Immunology_Janeway

Immunology_Janeway. 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 double-strand 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.


Immunology_Janeway

Immunology_Janeway. 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 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.


Immunology_Janeway

Immunology_Janeway. Zn2+ Zn2+ J region V region heptamer nonamer 23-bp RSS 12-bp RSS A 12-bp RSS bound to one RAG-1 favors binding of a 23-bp RSS to the other RAG-1 Fig. 5.10 The 12/23 base pair rule may result from asymmetric binding of RSSs to the RAG-1:RAG-2 dimer.


Immunology_Janeway

Immunology_Janeway. hairpins by 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 single-strand 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.


Immunology_Janeway

Immunology_Janeway. 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.


Immunology_Janeway

Immunology_Janeway. 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


Immunology_Janeway

Immunology_Janeway. 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.


Immunology_Janeway

Immunology_Janeway. 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


Immunology_Janeway

Immunology_Janeway. 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.


Immunology_Janeway

Immunology_Janeway. 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.


Immunology_Janeway

Immunology_Janeway. 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 heavyand 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


Immunology_Janeway

Immunology_Janeway. 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.


Immunology_Janeway

Immunology_Janeway. 5-7 The multiple inherited gene segments are used in different combinations.


Immunology_Janeway

Immunology_Janeway. 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


Immunology_Janeway

Immunology_Janeway. 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 heavyand 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.


Immunology_Janeway

Immunology_Janeway. 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


Immunology_Janeway

Immunology_Janeway. 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.


Immunology_Janeway

Immunology_Janeway. 5-8 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.


Immunology_Janeway

Immunology_Janeway. 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


Immunology_Janeway

Immunology_Janeway. Fig. 5.11 The introduction of Pand 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


Immunology_Janeway

Immunology_Janeway. 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 Pand N-nucleotides (indicated by light blue shading) in the final coding joint. The randomness of insertion of Pand 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.


Immunology_Janeway

Immunology_Janeway. TCGCCGGCAGATATATATGCGCATATPPNDJThe gaps are ÿlled by DNA synthesis and ligation to form coding joint TCCGGCAATGCTTAADJUnpaired nucleotides are removed by an exonuclease TCCGGCTAAATGCTTAAAATGCTCCGGCTATTAAATGCTTAADJPairing of strands DJN-nucleotide additions by TdT JDArtemis:DNA-PK complex opens DNA hairpins, generating palindromic P-nucleotides ATATATGCJDRAG complex generates DNA hairpin at coding ends RSSs brought together ATATATGCGCGCATATATGCGCGCGCATATATGCGCJD 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.


Immunology_Janeway

Immunology_Janeway. In heavy-chain gene rearrangements and in a proportion of human light-chain 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 single-stranded 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 light-chain genes, which undergo rearrangement after heavy-chain genes, when TdT expression has been shut off, as we


Immunology_Janeway

Immunology_Janeway. are common in heavy-chain V–D and D–J junctions. N-nucleotides are less common in light-chain 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.


Immunology_Janeway

Immunology_Janeway. 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.


Immunology_Janeway

Immunology_Janeway. 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.


Immunology_Janeway

Summary.

Immunology_Janeway. Summary.


Immunology_Janeway

Immunology_Janeway. 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 Pand


Immunology_Janeway

Immunology_Janeway. 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 Pand N-nucleotides and by the variable deletion of nucleotides at the ends of 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 lightand 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.


Immunology_Janeway

Immunology_Janeway. 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.


Immunology_Janeway

Immunology_Janeway. 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 homologous 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


Immunology_Janeway

Immunology_Janeway. 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


Immunology_Janeway

Immunology_Janeway. Fig. 5.12 The germline organization of the human T-cell receptor α and β loci.


Immunology_Janeway

Immunology_Janeway. 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 cluster of 52 functional Vβ gene segments located distant from two separate clusters that each contain a single D gene segment together with six or seven J gene segments and a single C gene. Each TCRβ C gene has separate exons encoding


Immunology_Janeway

Immunology_Janeway. located distant from two separate clusters that each contain a single D gene segment together with six or seven J gene segments and a single C gene. Each TCRβ C gene has separate exons encoding the constant domain, the hinge, the transmembrane region, and the cytoplasmic region (not shown). The TCRα locus is interrupted between the J and V gene segments by another T-cell receptor locus—the TCRδ locus (not shown here; see Fig. 5.17).


Immunology_Janeway

Immunology_Janeway. The TCRαand β-chain genes are composed of discrete segments that are joined by somatic recombination during development of the T cell. Functional αand β-chain genes are generated in the same way that complete immunoglobulin genes are created. For the α chain (upper part of figure), a Vα gene segment rearranges to a Jα gene segment to create a functional V-region exon. Transcription and splicing of the VJα exon to Cα generates the mRNA that is translated to yield the T-cell receptor α-chain protein. For the β chain (lower part of figure), like the immunoglobulin heavy chain, the variable domain is encoded in three gene segments, Vβ, Dβ, and Jβ. Rearrangement of these gene segments generates a functional VDJβ V-region exon that is transcribed and spliced to join to Cβ; the resulting mRNA is translated to yield the T-cell receptor β chain. The α and β chains pair soon after their synthesis to yield the α:β T-cell receptor heterodimer. Not all J gene segments are shown, and the leader


Immunology_Janeway

Immunology_Janeway. is translated to yield the T-cell receptor β chain. The α and β chains pair soon after their synthesis to yield the α:β T-cell receptor heterodimer. Not all J gene segments are shown, and the leader sequences preceding each V gene segment are omitted for simplicity.


Immunology_Janeway

Immunology_Janeway. Fig. 5.14 Recombination signal sequences flank T-cell receptor gene segments. As in the immunoglobulin gene loci (see Fig. 5.6), the individual gene segments at the TCRα and TCRβ loci are flanked by heptamer–spacer–nonamer recombination signal sequences (RSSs). RSS motifs containing 12-bp spacers are depicted here as orange arrowheads, and those containing 23-bp spacers are shown in purple. Joining of gene segments almost always follows the 12/23 rule. Because of the disposition of heptamer and nonamer RSSs in the TCRβ and TCRδ loci, direct Vβ to Jβ joining is in principle allowed by the 12/23 rule (unlike in the immunoglobulin heavy-chain gene), although this occurs very rarely owing to other types of regulation. Fig. 5.13 T-cell receptor αand β-chain gene rearrangement and expression.


Immunology_Janeway

Immunology_Janeway. gene segments (Fig. 5.14; see Section 5-4) and are recognized by the same enzymes. The DNA circles resulting from gene rearrangement (see Fig. 5.7) are known as T-cell receptor excision circles (TRECs) and are used as markers for T cells that have recently emigrated from the thymus. All known defects in genes that control V(D)J recombination affect T cells and B cells equally, and animals with these genetic defects lack functional B and T lymphocytes altogether (see Section 5-5). A further shared feature of immunoglobulin and T-cell receptor gene rearrangement is the presence of Pand N-nucleotides in the junctions between the V, D, and J gene segments of the rearranged TCRβ gene. In T cells, Pand N-nucleotides are also added between the V and J gene segments of all rearranged TCRα genes, whereas only about half of the V–J joints in immunoglobulin light-chain genes are modified by N-nucleotide addition, and these are often left without any P-nucleotides as well (Fig. 5.15; see Section


Immunology_Janeway

Immunology_Janeway. whereas only about half of the V–J joints in immunoglobulin light-chain genes are modified by N-nucleotide addition, and these are often left without any P-nucleotides as well (Fig. 5.15; see Section 5-8).


Immunology_Janeway

Immunology_Janeway. The main differences between the immunoglobulin genes and those encoding T-cell receptors reflect the differences in how B cells and T cells function. All the effector functions of B cells depend upon secreted antibodies whose different heavy-chain C-region isotypes trigger distinct effector mechanisms. The effector functions of T cells, in contrast, depend upon cell–cell contact and are not mediated directly by the T-cell receptor, which serves only for antigen Fig. 5.15 The number of human T-cell receptor gene segments and the sources of T-cell receptor diversity compared with those of immunoglobulins. Note that only about half of human κ chains contain N-nucleotides. Somatic hypermutation as a source of diversity is not included in this figure because it does not occur in T cells.


Immunology_Janeway

Immunology_Janeway. recognition. Thus, the C regions of the TCRα and TCRβ loci are much simpler than those of the immunoglobulin heavy-chain locus. There is only one Cα gene, and although there are two Cβ genes, they are very closely homologous and there is no known functional distinction between their products. The T-cell receptor C-region genes encode only transmembrane polypeptides.


