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Genetics of Antibody Diversity and Function

Genetics of Antibody Diversity and Function
Antibody genes are produced by somatic recombination
The immunoglobulin repertoire is encoded for by multiple germline gene segments that undergo somatic diversification in developing B‐cells. Hence, although the basic components needed to generate an immunoglobulin repertoire are inherited, an individual’s mature antibody repertoire is essentially formed during their lifetime by alteration of the inherited germline genes. The first evidence that immunoglobulin genes rearrange by somatic recombination was reported by Hozumi and Tonegawa in 1976 (Milestone 3.2). Because somatic recombination involves rearrangement of DNA in somatic rather than gamete cells, the newly recombined genes are not inherited. As a result, the primary immunoglobulin repertoire will differ slightly from one individual to the next, and will be further modified during an individual’s lifetime by their exposure to different antigens.

Milestone 3.2 The 1987 Nobel Prize in Physiology or Medicine
Susumu Tonegawa was awarded the 1987 Nobel Prize in Physiology or Medicine for “his discovery of the genetic principle for generation of antibody diversity.” In his 1976 paper, Tonegawa used Southern blot analysis of restriction enzyme digested DNA from lymphoid and nonlymphoid cells to show that the immunoglobulin variable and constant genes are distant from each other in the germline genome. Embryo DNA showed two components when hybridized to RNA probes specific for: (i) both variable and constant regions and (ii) only the constant region, whereas both probes localized to a single band when hybridized to DNA from an antibody‐ producing plasmacytoma cell. He proposed that the differential hybridization patterns could be explained if the variable and constant genes were distant from each other in germline DNA, but came together to encode the complete immunoglobulin gene during lymphocyte differentiation.

Figure 3.20 The human immunoglobulin loci. Schematics of the human heavy chain (top) and light chain lambda (middle) and kappa (bottom) loci are shown. The human heavy chain locus on chromosome 14 consists of approximately 40 functional VH genes, 23 DH genes, and 6 JH genes, which are organized into clusters upstream of the constant regions. The human lambda locus on chromosome 22 consists of approximately 30 functional Vλ genes and 5 functional Jλ gene segments, with each J segment followed by a constant segment. The human kappa locus on chromosome 2 consists of about 40 functional Vκ genes and 5 functional Jκ genes, with the J segments clustered upstream of the constant region. L, leader sequence.

The immunoglobulin variable gene segments and loci
The variable light and heavy chain loci in humans contain multiple gene segments, which are joined, using somatic recombination, to produce the final V region exon. The human heavy chain variable region is constructed from the joining of three gene segments, V (variable), D (diversity), and J (joining), whereas the light chain variable gene is constructed by the joining of two gene segments, V and J. There are multiple V, D, and J segments at the heavy chain and light chain loci, as illustrated in Figure 3.20.
The human VH genes have been mapped to chromosome 14, although orphan IgH genes have also been identified on chromosomes 15 and 16. The human VH locus, as for other antibody gene segments, is highly polymorphic, and has likely evolved through the repeated duplication, deletion, and recom­ bination of DNA. Polymorphisms found within the germline repertoire are due to the insertion or deletion of gene segments or the occurrence of different alleles of the same segment. A number of pseudogenes, ranging from those that are more conserved and contain a few point mutations to those that are more divergent with extensive mutations, are also present in immunoglobulin loci. There are approximately 100 human VH genes, which can be grouped into seven families based on sequence homology. Members of a given family show approxi­ mately 80% sequence homology at the nucleotide level. The functional heavy chain repertoire is formed from approxi­ mately 40 functional VH genes, 23 DH genes and 6 JH genes. The human lambda locus maps to chromosome 22, with approximately 30 functional Vλ genes and 5 functional Jλ gene segments. The Vλ genes can be grouped into 10 families. The human kappa locus on chromosome 2 is composed of a total of approximately 40 functional Vk genes and 5 functional Jk genes. However, the kappa locus contains a large duplication of most of the Vk genes, and most of the Vk genes in this distal cluster, although functional, are seldom used. The numbers of V genes vary between individuals as a result of polymorphisms.
The immunoglobulin loci also contain regulatory elements (Figure 3.21) including enhancers at the 3′ end of each locus and also in between the J and C regions (intronic enhancer) of the IGH and IGK loci. Both 3′ and intronic enhancers are important for V(D)J recombination, whereas the 3′ enhancers are more important for the efficient transcription of rearranged Ig genes. Some Ig loci have additional enhancer elements. Each Ig V gene has its own leader sequence and a simple promoter that contains a conserved octamer motif and a TATA box.
Regulatory elements of immunoglobulin loci
Figure 3.21 Regulatory elements of immunoglobulin loci. Each VDJ segment encoding the variable region is associated with a leader sequence. Closely upstream is the TATA box of the promoter, which binds RNA polymerase II, and the octamer motif that is one of a number of short sequences that bind transacting regulatory transcription factors. The V region promoters are relatively inactive and only association with enhancers, which are also composites of short sequence motifs capable of binding nuclear proteins, will increase the transcription rate to levels typical of actively secreting B‐cells. Primary transcripts are initiated 20 nucleotides downstream of the TATA box and extend beyond the end of the constant region. These are spliced, cleaved at the 3′ end and polyadenylated to generate the translatable mRNA.

