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The Generation Of Diversity For Antigen Recognition

The Generation Of Diversity For Antigen Recognition
We know that the immune system has to be capable of recognizing virtually any pathogen that has arisen or might arise. The awesome genetic solution to this problem of anticipating an unpredictable future involves the generation of millions of different specific antigen receptors, probably vastly more than the lifetime needs of the individual. As this greatly exceeds the estimated number of 25 000–30 000 genes in the human body, there are some clever ways to generate all this diversity, particularly as the total number of V, D, J, and C genes in an individual human coding for antibodies and TCRs is only around 400. Let’s revisit the genetics of antibody diversity, and explore the enormous similarities, and occasional differences, seen with the mechanisms employed to generate TCR diversity.

Intrachain amplification of diversity
Random VDJ combination increases diversity geometrically
We saw in Chapter 3 that, just as we can use a relatively small number of different building units in a child’s construction set such as LEGO® to create a rich variety of architectural masterpieces, so the individual receptor gene segments can be viewed as building blocks to fashion a multiplicity of antigen specific receptors for both B‐ and T‐cells. The immunoglobulin light chain variable regions are created from V and J segments, and the heavy chain variable regions from V, D, and J segments. Likewise, for both the αβ and γδ T‐cell receptors the variable region of one of the chains (α or γ) is encoded by a V and a J segment, whereas the variable region of the other chain (β or δ) is additionally encoded by a D segment. As for immunoglobulin genes, the enzymes RAG‐1 and RAG‐2 recognize recombination signal sequences (RSSs) adjacent to the coding sequences of the TCR V, D, and J gene segments. The RSSs again consist of conserved heptamers and nonamers separated by spacers of either 12 or 23 base‐pairs and are found at the 3′ side of each V segment, on both the 5′ and 3′ sides of each D segment, and at the 5′ of each J segment. Incorporation of a D segment is always included in the rearrangement; Vβ cannot join directly to Jβ, nor Vδ directly to Jδ. To see how sequence diversity is generated for TCR, let us take the αβ TCR as an example (Table 4.2). Although the precise number of gene segments varies from one individual to another, there are typically around 75 gene segments and 60 Jα gene segments. If there were entirely random joining of any one V to any one J segment, we would have the possibility of generating 4500 VJ combinations (75 × 60). Regarding the TCR β‐chain, there are approximately 50 Vβ genes that lie upstream of two clusters of DβJβ genes, each of which is associated with a Cβ gene (Figure 4.11). The first cluster, associated with Cβ1, has a single Dβ1 gene and 6 Jβ1 genes, whereas the second cluster associated with Cβ2 again has a single Dβ gene (Dβ2) with 7 Jβ2
The Dβ1 segment can combine with any of the 50 Vβ genes and with any of the 13 Jβ1 and Jβ2 genes (Figure 4.11). β2 behaves similarly but can only combine with one of the 7 downstream Jβ2 genes. This provides 1000 different possible VDJ combinations for the TCR β‐chain. Therefore, although the TCR α and β chain V, D, and J genes add up arithmetically to just 200, they produce a vast number of different α and β variable regions by geometric recombination of the basic elements. But, as with immunoglobulin gene rearrangement, that is only the beginning.
Figure 4.11 Rearrangement of the T‐cell receptor β‐chain gene locus. In this example Dβ1 has rearranged to Jβ2.2, and then the Vβ2 gene selected out of the 50 or so (Vβn) Vβ genes. If the same V and D segments had been used, but this time Jβ1.4 had been employed, then the Cβ1 gene segment would have been utilized instead of Cβ2.