Immunology_Janeway

Immunology_Janeway. Another difference between the rearrangement of immunoglobulin genes and T-cell receptor genes is in the nature of the RSSs surrounding the D gene segments. For the immunoglobulin heavy chain, the D segment is surrounded by two RSSs, both with a 12-bp spacing (see Fig. 5.6), whereas the D segments in the TCRβ and TCRγ loci have a 5ʹ 12-bp RSS and a 3ʹ 23-bp RSS (see Fig. 5.14). The arrangement in the immunoglobulin locus naturally enforces the inclusion of D segments in the heavy-chain V region, since direct V to J joining would violate the 12/23 rule. However, in the T-cell receptor loci, direct V to J joining would not violate this rule, since the 23-bp RSS of the Vβ or Vγ segment is compatible with the 12-bp RSS of the J gene segment, and yet normally, little to no such direct joining is observed. Instead, regulation of gene rearrangements appears to be controlled by mechanisms beyond the 12/23 rule, and these mechanisms are still being investigated.


Immunology_Janeway

Immunology_Janeway. 5-10 T-cell receptors concentrate diversity in the third hypervariable region.


Immunology_Janeway

Immunology_Janeway. The three-dimensional structure of the antigen-recognition site of a T-cell receptor looks much like that of the antigen-recognition site of an antibody molecule (see Sections 4-10 and 4-7, respectively). In an antibody, the center of the antigen-binding site is formed by the CDR3 loops of the heavy and light chains. The structurally equivalent third hypervariable loops of the T-cell receptor α and β chains, to which the D and J gene segments contribute, also form the center of the antigen-binding site of a T-cell receptor; the periphery of the site consists of the CDR1 and CDR2 loops, which are encoded within the germline V gene segments for the αand β chains. The extent and pattern of variability in T-cell receptors and immunoglobulins reflect the distinct nature of their ligands. Whereas the antigen-binding sites of immunoglobulins must conform to the surfaces of an almost infinite variety of different antigens, and thus come in a wide variety of shapes and chemical properties, the


Immunology_Janeway

Immunology_Janeway. the antigen-binding sites of immunoglobulins must conform to the surfaces of an almost infinite variety of different antigens, and thus come in a wide variety of shapes and chemical properties, the ligand for the major class of human T-cell receptors (α:β) is always a peptide bound to an MHC molecule. As a group, the antigen-recognition sites of T-cell receptors


Immunology_Janeway

Immunology_Janeway. Fig. 5.16 The most variable parts of the T-cell receptor interact with the peptide of a peptide:MHC complex. The CDR loops of a T-cell receptor are shown as colored tubes, which in this figure are superimposed on the peptide:MHC complex (MHC, gray; peptide, yellow-green with O atoms in red and N atoms in blue). The CDR loops of the α chain are in green, while those of the β chain are in magenta. The CDR3 loops lie in the center of the interface between the T-cell receptor and the peptide:MHC complex, and make direct contact with the antigenic peptide. would therefore be predicted to have a less variable shape, with most of the variability focused on the bound antigenic peptide occupying the center of the surface in contact with the receptor. Indeed, the less variable CDR1 and CDR2 loops of a T-cell receptor mainly contact the relatively less variable MHC component of the ligand, whereas the highly variable CDR3 regions mainly contact the unique peptide component (Fig. 5.16).


Immunology_Janeway

Immunology_Janeway. The structural diversity of T-cell receptors is attributable mainly to combinatorial and junctional diversity generated during the process of gene rearrangement. It can be seen from Fig. 5.15 that most of the variability in T-cell receptor chains is in the junctional regions, which are encoded by V, D, and J gene segments and modified by Pand N-nucleotides. The TCRα locus contains many more J gene segments than either of the immunoglobulin light-chain loci: in humans, 61 Jα gene segments are distributed over about 80 kb of DNA, whereas immunoglobulin light-chain loci have only 5 J gene segments at most (see Fig. 5.15). Because the TCRαlocus has so many J gene segments, the variability generated in this region is even greater for T-cell receptors than for immunoglobulins. Thus, most of the diversity resides in the CDR3 loops that contain the junctional region and form the center of the antigen-binding site. 5-11 γ:δ T-cell receptors are also generated by gene rearrangement.


Immunology_Janeway

Immunology_Janeway. 5-11 γ:δ T-cell receptors are also generated by gene rearrangement. A minority of T cells bear T-cell receptors composed of γ and δ chains (see Section 4-20). The organization of the TCRγ and TCRδ loci (Fig. 5.17) resembles that of the TCRα and TCRβ loci, although there are important differences. The cluster of gene segments encoding the δ chain is found entirely within the TCRα locus, between the Vα and the Jα gene segments. Vδ genes are


Immunology_Janeway

Immunology_Janeway. Fig. 5.17 The organization of the T-cell receptor γand δ-chain loci in humans. The TCRγ and TCRδ loci, like the TCRα and TCRβ loci, have discrete V, D, and J gene segments, and C genes. Uniquely, the locus encoding the δ chain is located entirely within the α-chain locus. The three Dδ gene segments, four Jδ gene segments, and the single δ C gene lie between the cluster of Vα gene segments and the cluster of Jα gene segments. There are two Vδ gene segments (not shown) located near the δ C gene, one just upstream of the D regions and one in inverted orientation just downstream of the C gene. In addition, there are six Vδ gene segments interspersed among the Vα gene segments. Five are shared with Vα and can be used by either locus, and one is unique to the δ locus. The human TCRγ locus resembles the TCRβ locus in having two C genes, each with its own set of J gene segments. The mouse γ locus (not shown) has a more complex organization and there are three functional clusters of γ gene


Immunology_Janeway

Immunology_Janeway. the TCRβ locus in having two C genes, each with its own set of J gene segments. The mouse γ locus (not shown) has a more complex organization and there are three functional clusters of γ gene segments, each containing V and J gene segments and a C gene. Rearrangement at the γ and δ loci proceeds as for the other T-cell receptor loci, with the exception that during TCRδ rearrangement two D segments can be used in the same gene. The use of two D segments greatly increases the variability of the δ chain, mainly because extra N-region nucleotides can be added at the junction between the two D gene segments as well as at the V–D and D–J junctions.


Immunology_Janeway

Immunology_Janeway. interspersed with the Vα genes but are located primarily in the 3ʹ region of the locus. Because all Vα gene segments are oriented such that rearrangement will delete the intervening DNA, any rearrangement at the α locus results in the loss of the δ locus (Fig. 5.18). There are substantially fewer V gene segments at the TCRγ and TCRδ loci than at either the TCRα or TCRβ loci or any of the immunoglobulin loci. Increased junctional variability in the δ chains may compensate for the small number of V gene segments and has the effect of focusing almost all the variability in the γ:δreceptor in the junctional region. As we have seen for the α:β T-cell receptors, the amino acids encoded by the junctional regions lie at the center of the T-cell receptor binding site. T cells bearing γ:δ receptors are a distinct lineage of T cells, and as discussed in Chapter 4, some γ:δ T cells recognize nonclassical MHC class I molecules and other molecules whose expression may be an indication of cellular


Immunology_Janeway

Immunology_Janeway. are a distinct lineage of T cells, and as discussed in Chapter 4, some γ:δ T cells recognize nonclassical MHC class I molecules and other molecules whose expression may be an indication of cellular damage or infection. As we saw in Section 4-20, the CDR3 of a γ:δ T cell is frequently longer than the CDR3 in an α:β T-cell receptor; this permits the CDR of γ:δ T cell receptors to interact directly with ligand and also contributes to the great diversity of these receptors. We will discuss the regulation of the fate choice between the α:βand γ:δ T-cell lineages in Chapter 8.


Immunology_Janeway

Immunology_Janeway. Summary. T-cell receptors are structurally similar to immunoglobulins and are encoded by homologous genes. T-cell receptor genes are assembled by somatic recombination from sets of gene segments in the same way that the immunoglobulin genes are. Diversity is, however, distributed differently in immunoglobulins and T-cell receptors: the T-cell receptor loci have roughly the same number of V gene segments as the immunoglobulin loci but more J gene segments, and there is greater diversification of the junctions between gene segments during the process of gene rearrangement. Thus, the greatest diversity of the T-cell receptor is in the central part of the receptor, within the CDR3, which in the case of α:β T-cell receptors contacts the bound peptide fragment of the ligand. Most of the diversity among γ:δ T-cell receptors is also within the CDR3, which is frequently longer than the CDR3 of α:β T-cell receptors and can also directly interact with ligands recognized by the γ:δ T cells.


Immunology_Janeway

Immunology_Janeway. Structural variation in immunoglobulin constant regions.