Overview of V(D)J recombination
Figure 3.22 Overview of V(D)J recombination. Diversity (D) and joining (J) gene segments in the germline DNA are joined together through somatic recombination at the heavy chain locus. The variable (V) gene segment is then joined to the recombined D–J gene to produce the fully recombined heavy chain exon. At the light chain loci, somatic recombination occurs with V and J segments only. The recombined DNA is transcribed, and the primary RNA transcript is then spliced, bringing together the V and constant (C) regions. The spliced mRNA molecule is translated to produce the immunoglobulin protein. The contribution of the different gene segments to the polypeptide sequence is illustrated for one of the heavy chains. H, hinge.

V(D)J recombination and combinatorial diversity
The joining of these gene segments, illustrated in Figure 3.22, is known as V(D)J recombination. V(D)J recombination is a highly regulated and ordered event. The light chain exon is constructed from a single V‐to‐J gene segment join. However, at the heavy chain locus, a D segment is first joined to a J segment, and then the V segment is joined to the combined DJ sequence. The rearranged DNA is transcribed, the RNA transcript is spliced to bring together the V region exon and the C region exon, and lastly the spliced mRNA is translated to produce the final immunoglobulin protein.
Numerous unique immunoglobulin genes can be made by joining different combinations of the V, D, and J segments at the heavy and light chain loci. The creation of diversity in the immunoglobulin repertoire through this joining of various gene segments is known as combinatorial diversity. Additional diversity is created by the pairing of different heavy chains with different lambda or kappa light chains. For example, the potential heavy chain repertoire is very approximately 40 VH × 23 DH × 6 JH = 5500 different combinations. Similarly, there are very approximately 150 (30 Vλ× 5 Jλ) and 200 (40 Vk × 5 Jk) different combinations, for a total of 350 light chain combinations. If we consider that each heavy chain could potentially pair with each light chain, then the diversity of the immunoglobulin repertoire would be quite large, on the order of 2 million possible combinations. However, V genes rearrange at very different frequencies, so there is enormous variation in the likelihood of different combinations. Additional diversity is also generated during gene segment recombination and by somatic hypermutation, as explained in the following sections. In this manner, although the number of germline gene segments appears limited in size, an incredibly diverse immunoglobulin repertoire can be generated.
The recombination signal sequence
Figure 3.23 The recombination signal sequence. The recombination signal sequence (RSS) is made up of conserved heptamer and nonamer sequences, separated by an unconserved 12‐ or 23‐nucleotide spacer. Efficient recombination occurs between segments with a 12‐nucleotide spacer and a 23‐nucleotide spacer. RSSs with 23‐nucleotide spacers flank the V and J segments of the heavy chain locus, the J segments of the kappa locus and the V segments of the lambda locus, whereas RSSs with 12‐nucleotide spacers flank the D segments of the heavy chain locus, the V segments of the kappa locus and the J segments of the lambda locus.

