from exhaustive cloning and sequencing of DNA from one individual and exclude all pseudogenes (mutated and nonfunctional versions of a gene sequence). As a result of genetic polymorphism, the numbers will not be the same for all people. © Garland Science design by blink studio limited The human V gene segments can be grouped into families in which each member shares at least 80% DNA sequence identity with all others in the λ light-chain locus L1 V λ 1 L2 V λ 2 Jλ 1 Cλ1 L Vλ×30 Jλ2 κ light-chain locus L1 V κ 1 L 2 Vκ 2 L3 V κ 3 J κ 1–5 C λ2 J λ4 Cλ 4 L V κ ×38 Cκ heavy-chain locus L1 V H 1 L2 L3 V H 3 VH 2 D H 1–23 Immunobiology | chapter 5 | 05_003 Murphy et al | Ninth edition © Garland Science design by blink studio limited LH VH ×40 J H 1–6 Cμ ERRNVPHGLFRVRUJ Fig. 5.5 The germline organization of the immunoglobulin heavy- and light-chain loci in the human genome. Depending on the individual, the genetic locus for the λ light chain (chromosome 22) has between 29 and 33 functional Vλ gene segments and four or five pairs of functional Jλ gene segments and Cλ genes. The κ locus (chromosome 2) is organized in a similar way, with about 38 functional Vκ gene segments accompanied by a cluster of five Jκ gene segments but with a single Cκ gene. In approximately 50% of individuals, the entire cluster of Vκ gene segments has undergone an increase by duplication (not shown, for simplicity). The heavy-chain locus (chromosome 14) has about 40 functional VH gene segments and a cluster of around 23 DH segments lying between these VH gene segments and 6 JH gene segments. The heavy-chain locus also contains a large cluster of CH genes (see Fig. 5.19). For simplicity, all V gene segments have been shown in the same chromosomal orientation; only the first CH gene (for Cμ) is shown, without illustrating its separate exons; and all pseudogenes have been omitted. This diagram is not to scale: the total length of the heavy-chain locus is more than 2 megabases (2 million bases), whereas some of the D gene segments are only 6 bases long. 177 178 Chapter 5: The Generation of Lymphocyte Antigen Receptors family. Both the heavy-chain and the κ-chain V gene segments can be subdivided into seven families, and there are eight families of Vλ gene segments. The families can be grouped into clans, made up of families that are more similar to each other than to families in other clans. Human VH gene segments fall into three clans. All the VH gene segments identified from amphibians, reptiles, and mammals also fall into the same three clans, suggesting that these clans existed in a common ancestor of these modern animal groups. Thus, the V gene segments that we see today have arisen by a series of gene duplications and diversification through evolutionary time. 5-4 Rearrangement of V, D, and J gene segments is guided by flanking DNA sequences. For a complete immunoglobulin or T-cell receptor chain to be expressed, DNA rearrangements must take place at the correct locations relative to the V, D, or J gene segment coding regions. In addition, these DNA rearrangements must be regulated such that a V gene segment is joined to a D or a J and not joined to another V gene segment. DNA rearrangements are guided by conserved noncoding DNA sequences, called recombination signal sequences (RSSs), that are found adjacent to the points at which recombination takes place. The structure and arrangements of the RSSs are shown in Fig. 5.6 for the λ and κ light-chain loci and the heavy-chain loci. An RSS consists of a conserved block of seven nucleotides—the heptamer 5ʹCACAGTG3ʹ, which is always contiguous with the coding sequence; followed by a nonconserved region known as the spacer, which is either 12 or 23 base pairs (bp) long; followed by a second conserved block of nine nucleotides, the nonamer 5ʹACAAAAACC3ʹ. Fig. 5.6 Recombination signal sequences are conserved heptamer and nonamer sequences that flank the gene segments encoding the V, D, and J regions of immunoglobulins. Recombination signal sequences (RSSs) are composed of heptamer (CACAGTG) and nonamer (ACAAAAACC) sequences that are separated by either 12 bp or approximately 23 bp of nucleotides. The heptamer–12-bp spacer–nonamer motif is depicted here as an orange arrowhead; the motif that includes the 23-bp spacer is depicted as a purple arrowhead. Joining of gene segments almost always involves a 12-bp and a 23-bp RSS—the 12/23 rule. The arrangement of RSSs in the V (red), D (green), and J (yellow) gene segments of heavy (H) and light (λ and κ) chains of immunoglobulin is shown here. The RAG-1 recombinase (see Section 5-5) cuts the DNA precisely between the last nucleotide of the V gene segment and the first C of the heptamer; or between the last G of the heptamer and the first nucleotide of the D or J gene segment. Note that according to the 12/23 rule, the arrangement of RSSs in the immunoglobulin heavy-chain gene segments precludes direct V-to-J joining. The sequences given here are the consensus sequences, but they can vary substantially from one gene segment to another, even in the same individual, as there is some flexibility in the recognition of these sequences by the enzymes that carry out the