Immunology_Janeway

Immunology_Janeway. This chapter so far has focused on the mechanisms of assembly of the V regions for immunoglobulins and T-cell receptors. We now turn to the C regions. The C regions of T-cell receptors act only to support the V regions and anchor the receptor into the membrane, and they do not vary after assembly of a complete receptor gene. Immunoglobulins, in contrast, can be made as both a trans-membrane receptor and a secreted antibody, and they can be made in several different classes, depending on the different C regions used by the heavy chain. The light-chain C regions (CL) provide only structural attachment for V regions, and there seem to be no functional differences between λand κ light chains. The heavy-chain locus encodes different C regions (CH) that are present as separate genes located downstream of the V-region segments. Initially, naive B cells use only the first two of these, the Cμ and Cδ genes, which are expressed along with the associated assembled V-region sequence to produce


Immunology_Janeway

Immunology_Janeway. of the V-region segments. Initially, naive B cells use only the first two of these, the Cμ and Cδ genes, which are expressed along with the associated assembled V-region sequence to produce transmembrane IgM and IgD on the surface of the naive B cell.


Immunology_Janeway

Immunology_Janeway. In this section, we introduce the different heavy-chain isotypes and discuss some of their special properties as well as the structural features that distinguish the CH regions of antibodies of the five major classes. We explain how Fig. 5.18 Deletion of the TCRδ locus is induced by rearrangement of a Vα to Jα gene segment. The TCRδ locus is entirely contained within the chromosomal region containing the TCRα locus. When any V region in the Vα/Vδ region rearranges to any one of the Jα segments, the intervening region, and the entire Vδ locus, is deleted. Thus, Vα rearrangement prevents any continued expression of a Vδ gene and precludes lineage development down the γ:δ pathway.


Immunology_Janeway

Immunology_Janeway. naive B cells express both Cμ and Cδ isotypes at the same time and how the same antibody gene can generate both membrane-bound immunoglobulin and secreted immunoglobulin through alternative mRNA splicing. During an antibody response, activated B cells can switch to the expression of CH genes other than Cμ and Cδ by a type of somatic recombination known as class switching (discussed in Chapter 10) that links different heavy-chain C regions (CH) to the rearranged VDJH gene segment. 5-12 Different classes of immunoglobulins are distinguished by the structure of their heavy-chain constant regions.


Immunology_Janeway

Immunology_Janeway. 5-12 Different classes of immunoglobulins are distinguished by the structure of their heavy-chain constant regions. The five main classes of immunoglobulins are IgM, IgD, IgG, IgE, and IgA, all of which can occur as transmembrane antigen receptors or secreted antibodies (Fig. 5.19). In humans, IgG is found as four subclasses (IgG1, IgG2, IgG3, and IgG4), named by decreasing order of their abundance in serum, and IgA antibodies are found as two subclasses (IgA1 and IgA2). The different heavy chains that define these classes are known as isotypes and are designated by the lowercase Greek letters μ, δ, γ, ε, and α. The different heavy chains are encoded by different immunoglobulin CH genes located in a gene cluster that is 3ʹ of the JH segments as illustrated in Fig. 5.19. Figure 5.20 lists the major physical and functional properties of the different human antibody classes.


Immunology_Janeway

Immunology_Janeway. The functions of the immunoglobulin classes are discussed in detail in Chapter 10, in the context of the humoral immune response; here, we just touch on them briefly. IgM is the first class of immunoglobulin produced after activation of a B cell, and the IgM antibody is secreted as a pentamer (see Section 5-14 and Fig. 5.21). This accounts for the high molecular weight of IgM and the fact that it is normally present in the bloodstream but not in tissues. Being a pentamer


Immunology_Janeway

Immunology_Janeway. The general structure of the main immunoglobulin isotypes (above in upper panel) is indicated, with each rectangle denoting an immunoglobulin domain. These isotypes are encoded by separate heavy-chain C-region genes arranged in a cluster in both mouse and human (lower panel). The constant region of the heavy chain for each isotype is indicated by the same color as the C-region gene segment that encodes it. IgM and IgE lack a hinge region but each contains an extra heavy-chain domain. Note the differences in the number and location of the disulfide bonds (black lines) linking the chains. The isotypes also differ in the distribution of N-linked carbohydrate groups, shown as hexagons. In humans, the gene cluster shows evidence of evolutionary duplication of a unit consisting of two γ genes, an ε gene, and an α gene. One of the ε genes is a pseudogene (ψ); hence only one subtype of IgE is expressed. For simplicity, other pseudogenes are not illustrated, and the exon details within each C


Immunology_Janeway

Immunology_Janeway. an ε gene, and an α gene. One of the ε genes is a pseudogene (ψ); hence only one subtype of IgE is expressed. For simplicity, other pseudogenes are not illustrated, and the exon details within each C gene are not shown. The classes of immunoglobulins found in mice are called IgM, IgD, IgG1, IgG2a, IgG2b, IgG3, IgA, and IgE.


Immunology_Janeway

Immunology_Janeway. Fig. 5.20 The physical and functional properties of the human immunoglobulin isotypes. IgM is so called because of its size: although monomeric IgM is only 190 kDa, it normally forms pentamers, known as macroglobulin (hence the M), of very large molecular weight (see Fig. 5.23). IgA dimerizes to give an approximate molecular weight of around 390 kDa in secretions. IgE antibody is associated with immediate-type hypersensitivity. When fixed to tissue mast cells, IgE has a much longer half-life than its half-life in plasma shown here. The relative activities of the various isotypes are compared for several functions, ranging from inactive (–) to most active (++++). also increases the avidity of IgM for antigens before its affinity is increased through the process of affinity maturation.


Immunology_Janeway

Immunology_Janeway. also increases the avidity of IgM for antigens before its affinity is increased through the process of affinity maturation. IgG isotypes produced during an immune response are found in the bloodstream and in the extracellular spaces in tissues. IgM and most IgG isotypes can interact with the complement component C1 to activate the classical complement pathway (described in Section 2-7). IgA and IgE do not activate complement. IgA can be found in the bloodstream, but it also acts in the defense of mucosal surfaces; it is secreted into the gut and respiratory tract, and also into mother’s milk. IgE is particularly involved in defense against multicellular parasites (for example, schistosomes), but it is also the antibody involved in common allergic diseases such as allergic asthma. IgG and IgE are always monomers, but IgA can be secreted as either a monomer or a dimer.


Immunology_Janeway

Immunology_Janeway. Sequence differences in the constant regions of the immunoglobulin heavy chains produce the distinct characteristics of each antibody isotype. These characteristics include the number and location of interchain disulfide bonds, the number of attached carbohydrate groups, the number of C domains, and the length of the hinge region (see Fig. 5.19). IgM and IgE heavy chains contain an extra C domain that replaces the hinge region found in γ, δ, and α chains. The absence of the hinge region does not imply that IgM and IgE molecules lack flexibility; electron micrographs of IgM molecules binding to ligands show that the Fab arms can bend relative to the Fc portion. However, such a difference in structure may have functional consequences that are not yet characterized. Different isotypes and subtypes also differ in their ability to engage various effector functions, as described below. 5-13 The constant region confers functional specialization on the antibody.


Immunology_Janeway

Immunology_Janeway. 5-13 The constant region confers functional specialization on the antibody. Antibodies can protect the body in a variety of ways. In some cases it is enough for the antibody simply to bind antigen. For instance, by binding tightly to a toxin or virus, an antibody can prevent it from recognizing its receptor on a host cell (see Fig. 1.25). The antibody V regions are sufficient for this activity. The C region is essential, however, for recruiting the help of other cells and molecules to destroy and dispose of pathogens to which the antibody has bound.


Immunology_Janeway

Immunology_Janeway. The Fc region contains all C regions of an antibody and has three main effector functions: Fc-receptor binding, complement activation, and regulation of secretion. First, the Fc region of certain isotypes binds to specialized Fc receptors expressed by immune effector cells. Fcγ receptors expressed on the surface of macrophages and neutrophils bind the Fc portions of IgG1 and IgG3 antibodies, facilitating the phagocytosis of pathogens coated with these antibodies. The Fc region of IgE binds to a high-affinity Fcε receptor on mast cells, basophils, and activated eosinophils, triggering the release of inflammatory mediators in response to antigens. We will return to this topic in Section 10-19.


Immunology_Janeway

Immunology_Janeway. Second, the Fc regions in antigen:antibody complexes can bind to the C1q complement protein (see Section 2-7) and initiate the classical complement cascade, which recruits and activates phagocytes to engulf and destroy pathogens. Third, the Fc portion can deliver antibodies to places they would not reach without active transport. These include transport of IgA into mucous secretions, tears, and milk, and the transfer of IgG from the pregnant mother into the fetal blood circulation. In both cases, the Fc portion of IgA or IgG engages a specific receptor, the neonatal Fc receptor (FcRn), that actively transports the immunoglobulin through cells to reach different body compartments. Podocytes in the kidney glomerulus express FcRn to help remove IgG that has been filtered from the blood and accumulated at the glomerular basement membrane.