Playing with the junctions
Another ploy to squeeze more variation out of the germline repertoire that is used by both the TCR and the immunoglobulin genes (see Figure 3.25) involves variable boundary recombinations of V, D, and J to produce different junctional sequences (Figure 4.12.).
As discussed in Chapter 3, further diversity results from the generation of palindromic sequences (P‐elements) arising from the formation of hairpin structures during the recombination process and from the insertion of nucleotides at the N region between the V, D, and J segments, a process associated with the expression of terminal deoxynucleotidyl transferase. While these mechanisms add nucleotides to the sequence, yet more diversity can be created by nucleases chewing away at the exposed strand ends to remove nucleotides. These maneuvers again greatly increase the repertoire, especially important for the TCR γ and δ genes, which are otherwise rather limited in number.
Additional mechanisms relate specifically to the D‐region sequence: particularly in the case of the TCR δ genes, where the D segment can be read in three different reading frames and two D segments can join together. Such DD combinations produce a longer third complementarity determining region (CDR3) than is found in other TCR or antibody molecules.
As the CDR3 in the various receptor chains is essentially composed of the regions between the V(D)J segments, where junctional diversity mechanisms can introduce a very high degree of amino acid variability, one can see why it is that this hypervariable loop usually contributes the most to determining the fine antigen‐binding specificity of these molecules.
Figure 4.12 Junctional diversity between a TCR V α and J α germline segment producing three variant protein sequences. The nucleotide triplet that is spliced out is colored the darker blue. For TCR β chain and Ig heavy chain genes junctional diversity can apply to V, D, and J segments.

Receptor editing
Recent observations have established that lymphocytes are not necessarily stuck with the antigen receptor they initially make: if they don’t like it they can change it. The replacement of an undesired receptor with one that has more acceptable characteristics is referred to as receptor editing. This process has been described for both immunoglobulins and for TCR, allowing the replacement of either nonfunctional rearrangements or autoreactive specificities. Furthermore, receptor editing in the periphery may rescue low‐affinity B‐cells from apoptotic cell death by replacing a low‐affinity receptor with a selectable one of higher affinity. That this does indeed occur in the periphery is strongly supported by the finding that mature B‐cells in germinal centers can express RAG‐1 and RAG‐2 that mediate the rearrangement process.
But how does this receptor editing work? Well, in the case of the receptor chains that lack D gene segments, namely the immunoglobulin light chain and the TCR α chain, a secondary rearrangement may occur by a V gene segment upstream of the previously rearranged VJ segment recombining to a 3′ J gene sequence, both of these segments having intact RSSs that are compatible (Figure 4.13a). However, for immunoglobulin heavy chains and TCR β deletes all of the D segment‐associated RSSs (Figure 4.13b). Because VH and JH both have 23 basepair spacers in their RSSs, they cannot recombine: that would break the 12/23 rule. This apparent obstacle to receptor editing of these chains may be overcome by the presence of a sequence near the 3′ end of the V coding sequences that can function as a surrogate RSS, such that the new V segment would simply replace the previously rearranged V, maintaining the same D and J sequence (Figure 4.13b). This is probably a relatively inefficient process and receptor editing may therefore occur more readily in immunoglobulin light chains and  TCR α chains than in immunoglobulin heavy chains and TCR β chains. Indeed, it has been suggested that the TCR α chain may undergo a series of rearrangements, continuously deleting previously functionally rearranged VJ segments until a selectable TCR is produced.
Receptor editing
Figure 4.13 Receptor editing. (a) For immunoglobulin light chain or TCR α chain the recombination signal sequences (RSSs; heptamer– nonamer motifs) at the 3′ end of each variable (V) segment and the 5′ of each joining (J) segment are compatible with each other and therefore an entirely new rearrangement can potentially occur as shown. This would result in a receptor with a different light chain variable sequence (in this example Vκ37Jκ4 replacing Vκ39Jκ3) together with the original heavy chain. (b) With respect to the immunoglobulin heavy chain or TCR β chain the organization of the heptamer–nonamer sequences in the RSS precludes a V segment directly recombining with the J segment. This is the so‐called 12/23 rule whereby the heptamer–nonamer sequences associated with a 23 base‐pair spacer (colored violet) can only base‐pair with heptamer–nonamer sequences containing a 12 base‐pair spacer (colored red). The heavy chain V and J both have an RSS with a 23 base‐pair spacer and so this is a nonstarter. Furthermore, all the unrearranged D segments have been deleted so that there are no 12 base‐pair spacers remaining. This apparent bar to secondary rearrangement is probably overcome by the presence of an RSS‐like sequence near the 3′ end of the V gene coding sequences, so that only the V gene segment is replaced (in the example shown, the sequence VH38DH3JH2 replaces VH40DH3JH2).