Recombination signal sequences
The recombination signal sequence (RSS) helps to guide recombination between appropriate gene segments. The RSS (Figure 3.23) is a noncoding sequence that flanks coding gene segments. It is made up of a conserved heptamer and nonamer sequences, which are separated by an unconserved 12‐ or 23‐nucleotide spacer. Efficient recombination occurs between segments with a 12‐nucleotide spacer and a 23‐nucleotide spacer. This “12/23” rule helps make certain that appropriate gene segments are joined together.
At the VH locus, the V and J segments are flanked by RSSs with a 23‐nucleotide spacer, whereas the D segments are flanked by RSSs with a 12‐nucleotide spacer. At light chain loci, the Vk segments are flanked by RSSs with 12‐nucleotide spacers, Jk segments are flanked by RSSs with 23‐nucleotide spacers, and this arrangement is reversed in the lambda locus.
The V(D)J recombinase
Figure 3.24 The V(D)J recombinase. In the initial steps of V(D)J recombination, the RAG‐1 and RAG‐2 proteins associate with the recombination signal sequences. A single‐stranded nick is then introduced between the 5′‐heptameric end of the recombination signal sequence and the coding segment, giving rise to a free 3′‐OH group that mediates a transesterification reaction. This reaction leads to the formation of DNA hairpins at the coding ends. Hairpin cleavage and resolution of the post‐cleavage complex by nonhomologous end‐s results in the formation of separate coding and signal joints, in the final steps of V(D)J recombination.

The recombinase machinery
The V(D)J recombinase is a complex of enzymes that mediates somatic recombination of immunoglobulin gene segments (Figure 3.24). The gene products of recombination‐activating genes 1 and 2 (RAG‐1 and RAG‐2) are lymphocyte‐specific enzymes essential for V(D)J recombination. In the initial steps of V(D)J recombination, the RAG complex binds the recombination signal sequences and, in association with high mobility group (HMG) proteins that are involved in DNA bending, the two recombination signal sequences are brought together. In contrast to the lymphoid‐specific RAG enzymes, HMG proteins are ubiquitously expressed.
Next, a single‐stranded nick is introduced between the 5′‐heptameric end of the recombination signal sequence and the coding segment. This nick results in a free 3′ OH group, which attacks the opposite, anti‐parallel DNA strand in a transesterification reaction. This attack gives rise to a double‐ stranded DNA break that leads to the formation of covalently sealed hairpins at the two coding ends and the formation of blunt signal ends. At this stage a post‐cleavage complex is formed, in which the RAG recombinase remains associated with the DNA ends.
The DNA break is finally repaired by nonhomologous end‐joining machinery. The recombination signal sequences are joined precisely to generate the signal joint. By contrast, nucleotides can be lost or added during repair of the coding ends (Figure 3.25). Junctional diversity is the diversification of variable region exons due to this imprecise joining of the coding ends.
First, a small number of nucleotides are often deleted from the coding end by an unknown exonuclease. Also, junctional diversity involves the potential addition of two types of nucleotides, P‐nucleotides and N‐nucleotides. The palindromic sequences that result from the asymmetric cleavage and template‐mediated fill‐in of the coding hairpins are referred to as P‐nucleotides. N‐nucleotides are generated by the nontemplated addition of nucleotides to the coding ends, which is mediated by the enzyme terminal deoxynucleotidyl transferase (TdT). Although P‐ and N‐nucleotides and deletion of the coding end and nucleotides serve to greatly diversify the immunoglobulin repertoire, the addition of these nucleotides may, as for other events in antibody gene assembly, result in the genera­ tion of receptor genes that are out of frame.
Similar to the RAG recombinase complex, the DNA repair machinery works as a protein complex. However, unlike the RAG recombinase, the nonhomologous end‐joining proteins are ubiquitously expressed. In the first steps of DNA repair, the Ku70 and Ku80 proteins form a heterodimer that binds the broken DNA ends. The Ku complex recruits the catalytic subunit of DNA‐dependent protein kinase (DNA‐PKcs), a serine‐threonine protein kinase. The activated DNA‐PKcs then recruits and phosphorylates XRCC4 and Artemis. Artemis is an endonuclease that opens the hairpin coding ends. Finally, DNA ligase IV binds XRCC4 to form an end‐ligation complex, and this complex mediates the final ligation and fill‐in steps needed to form the coding and signal joints.
Junctional diversity further diversifies the immune repertoire
Figure 3.25  Junctional diversity further diversifies the immune repertoire. The immunoglobulin repertoire is further diversified during cleavage and resolution of the coding‐end hairpins by deletion of a variable number of coding‐end nucleotides, the addition of N‐nucleotides by terminal deoxynucleotidyl transferase (TdT), and palindromic (P) nucleotides that arise owing to template‐mediated fill‐in of the asymmetrically cleaved coding hairpins. TdT randomly adds nucleotides to the DNA ends (N‐nucleotides), and the single‐stranded ends pair, possibly but not necessarily, through complementary nucleotides (TG on top strand and AC on bottom strand). Exonuclease trimming, to remove unpaired nucleotides, and the DNA repair machinery act to repair the DNA joint.