Immunology_Janeway

Immunology_Janeway. The role of the Fc portion in these effector functions has been demonstrated by studying immunoglobulins that have had one or more Fc domains cleaved off enzymatically or modified genetically. Many microorganisms have responded to the destructive potential of the Fc portion by evolving proteins that either bind it or cleave it, and so prevent the Fc region from working; examples are Protein A and Protein G of Staphylococcus and Protein D of Haemophilus. Researchers have exploited these proteins to help map the Fc region and also as immunological reagents. Not all immunoglobulin classes have the same capacity to engage each of the effector functions (see Fig. 5.20). For example, IgG1 and IgG3 have a higher affinity than IgG2 for the most common type of Fc receptor. 5-14 IgM and IgD are derived from the same pre-mRNA transcript and are both expressed on the surface of mature B cells.


Immunology_Janeway

Immunology_Janeway. 5-14 IgM and IgD are derived from the same pre-mRNA transcript and are both expressed on the surface of mature B cells. The immunoglobulin CH genes form a large cluster spanning about 200 kb to the 3ʹ side of the JH gene segments (see Fig. 5.19). Each CH gene is split into several exons (not shown in the figure), with each exon corresponding to an individual immunoglobulin domain in the folded C region. The gene encoding the μ C region lies closest to the JH gene segments, and therefore closest to the assembled VH-region exon (VDJ exon) after DNA rearrangement. Once rearrangement is completed, transcription from a promoter just 5ʹ to the rearranged VDJ exon produces a complete μ heavy-chain transcript. Any JH gene segments remaining between the assembled V gene and the Cμ gene are removed during RNA processing to generate the mature mRNA. The μ heavy chains are therefore the first to be expressed, and IgM is the first immunoglobulin to be produced during B-cell development.


Immunology_Janeway

Immunology_Janeway. Immediately 3ʹ to the μ gene lies the δ gene, which encodes the C region of the IgD heavy chain (see Fig. 5.19). IgD is coexpressed with IgM on the surface of almost all mature B cells, but is secreted in only small amounts by plasma cells. The unique function of IgD is still unclear and a matter of active research. Because IgD has hinge regions that are more flexible than those in IgM, IgD has been suggested to be an auxiliary receptor that may facilitate the binding of antigens by naive B cells. Mice lacking the Cδ exons show normal B-cell development and can generate largely normal antibody responses, but show a delay in the process of affinity maturation of antibody for antigens. We return to this topic in Chapter 10, when we discuss somatic hypermutation.


Immunology_Janeway

Immunology_Janeway. B cells expressing IgM and IgD have not undergone class switching, which requires irreversible changes to the DNA. Instead, these B cells produce a long primary mRNA transcript that is differentially spliced to yield either of two distinct mRNA molecules (Fig. 5.21). In one transcript, the VDJ exon is spliced to the Cμ exons and undergoes polyadenylation from a nearby site (pA1), to encode a complete IgM molecule. The second RNA transcript extends well beyond this site and includes the downstream Cδ exons. In this transcript, the VDJ exon is spliced to these Cδ exons and polyadenylation occurs at a separate site downstream (pA2). This transcript encodes an IgD molecule.


Immunology_Janeway

Immunology_Janeway. It has been known since the 1980s that the processing of the long mRNA transcript is developmentally regulated, with immature B cells making mostly the μ transcript and mature B cells making mostly the δ along with some μ, although until recently there was little to no molecular explanation. A recent forward genetic screen of N-ethyl-N-nitrosourea (ENU)-induced mutagenesis in mice identified a gene involved in IgD expression that regulates the alternative splicing process. The gene encodes ZFP318, a protein structurally related to the U1 small nuclear ribonucleoprotein of the spliceosome, the RNA– protein complex that is required for mRNA splicing. ZFP318 is not expressed in immature B cells, where the IgD transcript is not produced, but becomes expressed in mature and activated B cells that coexpress IgD with IgM. ZFP318 is required for alternative splicing of the long pre-mRNA from the VDJ exon to the Cδ exons, as mice with a fully inactivated ZFP318 gene fail to express IgD and


Immunology_Janeway

Immunology_Janeway. that coexpress IgD with IgM. ZFP318 is required for alternative splicing of the long pre-mRNA from the VDJ exon to the Cδ exons, as mice with a fully inactivated ZFP318 gene fail to express IgD and express increased levels of IgM. While the precise mechanism is unclear, it seems likely that ZFP318 may act directly on the pre-mRNA transcript during elongation, by suppressing splicing of the VDJ exon to the Cμ exons, allowing transcript elongation and promoting splicing to the Cδ exons. In short, expression of ZFP318 promotes IgD expression, although how ZFP318 expression itself is regulated in immature and mature B cells is still unknown.


Immunology_Janeway

Immunology_Janeway. 5-15 Transmembrane and secreted forms of immunoglobulin are generated from alternative heavy-chain mRNA transcripts. Each of the immunoglobulin isotypes can be produced either as a membrane-bound receptor or as secreted antibodies. B cells initially express the transmembrane form of IgM; after stimulation by antigen, some of their In mature B cells, transcription initiated at the VH promoter extends through both Cμ and Cδ exons. This long primary transcript is then processed by cleavage and polyadenylation (AAA), and by splicing. Cleavage and polyadenylation at the μ site (pA1) and splicing between Cμ exons yields an mRNA encoding the μ heavy chain (left panel). Cleavage and polyadenylation at the δ site (pA2) and a different pattern of splicing that joins the V region exon to the Cδ exons and removes the Cμ exons yields mRNA encoding the complete δ heavy chain (right panel). For simplicity we have not shown all the individual C-region exons.


Immunology_Janeway

Immunology_Janeway. progeny differentiate into plasma cells producing IgM antibodies, whereas others undergo class switching to express transmembrane immunoglobulins of a different class, followed by the production of secreted antibody of the new class. The membrane-bound forms of all immunoglobulin classes are monomers comprising two heavy and two light chains. IgM and IgA polymerize only when they have been secreted. The membrane-bound form of immunoglobulin heavy chain has at the carboxy terminus a hydrophobic transmembrane domain of about 25 amino acid residues that anchors it to the surface of the B lymphocyte. The secreted form replaces this transmembrane domain with a carboxy terminus composed of a hydrophilic secretory tail. These two forms of carboxy termini are encoded by different exons found at the end of each CH gene as these exons undergo alternative RNA processing.


Immunology_Janeway

Immunology_Janeway. For example, the IgM heavy-chain gene contains four exons—Cμ1 to Cμ4— that encode its four heavy-chain Ig domains (Fig. 5.22). The end of the Cμ4 exon also encodes the carboxy terminus for the secreted form. Two additional downstream exons, M1 and M2, encode the transmembrane forms. If the primary transcript is cleaved at the polyadenylation site (pAs) located just downstream of the Cμ4 exon but before the last two exons, then only the secreted molecule can be produced. If the polymerase transcribes through this first polyadenylation site, then splicing can occur from a non-consensus splice-donor site within the Cμ4 exon to the M1 exon. In this case, polyadenylation occurs at a downstream site (pAm) and the cell-surface form of immunoglobulin can be produced. This alternative splicing is incompletely understood, but


Immunology_Janeway

Immunology_Janeway. AAA AAA L VDJ C˜1 C˜2 C˜3 C˜4SCpAs pAmM1 M2 carboxy terminusfor transmembrane IgM carboxy terminusfor secreted IgM AAA AAA Rearranged DNA mRNA Protein Transcription Different forms of RNA processing Translation, protein processing Primary transcript RNA Secreted IgMTransmembrane IgM L VDJ C˜1 C˜2 C˜3 C˜4SC pAs pAmM1 M2


Immunology_Janeway

Immunology_Janeway. Fig. 5.22 Transmembrane and secreted forms of immunoglobulins are derived from the same heavy-chain sequence by alternative RNA processing. At the end of the heavy-chain C gene, there are two exons (M1 and M2, yellow) that together encode the transmembrane region and cytoplasmic tail of the transmembrane form. Within the last C-domain exon, a secretion-coding (SC) sequence (orange) encodes the carboxy terminus of the secreted form. In the case of IgD, the SC sequence is in a separate exon (not shown), but for the other isotypes, including IgM as shown here, the SC sequence is contiguous with the last C-domain exon. The events that dictate whether a heavy-chain RNA will result in a secreted or a transmembrane immunoglobulin occur during processing of the pre-mRNA transcript. Each heavy-chain C gene has two potential polyadenylation sites (shown as pAs and pAm). Left panel: the transcript is cleaved and polyadenylated (AAA) at the second site (pAm). Splicing occurs from a site located


Immunology_Janeway

Immunology_Janeway. C gene has two potential polyadenylation sites (shown as pAs and pAm). Left panel: the transcript is cleaved and polyadenylated (AAA) at the second site (pAm). Splicing occurs from a site located within the last Cμ4 exon just upstream of the SC sequence (orange), to a second site at the 5ʹ end of the M1 exons (yellow). This results in removal of the SC sequence and joining of the Cμ4 exon to the exons M1 and M2 and generates the transmembrane form of the heavy chain. Right panel: polyadenylation occurs at the first poly(A) addition site (pAs), and transcription terminates before the exons M1 and M2, preventing the generation of the transmembrane form of the heavy chain, and producing the secreted form.