recombination. The spacers vary in sequence, but their conserved lengths correspond to one turn (12 bp) or two turns (23 bp) of the DNA double helix. This is thought to bring the heptamer and nonamer sequences to the same side of the DNA helix to allow interactions with proteins that catalyze recombination, but this concept still lacks structural proof. The heptamer– spacer–nonamer sequence motif—the RSS—is always found directly adjacent to the coding sequence of V, D, or J gene segments. Recombination normally occurs between gene segments located on the same chromosome. A gene segment flanked by an RSS with a 12-bp spacer typically can be joined only to one flanked by a 23-bp spacer RSS. This is known as the 12/23 rule. Recombination signal sequence (RSS) with 23-base-pair spacer CACAGTG heptamer 23 GTGTCAC λ chain κ chain H chain Vλ Recombination signal sequence (RSS) with 12-base-pair spacer ACAAAAACC nonamer GGTTTTTGT nonamer TGTTTTTGG CCAAAAACA 23 12 VH 23 Immunobiology | chapter 5 | 05_004 Murphy et al | Ninth edition ERRNVPHGLFRVRUJ © Garland Science design by blink studio limited Jλ RSS 12 DH 12 CACTGTG heptamer GTGACAC 12 RSS Vκ 12 23 Jκ 23 JH Primary immunoglobulin gene rearrangement. It is important to recognize that the pattern of 12- and 23-bp spacers used by the various gene segments is different between the λ, κ, and heavy-chain loci (see Fig. 5.6). Thus, for the heavy chain, a DH gene segment can be joined to a JH gene segment and a VH gene segment to a DH gene segment, but VH gene segments cannot be joined to JH gene segments directly, as both VH and JH gene segments are flanked by 23-bp spacers. However, they can be joined with a DH gene segment between them, as DH segments have 12-bp spacers on both sides (see Fig. 5.6). In the antigen-binding region of an immunoglobulin, CDR1 and CDR2 are encoded directly in the V gene segment (see Fig. 5.2). CDR3 is encoded by the additional DNA sequence that is created by the joining of the V and J gene segments for the light chain, and the V, D, and J gene segments for the heavy chain. Further diversity in the antibody repertoire can be supplied by CDR3 regions that result from the joining of one D gene segment to another D gene segment, before being joined by a J gene segment. Such D–D joining is infrequent and seems to violate the 12/23 rule, suggesting that such violations of the 12/23 rule can occur at low frequency. In humans, D–D joining is found in approximately 5% of antibodies and is the major mechanism accounting for the unusually long CDR3 loops found in some heavy chains. The mechanism of DNA rearrangement is similar for the heavy- and lightchain loci, although only one joining event is needed to generate a light-chain gene but two are required for a heavy-chain gene. When two gene segments are in the same transcriptional orientation in the germline DNA, their rearrangement involves the looping out and deletion of the DNA between them (Fig. 5.7, left panels). By contrast, when the gene segments have opposite transcriptional orientations, the rearrangement retains the intervening DNA in the chromosome but with an inverted orientation (see Fig. 5.7, right panels). This mode of recombination is less common, but it accounts for about half of all Vκ to Jκ joins in humans because the orientation of half the Vκ gene segments is opposite to that of the Jκ gene segments. 5-5 The reaction that recombines V, D, and J gene segments involves both lymphocyte-specific and ubiquitous DNAmodifying enzymes. The overall enzymatic mechanisms involved in V-region rearrangement, or V(D)J recombination, are illustrated in Fig. 5.8. Two RSSs are brought together by interactions between proteins that specifically recognize the length of the spacers and thus enforce the 12/23 rule for recombination. The DNA molecule is then precisely cleaved by endonuclease activity at two locations and is then rejoined in a different configuration. The ends of the heptamer sequences are joined in a head-to-head fashion to form a signal joint. In the majority of cases, no nucleotides are lost or added between the two heptamer sequences, creating a double-heptamer sequence 5ʹCACAGTGCACAGTG3ʹ within the DNA molecule. When the joining segments are in the same orientation, the signal joint is contained in a circular piece of extrachromosomal DNA (see Fig. 5.7, left panels), which is lost from the genome when the cell divides. The V and J gene segments, which remain on the chromosome, join to form what is called the coding joint. When the joining segments are in the opposite relative orientation to each other within the chromosome (see Fig. 5.7, right panels), the signal joint is also retained within the chromosome, and the region of DNA between the V gene segment and the RSS of the J gene segment is inverted to form the coding joint. This situation leads to rearrangement by inversion. As we shall see later, the coding joint junction is imprecise, meaning that nucleotides can be added or lost between joined segments during the rearrangement process. This imprecise nature of coding joint formation adds to the variability in the V-region sequence, called junctional diversity. ERRNVPHGLFRVRUJ MOVIE 5.1 179 