Regulating V(D) J recombination
V(D)J recombination and the recombinase machinery must be carefully regulated to avoid wreaking havoc on the cellular genome. For instance, aberrant V(D)J recombination is implicated in certain B‐cell lymphomas. V(D)J recombination is largely regulated by controlling expression of the recombination machinery and the accessibility of gene segments and nearby enhancers and promoters. As previously mentioned, RAG‐1 and RAG‐2 activity is specific to lymphoid cells, and further regulation is imposed by downregulating RAG activity during appropriate stages of B‐cell development. Differential accessibility of gene segments to the recombinase machinery, which can be achieved by altering chromatin structure, also plays a role in making certain that appropriate gene segments are recombined in an appropriate order. Cis‐acting transcrip­ tional control elements, such as enhancers and promoters, also help regulate recombination. Although it is not a hard and fast rule, transcription from certain regulatory elements seems to correlate with rearrangement of the adjacent genes. This sterile, or nonproductive, transcription may somehow help target required proteins or modulate gene accessibility. Finally, in addition to directing recombination between appropriate gene segments, the precise sequences of the RSS itself, as well as the sequences of the gene segments themselves, can influence the efficiency of the recombination reaction.

Somatic hypermutation
Following antigen activation, the variable regions of immunoglobulin heavy and light chains are further diversified by somatic hypermutation. Somatic hypermutation involves the introduction of nontemplated point mutations into V regions of rapidly proliferating B‐cells in the germinal centers of lymphoid follicles. Antigen‐driven somatic hypermutation of variable immunoglobulin genes can result in an increase in binding affinity of the B‐cell receptor for its cognate ligand. As B‐cells with higher affinity immunoglobulins can more successfully compete for limited amounts of antigen present, an increase in the average affinity of the antibodies produced during an immune response is observed. This increase in the average affinity of immunoglobulins is known as affinity maturation.
Somatic hypermutation occurs at a high rate, thought to be on the order of about 1 × 10−3 mutations per base‐pair per generation, which is approximately 106 times higher than the mutation rate of cellular housekeeping genes. There is a bias for transition mutations, and the “mutation hotspots” in variable regions map to RGWY motifs (R = purine,  Y = pyrimidine, W= A or T). The exact mechanisms by which mutations are introduced and preferentially targeted to appropriate V regions, while constant regions of the immunoglobulin loci remain protected, is not clearly understood and is the subject of current research. Transcription through the target V region seems required, but is not sufficient, for somatic hypermutation. Additionally, the enzyme activation‐induced cytidine deaminase (AID) has been demonstrated to be essential for both somatic hypermutation and class switch recombination.
AID is a cytidine deaminase capable of carrying out targeted deamination of C to U, and shows strong homology with the RNA‐editing enzyme APOBEC‐1. It appears that AID directly deaminates DNA to produce U : G mismatches. The exact mechanism by which AID can differentially regulate somatic hypermutation and class switch recombination is currently being studied, and may depend on interactions of specific cofactors with specific domains of AID.
Therefore, diversity within the immunoglobulin repertoire is generated by: (i) the combinatorial joining of gene segments; (ii) junctional diversity; (iii) combinatorial pairing of heavy and light chains; and (iv) somatic hypermutation of V regions.
Immunoglobulin diversification using gene conversion
Figure 3.26 Immunoglobulin diversification using gene conversion. V(D)J recombination in chicken B‐cells results in assembly of a single variable region exon. In the process of gene conversion, sequences of pseudogenes, located upstream of the functional gene segments, are copied into the recombined variable exons at the light and heavy chain loci in rapidly proliferating B‐cells in the bursa of Fabricius. This results in a diversified antibody repertoire.