Immunology_Janeway

Immunology_Janeway. may involve the regulation of RNA polymerase activity as the polymerase transcribes through the IgM locus. One factor that regulates the polyadenylation of RNA transcripts is a cleavage stimulation factor subunit, CstF-64, which favors production of the transcript for secreted IgM. The transcription elongation factor ELL2, which is induced in plasma cells, also promotes polyadenylation at the pAs site and favors the secreted form. CstF-64 and ELL2 co-associate with RNA polymerase within the immunoglobulin locus. This differential RNA processing is illustrated for Cμ in Fig. 5.22, but it occurs in the same way for all isotypes. In activated B cells that differentiate to become antibody-secreting plasma cells, most of the transcripts are spliced to yield the secreted rather than the transmembrane form of whichever heavy-chain isotype the B cell is expressing. 5-16 IgM and IgA can form polymers by interacting with the J chain.


Immunology_Janeway

Immunology_Janeway. 5-16 IgM and IgA can form polymers by interacting with the J chain. Although all immunoglobulin molecules are constructed from a basic unit of two heavy and two light chains, both IgM and IgA can form multimers of these basic units (Fig. 5.23). C regions of IgM and IgA can include a ‘tailpiece’ of 18 amino acids that contains a cysteine residue essential for polymerization. A separate 15-kDa polypeptide chain called the J chain promotes polymerization by linking to the cysteine of this tailpiece, which is found only in the secreted forms of the μand α chains. (This J chain should not be confused with the immunoglobulin J region encoded by a J gene segment; see Section 5-2.) In the case of IgA, dimerization is required for transport through epithelia, as we will discuss in Chapter 10. IgM molecules are found as pentamers, and occasionally hexamers (without J chain), in plasma, whereas IgA is found mainly as a dimer in mucous secretions but as a monomer in plasma.


Immunology_Janeway

Immunology_Janeway. Immunoglobulin polymerization is also thought to be important in the binding of antibody to repetitive epitopes. An antibody molecule has at least two identical antigen-binding sites, each of which has a given affinity, or binding strength, for antigen. If the antibody attaches to multiple identical epitopes on a target antigen, it will dissociate only when all binding sites dissociate. The dissociation rate of the whole antibody will therefore be much slower than the dissociation rate for a single binding site; multiple binding sites thus give the antibody a greater total binding strength, or avidity. This consideration is particularly relevant for pentameric IgM, which has 10 antigen-binding sites.


Immunology_Janeway

Immunology_Janeway. Fig. 5.23 The IgM and IgA molecules can form multimers. IgM and IgA are usually synthesized as multimers in association with an additional polypeptide chain, the J chain. In dimeric IgA (left panel), the monomers have disulfide bonds to the J chain as well as to each other. In pentameric IgM (right panel), the monomers are cross-linked by disulfide bonds to each other and to the J chain. IgM can also form hexamers that lack a J chain (not shown).


Immunology_Janeway

Immunology_Janeway. IgM antibodies frequently recognize repetitive epitopes such as those on bacterial cell-wall polysaccharides, but individual binding sites are often of low affinity because IgM is made early in immune responses, before somatic hypermutation and affinity maturation. Multisite binding makes up for this, markedly improving the overall functional binding strength. This implies that binding of a single IgM pentamer to a target could be sufficient to mediate biological effector activity, whereas in the case of IgGs, two independent target molecules may need to be located in close proximity. Summary.


Immunology_Janeway

Immunology_Janeway. The classes of immunoglobulins are defined by their heavy-chain C regions, with the different heavy-chain isotypes being encoded by different C-region genes. The heavy-chain C-region genes are present in a cluster 3ʹ to the V, D, and J gene segments. A productively rearranged V-region exon is initially expressed in association with μ and δ CH genes, which are coexpressed in naive B cells by alternative splicing of an mRNA transcript that contains both the μ and δ CH exons. In addition, B cells can express any class of immunoglobulin as a membrane-bound antigen receptor or as a secreted antibody. This is achieved by differential splicing of mRNA to include exons that encode either a hydrophobic membrane anchor or a secretable tailpiece. The antibody that a B cell secretes upon activation thus recognizes the antigen that initially activated the B cell via its antigen receptor. The same V-region exon can subsequently be associated with any one of the other isotypes to direct the


Immunology_Janeway

Immunology_Janeway. thus recognizes the antigen that initially activated the B cell via its antigen receptor. The same V-region exon can subsequently be associated with any one of the other isotypes to direct the production of antibodies of different classes by the process of class switching, which is described in Chapter 10.


Immunology_Janeway

Immunology_Janeway. Evolution of the adaptive immune response.


Immunology_Janeway

Immunology_Janeway. The form of adaptive immunity that we have discussed so far in this book depends on the action of the RAG-1/RAG-2 recombinase to generate an enormously diverse clonally distributed repertoire of immunoglobulins and T-cell receptors. This system is found only in the jawed vertebrates, the gnathostomes, which split off from the other vertebrates around 500 million years ago. Adaptive immunity seems to have arisen abruptly in evolution. Even the cartilaginous fishes, the earliest group of jawed fishes to survive to the present day, have organized lymphoid tissue, T-cell receptors and immunoglobulins, and the ability to mount adaptive immune responses. The diversity generated within the vertebrate adaptive immune system was once viewed as unique among animal immune systems. But we now know that organisms as different as insects, echinoderms, and mollusks use a variety of genetic mechanisms to increase their repertoires of pathogen-detecting molecules, although they do not achieve true


Immunology_Janeway

Immunology_Janeway. that organisms as different as insects, echinoderms, and mollusks use a variety of genetic mechanisms to increase their repertoires of pathogen-detecting molecules, although they do not achieve true adaptive immunity. Nearer to home, it has been found that the surviving species of jawless vertebrates, the agnathans—the lampreys and hagfish—have a form of adaptive or ‘anticipatory’ immunity that is based on non-immunoglobulin ‘antibody’-like proteins and involves a system of somatic gene rearrangement that is quite distinct from RAG-dependent V(D)J rearrangement. So we should now view our adaptive immune system as only one solution, albeit the most powerful, to the problem of generating highly diverse systems for pathogen recognition.


Immunology_Janeway

Immunology_Janeway. 5-17 Some invertebrates generate extensive diversity in a repertoire of immunoglobulin-like genes. Until very recently, it was thought that invertebrate immunity was limited to an innate system that had a very restricted diversity in recognizing pathogens. This idea was based on the knowledge that innate immunity in vertebrates relied on around 10 distinct Toll-like receptors and a similar number of other receptors that also recognize PAMPs, and also on the assumption that the number of receptors in invertebrates was no greater. Recent studies have, however, uncovered at least two invertebrate examples of extensive diversification of an immunoglobulin superfamily member, which could potentially provide an extended range of recognition of pathogens.


Immunology_Janeway

Immunology_Janeway. In Drosophila, fat-body cells and hemocytes act as part of the immune system. Fat-body cells secrete proteins, such as the antimicrobial defensins (see Chapters 2 and 3), into the hemolymph. Another protein found in hemolymph is the Down syndrome cell adhesion molecule (Dscam), a member of the immunoglobulin superfamily. Dscam was originally discovered in the fly as a protein involved in specifying neuronal wiring. It is also made in fat-body cells and hemocytes, which can secrete it into the hemolymph, where it is thought to recognize invading bacteria and aid in their engulfment by phagocytes.