Recognition of the correct genomic regions by the RAG recombinase
A question that is only now being resolved is how the RAG‐1/ RAG‐2 recombinase selects the correct genomic regions to target for recombination. Clearly it would be disastrous were this complex able to access all DNA, randomly leaving double‐stranded breaks in its wake. One mechanism of protection is to induce RAG expression only where and when it is needed, but this does not explain how the RAG complex is targeted only to Ig and TCR loci in the cells in which it is expressed. This puzzle is explained by observations suggesting that alterations to histones – the proteins upon which DNA is packaged – flag particular loci for binding of the RAG complex. Recent studies have shown that histone H3 that has been modified by tri­ methylation on lysine at position 4 (H3K4me3) acts as a bind­ ing site for RAG‐2. Thus, genomic regions that are poised for VDJ recombination are located close to H3K4me3 histone “marks.” Consistent with this idea, experimental ablation of H3K4me3 marks results in greatly impaired V(D)J recombination. But the H3K4me3 mark is found at many more sites throughout the genome than there are antigen receptor loci, so how does the RAG‐1/RAG‐2 complex find the correct sites? The answer seems to be that the specificity of RAG‐1 for RSS sites, combined with that of RAG‐2 for H3K4me3 chromatin marks, may act as a clamp that guides the recombinase to the right locations. Binding of the RAG complex to the H3K4me3 mark may also activate the recombinase activity of RAG‐1 through an allosteric mechanism, increasing the catalytic activ­ ity of the complex when it has been positioned at the correct location.

Interchain amplification
The immune system took an ingenious step forward when two different types of chain were utilized for the recognition molecules because the combination produces not only a larger combining site with potentially greater affinity, but also new variability. Heavy–light chain pairing among immunoglobulins appears to be largely random and therefore two B‐cells can employ the same heavy chain but different light chains. This route to producing antibodies of differing specificity is easily seen in vitro where shuffling different recombinant light chains against the same heavy chain can be used to either fine‐tune, or sometimes even alter, the specificity of the final antibody. In general, the available evidence suggests that in vivo the major contribution to diversity and specificity comes from the heavy chain, perhaps not unrelated to the fact that the heavy chain CDR3 gets off to a head start in the race for diversity being, as it is, encoded by the junctions between three gene segments: V, D, and J.
This random association between TCR γ and δ chains, TCR α and β chains, and Ig heavy and light chains yields a further geometric increase in diversity. From Table 4.2 it can be seen that approximately 230 functional TCR and 153 functional Ig germline segments can give rise to 4.5 million and 2.3 million different combinations, respectively, by straight­forward associations without taking into account all of the fancy junctional mechanisms described above. Hats off to evolution!