180 Chapter 5: The Generation of Lymphocyte Antigen Receptors Fig. 5.7 V-region gene segments are joined by recombination. Top panel: in every V-region recombination event, the recombination signal sequences (RSSs) flanking the gene segments are brought together to allow recombination to take place. The 12-bp-spaced RSSs are shown in orange, the 23-bp-spaced RSSs in purple. For simplicity, the recombination of a light-chain gene is illustrated; for a heavychain gene, two separate recombination events are required to generate a functional V region. Left panels: in most cases, the two segments undergoing rearrangement (the V and J gene segments in this example) are arranged in the same transcriptional orientation in the chromosome, and juxtaposition of the RSSs results in the looping out of the intervening DNA. Recombination occurs at the ends of the heptamer sequences in the RSSs, creating the so-called signal joint and releasing the intervening DNA in the form of a closed circle. Subsequently, the joining of the V and J gene segments creates the coding joint in the chromosomal DNA. Right panels: in other cases, the V and J gene segments are initially oriented in opposite transcriptional directions. In this case, alignment of the RSSs requires the coiled configuration shown, rather than a simple loop, so that joining the ends of the two heptamer sequences now results in the inversion and integration of the intervening DNA into a new position on the chromosome. Again, the joining of the V and J segments creates a functional V-region exon. V gene segments may be in either forward or reverse transcriptional orientation relative to downstream gene segments Vn 23 23 L1 V1 Ln J 23 12 L2 V2 When a forward-oriented V gene segment recombines with a downstream gene segment, alignment of the two RSS regions loops out the intervening DNA When a reverse-oriented V gene segment recombines with a downstream gene segment, alignment of the RSS regions forms the intervening DNA into a coiled configuration L1 V1 L2 V2 Vn L2 Ln V2 J V1 Vn J Ln L1 After recombination this loop is excised from the chromosome, taking the two RSS regions with it deleted After recombination the coiled region is retained in the chromosome in an inverted orientation L1 V1 L2 V2 Vn L2 Ln V2 signal joint direction of transcription direction of transcription L1 V1 J coding joint inverted Ln Vn J Immunobiology | chapter 5 | 05_005 Murphy et al | Ninth edition © Garland Science design by blink studio limited The complex of enzymes that act in concert to carry out somatic V(D)J recombination is termed the V(D)J recombinase. The lymphoid-specific components of the recombinase are called RAG-1 and RAG-2, and they are encoded by two recombination-activating genes, RAG1 and RAG2. This pair of genes is essential for V(D)J recombination, and they are expressed in developing lymphocytes only while the lymphocytes are engaged in assembling their antigen receptors, as described in more detail in Chapter 8. Indeed, the RAG genes expressed together can confer on nonlymphoid cells such as fibroblasts the capacity to rearrange exogenous segments of DNA containing the appropriate RSSs; this is how RAG-1 and RAG-2 were initially discovered. ERRNVPHGLFRVRUJ Primary immunoglobulin gene rearrangement. Germline configuration V 23 12 RAG-1:2 binds RSS Synapsis of two RSSs J Cleavage of RSSs RAG-1/2 covalently closed DNA hairpin ends Coding joints Signal joints Ku70:Ku80 binds DNA ends Ku70:Ku80 binds DNA ends Ku70 Ku80 Ku80 Ku70 5´-phosphorylated blunt ends DNA-PK:Artemis opens hairpin Artemis DNA-PK TdT processes DNA ends terminal deoxynucleotidyl transferase (TdT) DNA ligase IV:XRCC4 ligates DNA ends DNA ligase IV:XRCC4 ligates DNA ends DNA ligase:XRCC4 DNA ligase:XRCC4 Imprecise coding joint Precise signal joint Immunobiology | chapter 5 | 05_006 Murphy et al | Ninth edition © Garland Science design by blink studio limited ERRNVPHGLFRVRUJ Fig. 5.8 Enzymatic steps in RAGdependent V(D)J rearrangement. Recombination of gene segments containing recombination signal sequences (RSSs, triangles) begins with the binding of a complex of RAG-1 (purple), RAG-2 (blue), and high-mobility-group (HMG) proteins (not shown) to one of the RSSs flanking the coding sequences to be joined (second row). The RAG complex then recruits the other RSS. In the cleavage step, the endonuclease activity of RAG makes single-stranded cuts in the DNA backbone precisely between each coding segment and its RSS. At each cutting point this creates a 3ʹ-OH group, which then reacts with a phosphodiester bond on the opposite DNA strand to generate a hairpin, leaving a blunt double-stranded break at the end of the RSS. These two types of DNA ends are resolved in different ways. At the coding ends (left panels), essential repair proteins such as Ku70:Ku80 (green) bind to the hairpin. Ku70:80 forms a ringlike structure as a heterodimer, but the monomers do not encircle the DNA. The DNA-PK:Artemis complex (purple) then joins the complex, and its endonuclease activity opens the DNA hairpin at a random site, yielding either two flush-ended DNA strands or a single-strand extension. The cut end is then modified by terminal deoxynucleotidyl transferase (TdT, pink) and exonuclease, which randomly add and remove nucleotides, respectively (this step is shown in more detail in Fig. 5.11). The two coding ends are finally ligated by DNA ligase IV in association with XRCC4 (turquoise). At the signal ends (right panels), Ku70:Ku80 binds to the RSS but the ends are not further modified. Instead, a complex of DNA ligase IV:XRCC4 joins the two ends precisely to form the signal joint. 