Immunology_Janeway

Immunology_Janeway. The Dscam protein contains multiple, usually 10, immunoglobulin-like domains. The gene that encodes Dscam has, however, evolved to contain a large number of alternative exons for several of these domains (Fig. 5.24). Exon 4 of the gene encoding the Dscam protein can be any 1 of 12 different exons, each specifying an immunoglobulin domain of differing sequence. Exon cluster 6 has 48 alternative exons, cluster 9 another 33, and cluster 17 a further 2: it is estimated that the Dscam gene could encode around 38,000 protein isoforms. A role for Dscam in immunity was proposed when it was found that in vitro phagocytosis of Escherichia coli by isolated hemocytes lacking Dscam was less efficient than by normal hemocytes. These observations suggest that at least some of this extensive repertoire of alternative exons may have evolved to diversify insects’ ability to recognize pathogens. This role for Dscam has been confirmed in the mosquito Anopheles gambiae, in which silencing of the Dscam


Immunology_Janeway

Immunology_Janeway. of alternative exons may have evolved to diversify insects’ ability to recognize pathogens. This role for Dscam has been confirmed in the mosquito Anopheles gambiae, in which silencing of the Dscam homolog AgDscam has been shown to weaken the mosquito’s normal resistance to bacteria and to the malaria parasite Plasmodium. There is also evidence from the mosquito that some Dscam exons have specificity for particular pathogens. It is not clear whether Dscam isoforms are expressed in a clonal manner.


Immunology_Janeway

Immunology_Janeway. Another invertebrate, this time a mollusk, uses a different strategy to diversify an immunoglobulin superfamily protein for use in immunity. The freshwater snail Biomphalaria glabrata expresses a small family of fibrinogen-related proteins (FREPs) thought to have a role in innate immunity. FREPs have one or two immunoglobulin domains at their amino-terminal end and a fibrinogen domain at their carboxy terminus. The immunoglobulin domains may interact with pathogens, while the fibrinogen domain may confer on the FREP lectin-like properties that help precipitate the complex. FREPs are produced by


Immunology_Janeway

Immunology_Janeway. Fig. 5.24 The Dscam protein of Drosophila innate immunity contains multiple immunoglobulin domains and is highly diversified through alternative splicing. The gene encoding Dscam in Drosophila contains several large clusters of alternative exons. The clusters encoding exon 4 (green), exon 6 (light blue), exon 9 (red), and exon 17 (orange) contain 12, 48, 33, and 2 alternative exons, respectively. For each of these clusters, only one alternative exon is used in the complete Dscam mRNA. There is some differential usage of exons in neurons, fat-body cells, and hemocytes. All three cell types use the entire range of alternative exons for exons 4 and 6. For exon 9, there is a restricted use of alternative exons in hemocytes and fat-body cells. The combinatorial use of alternative exons in the Dscam gene makes it possible to generate more than 38,000 protein isoforms. Adapted from Anastassiou, D.: Genome Biol. 2006, 7:R2.


Immunology_Janeway

Immunology_Janeway. hemocytes and secreted into the hemolymph. Their concentration increases when the snail is infected by parasites—it is, for example, the intermediate host for the parasitic schistosomes that cause human schistosomiasis.


Immunology_Janeway

Immunology_Janeway. The B. glabrata genome contains many copies of FREP genes that can be divided into approximately 13 subfamilies. A study of the sequences of expressed FREP3 subfamily members has revealed that the FREPs expressed in an individual organism are extensively diversified compared with the germline genes. There are fewer than five genes in the FREP3 subfamily, but an individual snail was found to generate more than 45 distinct FREP3 proteins, all with slightly different sequences. An analysis of the protein sequences suggested that this diversification was due to the accumulation of point mutations in one of the germline FREP3 genes. Although the precise mechanism of this diversification, and the cell type in which it occurs, are not yet known, it does suggest some similarity to somatic hypermutation that occurs in the immunoglobulins. Both the insect and Biomphalaria examples seem to represent a way of diversifying molecules involved in immune defense, but although they resemble in some


Immunology_Janeway

Immunology_Janeway. that occurs in the immunoglobulins. Both the insect and Biomphalaria examples seem to represent a way of diversifying molecules involved in immune defense, but although they resemble in some ways the strategy of an adaptive immune response, there is no evidence of clonal selection—the cornerstone of true adaptive immunity.


Immunology_Janeway

Immunology_Janeway. somatic gene rearrangement to diversify receptors built from LRR domains.


Immunology_Janeway

Immunology_Janeway. Since the early 1960s it has been known that certain jawless fishes, the hagfish and the lamprey, could mount a form of accelerated rejection of transplanted skin grafts and exhibit a kind of immunological delayed-type hypersensitivity. Their serum also seemed to contain an activity that behaved as a specific agglutinin, increasing in titer after secondary immunizations, in a similar way to an antibody response in higher vertebrates. Although these phenomena seemed reminiscent of adaptive immunity, there was no evidence of a thymus or of immunoglobulins, but these animals did have cells that could be considered to be genuine lymphocytes on the basis of morphological and molecular analysis. Analysis of the genes expressed by lymphocytes of the sea lamprey Petromyzon marinus revealed none related to T-cell receptor or immunoglobulin genes. However, these cells expressed large amounts of mRNAs from genes encoding proteins with multiple LRR domains, the same protein domain from which the


Immunology_Janeway

Immunology_Janeway. to T-cell receptor or immunoglobulin genes. However, these cells expressed large amounts of mRNAs from genes encoding proteins with multiple LRR domains, the same protein domain from which the pathogen-recognizing Toll-like receptors (TLRs) are built (see Section 3-5).


Immunology_Janeway

Immunology_Janeway. This might simply have meant that these cells are specialized for recognizing and reacting to pathogens, but the LRR proteins expressed had some surprises in store. Instead of being present in a relatively few forms (like the invariant TLRs), they were found to have highly variable amino acid sequences, with a large number of variable LRR units placed between less variable amino-terminal and carboxy-terminal LRR units. These LRR-containing proteins, called variable lymphocyte receptors (VLRs), have an invariant stalk region connecting them to the plasma membrane by a glycosylphosphatidylinositol linkage, and they can either be tethered to the cell or, at other times, like antibodies, be secreted into the blood.


Immunology_Janeway

Immunology_Janeway. Analysis of the expressed lamprey VLR genes indicates that they are assembled by a process of somatic gene rearrangement (Fig. 5.25). In the germline configuration, there are three incomplete VLR genes, VLRA, VLRB, and VLRC, each encoding a signal peptide, a partial amino-terminal LRR unit, and a partial carboxy-terminal LRR unit, but these three blocks of coding sequence are separated by noncoding DNA that contains neither typical signals for RNA splicing nor the RSSs present in immunoglobulin genes (see Section 5-4). Instead, the regions flanking the incomplete VLR genes include a large number of DNA ‘cassettes’ that contain LRR units—one, two, or three LRR domains at a time. Each mature lamprey lymphocyte expresses a complete and unique VLR gene, either VLRA, VLRB, or VLRC, which has undergone recombination of these flanking regions with the germline VLR gene.


Immunology_Janeway

Immunology_Janeway. The creation of a complete VLR gene is currently thought to occur during replication of lamprey lymphocyte DNA by a ‘copy-choice’ mechanism that is similar, but not identical, to gene conversion (described in Section 5-20). During DNA replication, LRR units flanking the VLR gene are copied into the VLR gene—presumably when a DNA strand being synthesized switches templates and copies sequences from one of these LRR units. Although final proof is still lacking, this template-switching mechanism may be triggered by enzymes of the AID-APOBEC family that are expressed by lamprey lymphocytes, and whose cytidine deaminase activity (CDA) could cause the single-strand DNA breaks that can start the copy-choice process. Lampreys possess two such enzymes: CDA1, which is expressed in VLRA-lineage lymphocytes, and CDA2, which is expressed in VLRB-lineage lymphocytes. It is not yet known if CDA1 or CDA2 is expressed in VLRC-expressing lymphocytes. The final VLR gene contains a complete


Immunology_Janeway

Immunology_Janeway. lymphocytes, and CDA2, which is expressed in VLRB-lineage lymphocytes. It is not yet known if CDA1 or CDA2 is expressed in VLRC-expressing lymphocytes. The final VLR gene contains a complete amino-terminal capping LRR subunit, followed by the addition of up to seven internal LRR domains, each 24 amino acids long, and the removal of the internal noncoding regions to complete the formation of the carboxy-terminal LRR domain (see Fig. 5.25).