Gene conversion and repertoire diversification
Although mice and humans use combinatorial and junctional diversity as a mechanism to generate a diverse repertoire, in many species, including birds, cattle, swine, sheep, horses, and rabbits, V(D)J recombination results in assembly and expres­ sion of a single functional gene. Repertoire diversification is then achieved by gene conversion, a process in which pseudo‐ V genes are used as templates to be copied into the assembled variable region exon. Further diversification may be achieved by somatic hypermutation.
The process of gene conversion was originally identified in chickens, in which immature B‐cells have the same variable region exon. During B‐cell development in the bursa of Fabricius, rapidly proliferating B‐cells undergo gene conversion to diversify the immunoglobulin repertoire (Figure 3.26). Stretches of sequences from germline variable region pseudogenes, located upstream of the functional V genes, are introduced into the VL and VH regions. This process takes place in the ileal Peyer’s patches of cattle, swine, and horses, and in the appendix of rabbits. These gut‐associated lymphoid tissues are the mammalian equivalent of the bursa in these species.

Class switch recombination
Antigen‐stimulated IgM expressing B‐cells in germinal centers of secondary lymphoid organs, such as the spleen and lymph nodes, undergo class switch recombination. Class switch recombination (CSR) allows the IgH constant region exon of a given antibody to be exchanged for an alternative exon, giv­ ing rise to the expression of antibodies with the same antigen specificity but of differing isotypes, and therefore of differing effector functions as described earlier. CSR occurs through a deletional DNA recombination event at the IgH locus (Figure 3.27), which has been extensively studied in mice. Constant region exons for IgD, IgG, IgE, and IgA isotypes are located downstream of the IgM (Cμ) exon, and CSR occurs between switch or S regions. S regions are repetitive sequences, which are often G‐rich on the nontemplate strand, that are found upstream of each CH exon except Cδ. Breaks are introduced into the DNA of two S regions and fusion of the S regions leads to a rearranged CH locus, in which the variable exon is joined to an exon for a new constant region. The DNA between the two switch regions is excised and forms an episomal circle. Finally, alternative splicing of the primary RNA transcript generated from the rearranged DNA gives rise to either membrane‐bound or secreted forms of the immunoglobulin.
Prior to recombination between switch regions, transcription is initiated from a promoter found upstream of an exon that precedes all CH genes capable of undergoing CSR, the intervening (I) exon. These germline transcripts include I, S, and C region exons, and do not appear to code for any functional protein. However, this germline transcription is required, although not sufficient, to stimulate CSR. The precise mecha­ nism responsible for CSR is the subject of current study, but work indicates that AID, described previously to be involved in somatic hypermutation, helps mediate CSR, along with some components of the nonhomologous end‐joining pathway and several other DNA repair pathways. The joining of S regions may be mediated by association with transcriptional promoters, enhancers, chromatin factors, DNA repair proteins, AID‐associated factors, or by interactions involving S region sequences themselves.
Class switch recombination allows expression of different antibody isotypes
Figure 3.27 Class switch recombination allows expression of different antibody isotypes. It involves DNA recombination at repetitive sequences termed switch or S regions, and is illustrated here for an IgM to IgG2a switch at the mouse heavy chain locus. Switching to an IgG2a isotype begins with germline transcription from the promoter upstream of the constant region exon and recombination between the Sμ and Sγ2a regions. This DNA recombination reaction brings the IgG2a constant region exon downstream of the variable region exon. The remaining switch regions and constant region exons are deleted and form an episomal circle. Transcription of the rearranged DNA yields IgG2a mRNA, which can be translated to give rise to the IgG2a immunoglobulin protein.