181 182 Chapter 5: The Generation of Lymphocyte Antigen Receptors The other proteins in the recombinase complex are members of the ubiquitously expressed nonhomologous end joining (NHEJ) pathway of DNA repair known as double-strand break repair (DSBR). In all cells, this process is responsible for rejoining the two ends at the site of a double-strand break in DNA. The DSBR joining process is imprecise, meaning that nucleotides are frequently gained or lost at the site of joining. This has evolutionary relevance as in most cells it would not be advantageous to gain or lose nucleotides when repairing DSBs. However, in lymphocytes, the imprecise nature of DSBR is critical for junctional diversity and adaptive immunity. Thus, this may be the driving pressure for NHEJ to mediate imprecise joining. One ubiquitous protein contributing to DSBR is Ku, which is a heterodimer (Ku70:Ku80); this forms a ring around the DNA and associates tightly with a protein kinase catalytic subunit, DNA-PKcs, to form the DNA-dependent protein kinase (DNA-PK). Another protein that associates with DNA-PKcs is Artemis, which has nuclease activity. The DNA ends are finally joined together by the enzyme DNA ligase IV, which forms a complex with the DNA repair protein XRCC4. DNA polymerases μ and λ participate in DNA-end fill-in synthesis. In addition, polymerase μ can add nucleotides in a template-independent manner. In summary, lymphocytes have adapted several enzymes used in common DNA repair pathways to help complete the process of somatic V(D)J recombination that is initiated by the RAG-1 and RAG-2 V(D)J recombinases. Crystal structure of RAG-1:RAG-2 complex RAG-2 RAG-2 Zn2+ Zn2+ RAG-1 RAG-1 NBD NBD Immunobiology | chapter 5 | 05_102 Murphy et al | Ninth edition © Garland Science design by blink studio limited The first reaction is an endonucleolytic cleavage that requires the coordinated activity of both RAG proteins. Initially, a complex of RAG-1 and RAG-2 proteins, together with high-mobility group chromatin protein HMGB1 or HMGB2, recognizes and aligns the two RSSs that are the target of the cleavage reaction. RAG-1 operates as a dimer, with RAG-2 acting as a cofactor (Fig. 5.9). RAG-1 specifically recognizes and binds the heptamer and the nonamer of the RSS and contains the Zn2+-dependent endonuclease activity of the RAG protein complex. As a dimer, RAG-1 seems to align the two RSSs that will undergo rearrangement. Recent models suggest that the 12/23 rule may be established because an essential asymmetric orientation of the RAG-1:RAG-2 complex induces a preference for binding to RSS elements of different types (Fig. 5.10). The bound RAG complex makes a single-strand DNA break at the nucleotide just 5ʹ of the heptamer of the RSS, thus creating a free 3ʹ-OH group at the end of the coding segment. This nucleophilic 3ʹ-OH group immediately attacks the phosphodiester bond on the opposite DNA strand, making a double-strand break and creating a DNA ‘hairpin’ at the coding region and a flush doublestrand break at the end of the heptamer sequence. This cutting process occurs twice, once for the each gene segment being joined, producing four ends: two hairpin ends at the coding regions and two flush ends at both heptamer sequences (see Fig. 5.8). These DNA ends do not float apart, however, but are held tightly in the complex until a joining step has been completed. The blunt ends of the heptamer sequence are precisely joined by a complex of DNA ligase IV and XRCC4 to form the signal joint. Formation of the coding joint is more complex. The two coding hairpin ends are each bound by Ku, which recruits the DNA-PKcs subunit. Artemis is recruited into this complex and is phosphorylated by DNA-PK. Artemis then opens the DNA hairpins by making a single-strand nick in the DNA. This nicking can happen at various points along the hairpin, which leads to sequence variability in the final joint. The DNA repair enzymes in the complex modify the opened Fig. 5.9 RAG-1 and RAG-2 form a heterotetramer capable of binding to two RSSs. Shown as ribbon diagrams, the RAG-1:RAG-2 complex contains two RAG-1 (green and blue) and two RAG-2 proteins (purple). The first 383 amino acids of RAG-1 were truncated before crystallization. The N-terminal nonamer binding domain (NBD) of the two RAG-1 proteins undergoes domain swapping and mediates dimerization of the two proteins. The remainder of the RAG-1 protein contains the endonuclease activity that is dependent on the binding of a Zn2+ ion. Each RAG-1 protein binds a separate RAG-2 protein. Courtesy of Martin Gellert. ERRNVPHGLFRVRUJ Primary immunoglobulin gene rearrangement. A flexible hinge connects the RAG-1 NBD domain to the remainder of the molecule RAG-2 Zn2+ V region RAG-2 J region