Immunology_Janeway

Immunology_Janeway. It is estimated that this somatic rearrangement mechanism can generate as much diversity in the VLR proteins as is possible for immunoglobulins. Indeed, the crystal structure of a VLR protein shows that the concave surface formed by the series of LRR repeats interacts with a variable insert in the carboxy-terminal LRR to form a surface capable of interacting with a great diversity of antigens. Thus, the diversity of the anticipatory repertoire of agnathans may be limited not by the numbers of possible receptors they can generate but by the number of lymphocytes present in any individual, as in the adaptive immune system of their evolutionary cousins, the gnathostomes. As noted above, each lamprey lymphocyte rearranges only one of the two germline VLR genes, expressing either a complete VLRA or VLRB or VLRC protein. The first two cell populations seem to have some characteristics of mammalian T and B lymphocytes,


Immunology_Janeway

Immunology_Janeway. Top panel: an incomplete germline copy of a lamprey VLR gene contains a framework (right) for the complete gene: the portion encoding the signal peptide (SP), part of an amino-terminal LRR unit (NT, dark blue), and a carboxy-terminal LRR unit that is split into two parts (LRR, light red; and CT, red) by intervening noncoding DNA sequences. Nearby flanking regions (left) contain multiple copies of VLR gene-‘cassettes’ with single or double copies of variable LRR domains (green) and cassettes that encode part of the amino-terminal LRR domains (light blue, yellow). Middle panel: somatic recombination causes various LRR units to be copied into the original VLR gene. This creates a complete VLR gene that contains the assembled amino-terminal LRR cassette (LRR NT) and first LRR (yellow), followed by several variable LRR units (green) and the completed carboxy-terminal LRR unit, and ends with the portion that encodes the stalk region of the VLR receptor. The cytidine deaminases PmCDA1 and


Immunology_Janeway

Immunology_Janeway. by several variable LRR units (green) and the completed carboxy-terminal LRR unit, and ends with the portion that encodes the stalk region of the VLR receptor. The cytidine deaminases PmCDA1 and PmCDA2 from the lamprey


Immunology_Janeway

Immunology_Janeway. P. marinus are candidates for enzymes that may initiate this gene rearrangement. Expression of the rearranged gene results in a complete receptor that can be attached to the cell membrane by glycosylphosphatidylinositol (GPI) linkage of its stalk. Bottom panel: an individual lymphocyte undergoes somatic gene rearrangement to produce a unique VLR receptor. These receptors can be tethered to the surface of the lymphocyte via the GPI linkage or can be secreted into the blood. Unique somatic rearrangement events in each developing lymphocyte generate a repertoire of VLR receptors of differing specificities. Adapted from Pancer, Z., and Cooper, M.D.: Annu. Rev. Immunol. 2006, 24:497–518.


Immunology_Janeway

Immunology_Janeway. In vertebrates, evolution of the locus results in a multipart antigen-receptor locus that can be rearranged by RAG-mediated somatic recombination Recombination separates the RAG genes from the TR-tagged gene segments Insertion of the transposon into a V-type Ig receptor gene splits the gene into two TR TRRAG1 gene excision site RAG2 gene RAG1 gene RSS RSS RAG2 gene Transposon-derived RAG1/2-like gene cluster in a deuterostome ancestor Transposase activity can excise the transposon at terminal repeat sequences and reinsert it at a new location in the genome J JVV V-type Ig-like domain Fig. 5.26 Integration of a transposon into a V-type immunoglobulin receptor gene is thought to have given rise to the T-cell receptor and immunoglobulin genes.


Immunology_Janeway

Immunology_Janeway. Fig. 5.26 Integration of a transposon into a V-type immunoglobulin receptor gene is thought to have given rise to the T-cell receptor and immunoglobulin genes. respectively, and VLRC cells appear more closely related to the VLRA lineage. For example, VLRA-expressing lymphocytes also express genes similar to some mammalian T-cell cytokine genes, suggesting an even closer similarity to our own RAG-dependent adaptive immune system than was previously appreciated. repertoire of immunoglobulin-like genes appeared abruptly in the cartilaginous fishes.


Immunology_Janeway

Immunology_Janeway. Within the vertebrates, we can trace the development of immune functions from the agnathans through the cartilaginous fishes (sharks, skates, and rays) to the bony fishes, then to the amphibians, to reptiles and birds, and finally to mammals. RAG-dependent V(D)J recombination has not been found in agnathans, other chordates, or any invertebrate. The origins of RAG-dependent adaptive immunity are now becoming clearer as the genome sequences of many more animals become available. The first clue was that RAG-dependent recombination shares many features with the transposition mechanism of DNA transposons—mobile genetic elements that encode their own transposase, an enzymatic activity that allows them to excise from one site in the genome and reinsert themselves elsewhere. The mammalian RAG complex can act as a transposase in vitro, and even the structure of the RAG genes, which lie close together in the chromosome and lack the usual introns of mammalian genes, is reminiscent of a


Immunology_Janeway

Immunology_Janeway. RAG complex can act as a transposase in vitro, and even the structure of the RAG genes, which lie close together in the chromosome and lack the usual introns of mammalian genes, is reminiscent of a transposon.


Immunology_Janeway

Immunology_Janeway. All this provoked speculation that the origin of RAG-dependent adaptive immunity was the invasion of a DNA transposon into a gene similar to an immunoglobulin or a T-cell receptor V-region gene, an event that would have occurred in some ancestor of the jawed vertebrates (Fig. 5.26). DNA transposons carry inverted repeated sequences at either end, which are bound by the transposase for transposition to occur. These terminal repeats are considered to be the ancestors of the RSSs in present-day antigen-receptor genes (see Section 5-4), while the RAG-1 protein is believed to have evolved from a transposase. Subsequent duplication, reduplication, and recombination of the immune-receptor gene and its inserted RSSs eventually led to the separation of the RAG genes from the rest of the relic transposon and to the multisegmented immunoglobulin and T-cell receptor loci of present-day vertebrates.


Immunology_Janeway

Immunology_Janeway. The ultimate origins of the RSSs and the RAG-1 catalytic core are now thought to lie in the Transib superfamily of DNA transposons, and genome sequencing has led to the discovery of sequences related to RAG1 in animals as distantly related to vertebrates as the sea anemone Nematostella. The origin of RAG2 is


Immunology_Janeway

Immunology_Janeway. Top panel: a DNA transposon in an ancestor of the deuterostomes (the large group of phyla to which the chordates belong) is thought to have had genes related to RAG1 and RAG2—prototype RAG1 (purple) and RAG2 (blue), which acted as its transposase. DNA transposons are bounded by terminal inverted repeat (TR) sequences. Second panel: to excise a transposon from DNA, the transposase proteins (purple and blue) bind the TRs, bringing them together, and the transposase enzymatic activity cuts the transposon out of the DNA, leaving a footprint in the host DNA that resembles the TRs. After excision from one site, the transposon reinserts elsewhere in the genome, in this case into a V-type immunoglobulin receptor (green). The enzymatic activity of the transposase enables the transposon to insert into DNA in a reaction that is the reverse of the excision reaction. Third panel: the integration of the RAG1/2-like transposon into the middle of the gene for a V-type immunoglobulin receptor splits


Immunology_Janeway

Immunology_Janeway. into DNA in a reaction that is the reverse of the excision reaction. Third panel: the integration of the RAG1/2-like transposon into the middle of the gene for a V-type immunoglobulin receptor splits the V exon into two parts. Fourth and fifth panels: in the evolution of the immunoglobulin and T-cell receptor genes, the initial integration event has been followed by DNA rearrangements that separate the transposase genes (now known as the RAG1 and RAG2 genes) from the transposon TRs, which we now term the recombination signal sequences (RSSs). The purple sea urchin (an invertebrate deuterostome) has a RAG1/2-like gene cluster (not shown) and expresses proteins similar to RAG-1 and RAG-2 proteins, but does not have immunoglobulins, T-cell receptors, or adaptive immunity. The RAG-like proteins presumably retain some other cellular function (so far unknown) in this animal.


Immunology_Janeway

Immunology_Janeway. more obscure, but a RAG1–RAG2-related gene cluster was recently discovered in sea urchins, invertebrate relatives of the chordates. Sea urchins themselves show no evidence of immunoglobulins, T-cell receptors, or adaptive immunity, but the proteins expressed by the sea-urchin RAG genes form a complex with each other and with RAG proteins from the bull shark (Carcharhinus leucas), a primitive jawed vertebrate, but not with those from mammals. This suggests that these proteins could indeed be related to the vertebrate RAGs, and that RAG-1 and RAG-2 were already present in a common ancestor of chordates and echinoderms (the group to which sea urchins belong), presumably fulfilling some other cellular function.


Immunology_Janeway

Immunology_Janeway. The origin of somatic gene rearrangement in the excision of a transposable element makes sense of an apparent paradox in the rearrangement of immune-system genes. This is that the RSSs are joined precisely in the excised DNA (see Section 5-5), which has no further function and whose fate is irrelevant to the cell, whereas the cut ends in the genomic DNA, which form part of the immunoglobulin or T-cell receptor gene, are joined by an error-prone process, which could be viewed as a disadvantage. However, when looked at from the transposon’s point of view, this makes sense, because the transposon preserves its integrity by this excision mechanism, whereas the fate of the DNA it leaves behind is of no significance to it. As it turned out, the error-prone joining in the primitive immunoglobulin gene generated useful diversity in antigen-recognition molecules and was strongly selected for. The RAG-based rearrangement system also provided something else that mutations could not—a means of


Immunology_Janeway

Immunology_Janeway. gene generated useful diversity in antigen-recognition molecules and was strongly selected for. The RAG-based rearrangement system also provided something else that mutations could not—a means of rapidly modifying the size of the coding region, not just its diversity.