Zn2+ RAG-1 flexible hinge A 12-bp RSS bound to one RAG-1 favors binding of a 23-bp RSS to the other RAG-1 Zn2+ RAG-1 12-bp RSS nonamer binding domains (NBD) Zn2+ heptamer 23-bp RSS nonamer Immunobiology | chapter 5 | 05_103 Murphy et al | Ninth hairpins by edition removing nucleotides, while at the same time the lymphoidspecific enzyme terminal deoxynucleotidyl transferase (TdT), which is also part of the recombinase complex, adds nucleotides randomly to the singlestrand ends. Addition and deletion of nucleotides can occur in any order, and one does not necessarily precede the other. Finally, DNA ligase IV joins the processed ends together, thus reconstituting a chromosome that includes the rearranged gene. This repair process creates diversity in the joint between gene segments while ensuring that the RSS ends are ligated without modification and that unintended genetic damage such as a chromosome break is avoided. Despite the use of some ubiquitous mechanisms of DNA repair, adaptive immunity based on the RAG-mediated generation of antigen receptors by somatic recombination seems to be unique to the jawed vertebrates, and its evolution is discussed in the last part of this chapter. © Garland Science design by blink studio limited Fig. 5.10 The 12/23 base pair rule may result from asymmetric binding of RSSs to the RAG-1:RAG-2 dimer. Left panel: This cartoon of the structure shown in Fig 5.9 illustrates the flexibility of the hinge connecting the NBD to the catalytic domain of RAG-1. Right panel: the NBD domain of RAG-1 interacts with the RSS nonamer sequence (blue), while the RSS heptamer sequence (red) is bound to the portion of RAG-1 that contains the Zn2+ endonuclease activity. In this cartoon model, the interaction of a 12-bp RSS with one of the RAG-1 subunits induces the NBD domain to rotate toward the catalytic domain of RAG-1, to accommodate the length of the RSS. Since the two NBD domains are coupled by domain swaps, this induced conformation pulls the other NBD away from its RAG-1 subunit, which then prefers binding of the 23-bp RSS. The endonucleolytic cleavage (arrows) of the DNA by RAG-1 occurs precisely at the junction between the heptamer and the respective V, D, or J gene segment. The in vivo roles of the enzymes involved in V(D)J recombination have been established through both natural and artificially induced mutations. Mice lacking TdT have about 10% of the normal level of non-templated nucleotides added to the joints between gene segments. This small remainder may result from the template-independent activity of DNA polymerase μ. Mice in which either of the RAG genes has been inactivated, or which lack DNA-PKcs, Ku, or Artemis, suffer a complete block in lymphocyte development at the gene-rearrangement stage or make only trivial numbers of B and T cells. They are said to suffer from severe combined immune deficiency (SCID). The original scid mutation was discovered some time before the components of the recombination pathway were identified and was subsequently identified as a mutation in DNA-PKcs. In humans, mutations in RAG1 or RAG2 that result in partial V(D)J recombinase activity are responsible for an inherited disorder called Omenn syndrome, which is characterized by an absence of circulating B cells and an infiltration of skin by activated oligoclonal T lymphocytes. Mice deficient in components of ubiquitous DNA repair pathways, such as DNA-PKcs, Ku, or Artemis, are defective in double-strand break repair in general and are therefore also hypersensitive to ionizing radiation (which produces double-strand breaks). Defects in Artemis in humans produce a combined immunodeficiency of B and T cells that is associated with increased radiosensitivity. SCID caused by mutations in DNA repair pathways is called irradiation-sensitive SCID (IR-SCID) to distinguish it from SCID due to lymphocyte-specific defects. Another genetic condition in which radiosensitivity is associated with some degree of immunodeficiency is ataxia telangiectasia, which is due to mutations in the protein kinase ATM (ataxia telangiectasia mutated), which are also associated with cerebellar degeneration and increased radiation sensitivity ERRNVPHGLFRVRUJ X-linked Severe Combined Immunodeficiency Ataxia Telangiectasia 183 184 Chapter 5: The Generation of Lymphocyte Antigen Receptors Omenn Syndrome and cancer risk. ATM is a serine/threonine kinase, like DNA-PKcs, and functions during V(D)J recombination by activating pathways that prevent the chromosomal translocations and large DNA deletions that can sometimes occur during resolution of DNA double-strand breaks. Some V(D)J recombination can occur in the absence of ATM, since the immune deficiencies seen in ataxia telangiectasia, which include low numbers of B and T cells and/or a deficiency in antibody class switching, are variable in their severity and are less severe than in SCID. Evidence that ATM and DNA-PKcs are partially redundant in their functions comes from the observation that B cells lacking both kinases show much more severely abnormal signal joining sequences compared with B cells lacking either enzyme alone. 