Immunology_Janeway

Immunology_Janeway. The next question is what sort of gene the transposon inserted into. Proteins containing Ig-like domains are ubiquitous throughout the plant, animal, and bacterial kingdoms, making this one of the most abundant protein super-families; in species whose genomes have been fully sequenced, the immunoglobulin superfamily is one of the largest families of protein domains in the genome. The functions of the members of this superfamily are very disparate, and they are a striking example of natural selection taking a useful structure— the basic Ig-domain fold—and adapting it to different purposes.


Immunology_Janeway

Immunology_Janeway. The immunoglobulin superfamily domains can be divided into four families on the basis of differences in structure and sequence of the immunoglobulin domain. These are V (resembling an immunoglobulin variable domain), C1 and C2 (resembling constant-region domains), and a type of immunoglobulin domain called an I domain (for intermediate). The target of the RSS-containing element is likely to have been a gene encoding a cell-surface receptor containing an Ig-like V domain, most probably a type similar to present-day VJ domains. These domains are found in some invariant receptor proteins and are so called because of the resemblance of one of the strands to a J segment. It is possible to imagine how transposon movement into such a gene could produce separate V and J gene segments (see Fig. 5.26). On the basis of phylogenetic analysis, agnathan paired receptors resembling Ag receptors, or APARs, which are encoded by a multigene family found in hagfish and lamprey, are currently the best


Immunology_Janeway

Immunology_Janeway. 5.26). On the basis of phylogenetic analysis, agnathan paired receptors resembling Ag receptors, or APARs, which are encoded by a multigene family found in hagfish and lamprey, are currently the best candidates for being relatives of the ancestor of the antigen receptor. Their DNA sequences predict single-pass transmembrane proteins with a single extracellular VJ domain and a cytoplasmic region containing signaling modules. APARs are expressed in leukocytes.


Immunology_Janeway

Immunology_Janeway. 5-20 Different species generate immunoglobulin diversity in different ways.


Immunology_Janeway

Immunology_Janeway. Most of the vertebrates we are familiar with generate a large part of their antigen receptor diversity in the same way as mice and humans, by putting together gene segments in different combinations. There are exceptions, however, even within the mammals. Some animals use gene rearrangement to always join together the same V and J gene segment initially, and then diversify this recombined V region. In birds, rabbits, cows, pigs, sheep, and horses, there is little or no germline diversity in the V, D, and J gene segments that are rearranged to form the genes for the initial B-cell receptors, and the rearranged V-region sequences are identical or similar in most immature B cells. These immature B cells migrate to specialized microenvironments—the bursa of Fabricius in the gut of chickens, and another intestinal lymphoid organ in rabbits. Here, B cells proliferate rapidly, and their rearranged immunoglobulin genes undergo further diversification.


Immunology_Janeway

Immunology_Janeway. In birds and rabbits this occurs mainly by gene conversion, a process by which short sequences in the expressed rearranged V-region gene are replaced with sequences from an upstream V gene segment pseudogene. The germline arrangement of the chicken heavy-chain locus is a single set of rearranging V, J, D, and C gene segments and multiple copies of V-segment pseudogenes. Diversity in this system is created by gene conversion in which sequences from the VH pseudogenes are copied into the single rearranged VH gene (Fig. 5.27). It seems that gene conversion is related to somatic hypermutation


Immunology_Janeway

Immunology_Janeway. Fig. 5.27 The diversification of chicken immunoglobulins occurs through gene conversion. In chickens, the immunoglobulin diversity that can be created by V(D)J recombination is extremely limited. Initially, there are only one active V, one J, and 15 D gene segments at the chicken heavy-chain locus and one active V and one J gene segment at the single light-chain locus (top left panel). Primary gene rearrangement can thus produce only a very limited number of receptor specificities (second panels). Immature B cells expressing this receptor migrate to the bursa of Fabricius, where the cross-linking of surface immunoglobulin (sIg) induces cell proliferation (second panels). The chicken genome contains numerous pseudogenes with a prerearranged VH–D structure. Gene conversion events introduce sequences from these adjacent V gene segment pseudogenes into the expressed gene, creating diversity in the receptors (third panels). Some of these gene conversions will inactivate the previously


Immunology_Janeway

Immunology_Janeway. sequences from these adjacent V gene segment pseudogenes into the expressed gene, creating diversity in the receptors (third panels). Some of these gene conversions will inactivate the previously expressed gene (not shown). If a B cell can no longer express sIg after such a gene conversion, it is eliminated. Repeated gene conversion events can continue to diversify the repertoire (bottom panels).


Immunology_Janeway

Immunology_Janeway. Sequences from V pseudogenes are introduced into rearranged V genes through gene conversion Multiple rounds of gene conversion can alter affnities of antibody for antigen C˜VDJ˜C°VJ°C˜VDJ˜C°VJ°C˜VDJ˜C°VJ°Immature chicken B cells. All have rearranged the same VH and V˜ genes V H D˜C˜J˜V°C°J°VH pseudogenes V° pseudogenes Germline chicken immunoglobulin genes in its mechanism, because gene conversion in a chicken B-cell line has been shown to require the enzyme activation-induced cytidine deaminase (AID). In Chapter 10, we will see that this same enzyme is involved in class switching and affinity maturation of the antibody response. For gene conversion, it is thought that single-strand cuts in DNA generated by the endonuclease apurinic/apyrimidinic endonuclease-1 (APE1) after the actions of AID are the signal that initiates a homology-directed repair process in which a homologous V pseudogene segment is used as the template for the DNA replication that repairs the V-region gene.


Immunology_Janeway

Immunology_Janeway. In sheep and cows, immunoglobulin diversification is the result of somatic hypermutation, which occurs in an organ known as the ileal Peyer’s patch. Somatic hypermutation, independent of T cells and a particular driving antigen, also contributes to immunoglobulin diversification in birds, sheep, and rabbits.


Immunology_Janeway

Immunology_Janeway. A more fundamentally different organization of immunoglobulin genes is found in the cartilaginous fish, the most primitive jawed vertebrates. Sharks have multiple copies of discrete VL–JL–CL and VH–DH–JH–CH cassettes, and activate rearrangement within individual cassettes (Fig. 5.28). Although this is somewhat different from the kind of combinatorial gene rearrangement of higher vertebrates, in most cases there is still a requirement for a RAG-mediated somatic rearrangement event. As well as rearranging genes, cartilaginous fish have multiple ‘rearranged’ VL regions (and sometimes rearranged VH regions) in the germline genome (see Fig. 5.28) and apparently generate diversity by activating the transcription of different copies. Even here, some diversity is also contributed by combinatorial means by the subsequent pairing of heavy and light chains.


Immunology_Janeway

Immunology_Janeway. This ‘germline-joined’ organization of the light-chain loci is unlikely to represent an intermediate evolutionary stage, because in that case the heavy-chain and light-chain genes would have had to independently acquire the capacity for rearrangement by convergent evolution. It is much more likely that, after the divergence of the cartilaginous fishes, some immunoglobulin loci became rearranged in the germline of various ancestors through activation of the RAG genes in germ cells, with the consequent inheritance of the rearranged loci by the offspring. In these species, the rearranged germline loci might confer some advantages, such as ensuring rapid responses to common pathogens by producing a preformed set of immunoglobulin chains. The IgM antibody isotype is thought to go back to the origins of adaptive immunity. It is the predominant form of immunoglobulin in cartilaginous


Immunology_Janeway

Immunology_Janeway. The IgM antibody isotype is thought to go back to the origins of adaptive immunity. It is the predominant form of immunoglobulin in cartilaginous Fig. 5.28 The organization of immunoglobulin genes is different in different species, but all can generate a diverse repertoire of receptors.


Immunology_Janeway

Immunology_Janeway. The organization of the immunoglobulin heavy-chain genes in mammals, in which there are separated clusters of repeated V, D, and J gene segments, is not the only solution to the problem of generating a diverse repertoire of receptors. Other vertebrates have found alternative solutions. In ‘primitive’ groups, such as the sharks, the locus consists of multiple repeats of a basic unit composed of a V gene segment, one or two D gene segments, a J gene segment, and a C gene segment. A more extreme version of this organization is found in the κ-like light-chain locus of some cartilaginous fishes such as the rays and the carcharhine sharks, in which the repeated unit consists of already rearranged VJ–C genes, from which a random choice is made for expression. In chickens, there is a single rearranging set of gene segments at the heavy-chain locus but there are multiple copies of pre-integrated VH–D segment pseudogenes. Diversity in this system is created by gene conversion, in which