5-6 The diversity of the immunoglobulin repertoire is generated by four main processes. The gene rearrangements that combine gene segments to form a complete V-region exon generate diversity in two ways. First, there are multiple different copies of each type of gene segment, and different combinations of gene segments can be used in different rearrangement events. This combinatorial diversity is responsible for a substantial part of the diversity of V regions. Second, junctional diversity is introduced at the joints between the different gene segments as a result of the addition and subtraction of nucleotides by the recombination process. A third source of diversity is also combinatorial, arising from the many possible different combinations of heavy- and light-chain V regions that pair to form the antigen-binding site in the immunoglobulin molecule. The two means of generating combinatorial diversity alone could give rise, in theory, to approximately 1.9 × 106 different antibody molecules, as we will see below. Coupled with junctional diversity, it is estimated that at least 1011 different receptors could make up the repertoire of receptors expressed by naive B cells, and diversity could even be several orders of magnitude greater, depending on how one calculates junctional diversity. Finally, somatic hypermutation, which we describe in Chapter 10, occurs only in B cells after the initiation of an immune response and introduces point mutations into the rearranged V-region genes. This process generates further diversity in the antibody repertoire that can be selected for enhanced binding to antigen. 5-7 The multiple inherited gene segments are used in different combinations. There are multiple copies of the V, D, and J gene segments, each of which can contribute to an immunoglobulin V region. Many different V regions can therefore be made by selecting different combinations of these segments. For human κ light chains, there are approximately 40 functional Vκ gene segments and 5 Jκ gene segments, and thus potentially 200 different combinations of complete Vκ regions. For λ light chains there are approximately 30 functional Vλ gene segments and 4 to 5 Jλ gene segments, yielding at least 120 possible Vλ regions (see Fig. 5.4). So, in all, 320 different light chains can be made as a result of combining different light-chain gene segments. For the heavy chains of humans, there are 40 functional VH gene segments, approximately 25 DH gene segments, and 6 JH gene segments, and thus around 6000 different possible VH regions (40 × 25 × 6 = 6000). During B-cell development, rearrangement at the heavy-chain gene locus to produce a heavy chain is followed by several rounds of cell division before light-chain gene rearrangement takes place, resulting in the same heavy chain being paired with different light chains in different cells. Because both the heavy- and the light-chain V regions contribute to antibody specificity, each of the 320 different light chains could be combined with each of the approximately 6000 heavy chains to give around 1.9 × 106 different antibody specificities. ERRNVPHGLFRVRUJ Primary immunoglobulin gene rearrangement. This theoretical estimate of combinatorial diversity is based on the number of germline V gene segments contributing to functional antibodies (see Fig. 5.4); the total number of V gene segments is larger, but the additional gene segments are pseudogenes and do not appear in expressed immunoglobulin molecules. In practice, combinatorial diversity is likely to be less than one might expect from the calculations above. One reason is that not all V gene segments are used at the same frequency; some are common in antibodies, while others are found only rarely. This bias for or against certain V gene segments relates to their proximity with intergenic control regions within the heavy-chain locus that activate V(D)J recombination in developing B cells. Also, not every heavy chain can pair with every light chain: certain combinations of VH and VL regions will not form a stable molecule. Cells in which heavy and light chains fail to pair may undergo further light-chain gene rearrangement until a suitable chain is produced or they will be eliminated. Nevertheless, it is thought that most heavy and light chains can pair with each other, and that this type of combinatorial diversity has a major role in forming an immuno­globulin repertoire with a wide range of specificities. 5-8 RSSs brought together D Variable addition and subtraction of nucleotides at the junctions between gene segments contributes to the diversity of the third hypervariable region. As noted earlier, of the three hypervariable loops in an immunoglobulin chain, CDR1 and CDR2 are encoded within the V gene segment. CDR3, however, falls at the joint between the V gene segment and the J gene segment, and in the heavy chain it is partly encoded by the D gene segment. In both heavy and light chains, the diversity of CDR3 is significantly increased by the addition and deletion of nucleotides at two steps in the formation of the junctions between gene segments. The added nucleotides are known as P-nucleotides and N-nucleotides, and their addition is illustrated in Fig. 5.11. P-nucleotides are so called because they make up palindromic sequences added to the ends of the gene segments. As described in Section 5-5, the RAG proteins generate DNA hairpins at the coding ends of the V, D, or J segments, after which Artemis catalyzes a single-stranded cleavage at a random point within the coding sequence but near where the hairpin was first formed. When this cleavage occurs at a different point from the initial break induced by the RAG1/2 complex, a single-stranded tail is formed from a few nucleotides of the coding sequence plus the complementary nucleotides from the other DNA Fig. 5.11 The introduction of P- and N-nucleotides diversifies the joints between gene segments during immunoglobulin gene rearrangement. The process is illustrated for a DH to JH rearrangement (first panel); however, the same steps occur in VH to DH and in VL to JL rearrangements. After formation of the DNA hairpins (second panel), the two heptamer sequences are ligated to form the signal joint (not shown here), while the Artemis:DNA-PK complex cleaves the DNA hairpin at a random site (indicated by the arrows) to yield a single-stranded DNA end (third panel). Depending on the site of cleavage, this single-stranded DNA may contain nucleotides that were originally complementary in the double-stranded DNA and which therefore form short DNA palindromes, such as TCGA and ATAT, as indicated by the light blue-shaded box. For example, the sequence GA at the end of the D segment shown is complementary to the preceding sequence TC. Such stretches of nucleotides that originate from the complementary strand are known as P-nucleotides. Where the enzyme terminal deoxynucleotidyl transferase (TdT) is present, nucleotides are added at random to the ends of the single-stranded segments (fourth panel); these nontemplated, or N, nucleotides are indicated by the shaded box. The two single-stranded ends then pair (fifth panel). Exonuclease trimming of unpaired nucleotides (sixth panel) and repair of the coding joint by DNA synthesis and ligation (bottom panel) leaves both P- and N-nucleotides (indicated by light blue shading) in the final coding joint. The randomness of insertion of P- and N-nucleotides makes an individual P–N region virtually unique and a valuable marker for following an individual B-cell clone as it develops, for instance in studies of somatic hypermutation. ERRNVPHGLFRVRUJ T C C A C A G T G A G G T G T C A C C A C T G T G T A G T G A C A C A T J RAG complex generates DNA hairpin at coding ends D T C T A A G A T J Artemis:DNA-PK complex opens DNA hairpins, generating palindromic P-nucleotides D T C G A A T A T J N-nucleotide additions by TdT D T C G A C T C A T A G C G A T A T J Pairing of strands D T C G A C T C T A A G C G A T A T J Unpaired nucleotides are removed by an exonuclease D T C G A C T C A G C G A T A T J The gaps are filled by DNA synthesis and ligation to form coding joint D T C G A C T C G C T A T A A G C T G A G C G A T A T P N P Immunobiology | chapter 5 | 05_007 Murphy et al | Ninth edition © Garland Science design by blink studio limited J 185 186 Chapter 5: The Generation of Lymphocyte Antigen Receptors strand (see Fig. 5.11). In many light-chain gene rearrangements, DNA repair enzymes then fill in complementary nucleotides on the single-stranded tails, which would leave short palindromic sequences (the P-nucleotides) at the joint if the ends were rejoined without any further exonuclease activity. In heavy-chain gene rearrangements and in a proportion of human lightchain gene rearrangements, however, N-nucleotides are added by a quite different mechanism before the ends are rejoined. N-nucleotides are so called because they are non-template-encoded. They are added by the enzyme TdT to the single-stranded ends of the coding DNA after hairpin cleavage. After the addition of up to 20 nucleotides, single-stranded stretches may have some complementary base pairs. Repair enzymes then trim off nonmatching nucleotides, synthesize complementary DNA to fill in the remaining singlestranded gaps, and ligate the new DNA to the palindromic region (see Fig. 5.11). TdT is maximally expressed during the period in B-cell development when the heavy-chain gene is being assembled, and so N-nucleotides are common in heavy-chain V–D and D–J junctions. N-nucleotides are less common in lightchain genes, which undergo rearrangement after heavy-chain genes, when TdT expression has been shut off, as we will explain further in Chapter 8 when discussing the specific developmental stages of B and T cells. Nucleotides can also be deleted at gene segment junctions. This is accomplished by exonucleases, and although these have not yet been identified, Artemis has dual endonuclease and exonuclease activity and so could well be involved in this step. Thus, a heavy-chain CDR3 can be shorter than even the smallest D segment. In some instances it is difficult, if not impossible, to recognize the D segment that contributed to CDR3 formation because of the excision of most of its nucleotides. Deletions may also erase the traces of P-nucleotide palindromes introduced at the time of hairpin opening. For this reason, many completed VDJ joins do not show obvious evidence of P-nucleotides. As the total number of nucleotides added by these processes is random, the added nucleotides often disrupt the reading frame of the coding sequence beyond the joint. Such frameshifts will lead to a nonfunctional protein, and DNA rearrangements leading to such disruptions are known as nonproductive rearrangements. As roughly two in every three rearrangements will be nonproductive, many B-cell progenitors never succeed in producing functional immunoglobulin and therefore never become mature B cells. Thus, junctional diversity is achieved only at the expense of considerable loss of cells during B-cell development. In Chapter 8, we return to this topic when we discuss the cellular stages of B-cell development and how they relate to the temporal sequence of rearrangement of the V, D, and J gene segments of the antigen receptor chains. Summary. The extraordinary diversity of the immunoglobulin repertoire is achieved in several ways. Perhaps the most important factor enabling this diversity is that V regions are encoded by separate gene segments (V, D, and J gene segments), which are brought together by a somatic recombination process— V(D)J recombination—to produce a complete V-region exon. Many different gene segments are present in the genome of an individual, thus providing a heritable source of diversity that this combinatorial mechanism can use. Unique lymphocyte-specific recombinases, the RAG proteins, are absolutely required to catalyze this rearrangement, and the evolution of RAG proteins coincided with the appearance of the modern vertebrate adaptive immune system. Another substantial fraction of the functional diversity of immunoglobulins comes from the imprecise nature of the joining process itself. Variability at the coding joints between gene segments is generated by the insertion of random numbers of P- and N-nucleotides and by the variable deletion of nucleotides at the ends of ERRNVPHGLFRVRUJ T-cell receptor gene rearrangement. some segments. These are brought about by the random opening of the hairpin by Artemis and by the actions of TdT. The association of different light- and heavy-chain V regions to form the antigen-binding site of an immunoglobulin molecule contributes further diversity. The combination of all of these sources of diversity generates a vast primary repertoire of antibody specificities. T-cell receptor gene rearrangement. The mechanism by which B-cell antigen receptors are generated is such a powerful means of creating diversity that it is not surprising that the antigen receptors of T cells bear structural resemblances to immunoglobulins and are generated by the same mechanism. In this part of the chapter we describe the organization of the T-cell receptor loci and the generation of the genes for the individual T-cell receptor chains. 5-9 The T-cell receptor gene segments are arranged in a similar pattern to immunoglobulin gene segments and are rearranged by the same enzymes. Like immunoglobulin light and heavy chains, T-cell receptor (TCR) α and β chains each consist of a variable (V) amino-terminal region and a constant (C) region (see Section 4-10). The organization of the TCRα and TCRβ loci is shown in Fig. 5.12. The organization of the gene segments is broadly homo­ logous to that of the immunoglobulin gene segments (see Sections 5-2 and 5-3). The TCRα locus, like the loci of the immunoglobulin light chains, contains V and J gene segments (Vα and Jα). The TCRβ locus, like the locus of the immuno­globulin heavy chain, contains D gene segments in addition to Vβ and Jβ gene segments. The T-cell receptor gene segments rearrange during T-cell development to form complete V-domain exons (Fig. 5.13). T-cell receptor gene rearrangement takes place in the thymus; the order and regulation of the rearrangements are dealt with in detail in Chapter 8. Essentially, however, the mechanics of gene rearrangement are similar for B and T cells. The T-cell receptor gene segments are flanked by 12-bp and 23-bp spacer recombination signal sequences (RSSs) that are homologous to those flanking immunoglobulin α-chain locus L1 Vα1 L3 Vα3 L2 Vα2 LVα ×70 – 80 Cα Jα x 61 β-chain locus L1 Vβ1 L3 Vβ3 L2 Vβ2 Dβ1 Jβ1 x 6 Immunobiology | chapter 5 | 05_008 Murphy et al | Ninth edition © Garland Science design by blink studio limited L Vβ ×52 Cβ1 Dβ2 Jβ2 x 7 Cβ2 ERRNVPHGLFRVRUJ Fig. 5.12 The germline organization of the human T-cell receptor α and β loci. The arrangement of the gene segments for the T-cell receptor resembles that at the immunoglobulin loci, with separate variable (V), diversity (D), and joining (J) gene segments, and constant (C) genes. The TCRα locus (chromosome 14) consists of 70–80 Vα gene segments, each preceded by an exon encoding the leader sequence (L). How many of these Vα gene segments are functional is not known exactly. A cluster of 61 Jα gene segments is located a considerable distance from the Vα gene segments. The Jα gene segments are followed by a single C gene, which contains separate exons for the constant and hinge domains and a single exon encoding the transmembrane and cytoplasmic regions (not shown). The TCRβ locus (chromosome 7) has a different organization, with a clus