Somatic hypermutation
As discussed in Chapter 3, there is inescapable evidence that immunoglobulin V‐region genes can undergo significant somatic hypermutation. Analysis of 18 murine λ myelomas revealed 12 with identical structure, four showing just one amino acid change, one with two changes and one with four changes, all within the hypervariable regions and indicative of somatic hypermutation of the single mouse λ germline gene. In another study, following immunization with pneumococcal antigen, a single germline T15 VH gene gave rise by mutation to several different VH genes all encoding phosphorylcholine antibodies (Figure 4.14).
A number of features of this somatic diversification phe­ nomenon are worth revisiting. The mutations are the result of single nucleotide substitutions, they are restricted to the variable as distinct from the constant region and occur in both framework and hypervariable regions. The mutation rate is remarkably high, approximately 1 × 10−3 per base‐pair per generation, which is approximately a million times higher than for other mammalian genes. In addition, the mutational mechanism is bound up in some way with class switch recombination as the enzyme activation‐induced cytidine deaminase (AID) is required for both processes and hypermutation is more frequent in IgG and IgA than in IgM antibodies, affecting both heavy (Figure 4.14) and light chains. However, VH genes are, on average more mutated than VL genes. This might be a consequence of receptor editing acting more frequently on light chains, as this would have the effect of wiping the slate clean with respect to light chain V gene mutations while maintaining already accumulated heavy chain V gene point mutations.
As we outlined in Chapter 3, AID initiates both class switch recombination as well as somatic hypermutation through deaminating deoxycytidine within certain DNA hotspots that are characterized by the presence of WRC sequences (W = A or T, R = purine, and C is the deoxycytidine that becomes deaminated). Although the target of AID was initially thought to be RNA, more recent evidence suggests that this enzyme works directly on DNA, although RNA editing is not ruled out. Deamination of deoxycytidine changes this base to a deoxyuracil that would normally be repaired by mismatch repair enzymes but, for reasons that are not yet fully understood, can result in removal of the mismatched uracil that generates a gap that is filled in by an errorprone polymerase to generate a point mutation at this position and can also mutate surrounding bases. It remains unclear how AID is targeted to the correct locations within V regions of rearranged Ig genes, to ensure that mutations are not inadvertently introduced at other loci, but similar to the RAG recombinase, this might involve specific histone modifications. Hyperacetylated versions of histones H3 and H4 appear to be more abundant in mutating V regions than in the C regions of Ig genes. This observation, coupled with observations that AID is recruited to actively transcribing Ig genes by proteins that bind to CAGGTG sequences found in all Ig transcriptional enhancers, suggests a possible mechanism. Thus, the combination of the CAGGTG sequence motif, coupled with the modified histones discussed above, may position AID at the correct locations from which to operate.
Somatic hypermutation does not appear to add significantly to the repertoire available in the early phases of the primary response, but occurs during the generation of memory and is responsible for tuning the response towards higher affinity.
Recently, data have been put forward suggesting that there is yet another mechanism for creating further diversity. This involves the insertion or deletion of short stretches of nucleotides within the immunoglobulin V gene sequence of both heavy and light chains. This mechanism would have an intermediate effect on antigen recognition, being more dramatic than single point mutation, but considerably more subtle than receptor editing. In one study, a reverse transcriptase‐polymerase chain reaction (RT‐PCR) was employed to amplify the expressed VH and VL genes from 365 IgG+ B‐cells and it was shown that 6.5% of the cells contained nucleotide insertions or deletions. The transcripts were left in‐frame and no stop codons were introduced by these modifications. The percentage of cells containing these alterations is likely to be an underestimate. All the insertions and deletions were in, or near to, CDR1 and/or CDR2. N‐region diversity of the CDR3 meant that it was not possible to analyze the third hypervariable region for insertions/deletions of this type and therefore these would be missed in the analysis. The fact that the alterations were associated with CDRs does suggest that the B‐cells had been subjected to selection by antigen. It was also notable that the insertions/deletions occurred at known hotspots for somatic point mutation, and the same error‐prone DNA polymerase responsible for somatic hypermutation may also be involved here. The sequences were often a duplication of an adjacent sequence in the case of insertions or a deletion of a known repeated sequence. This type of modification may, like receptor editing, play a major role in eliminating autoreactivity and also in enhancing antibody affinity.
T‐cell receptor genes, on the other hand, do not generally undergo somatic hypermutation. It has been argued that this would be a useful safety measure as T‐cells are positively selected in the thymus for weak reactions with self MHC, so that mutations could readily lead to the emergence of high‐ affinity autoreactive receptors and autoimmunity.
One may ask how it is that this array of germline genes is protected from genetic drift. With a library of 390 or so functional V, D, and J genes, selection would act only weakly on any single gene that had been functionally crippled by mutation and this implies that a major part of the library could be lost before evolutionary forces operated. One idea is that each subfamily of related V genes contains a prototype coding for an antibody indispensable for protection against some common pathogen, so that mutation in this gene would put the host at a disadvantage and would therefore be selected against. If any of the other closely related genes in its set became defective through mutation, this indispensable gene could repair them by gene conversion, a mechanism in which two genes interact in such a way that the nucleotide sequence of part or all of one becomes identical to that of the other. Although gene conver­ sion has been invoked to account for the diversification of MHC genes, it can also act on other families of genes to main­ tain a degree of sequence homogeneity. Certainly it is used extensively by, for example, chickens and rabbits, in order to generate immunoglobulin diversity. In the rabbit only a single germline VH gene is rearranged in the majority of B‐cells; this then becomes a substrate for gene conversion by one of the large number of VH pseudogenes. There are also large numbers of VH pseudogenes and orphan genes (genes located outside the gene locus, often on a completely different chromosome) in humans that actually outnumber the functional genes, although there is no evidence to date that these are used in gene conversion processes.

Figure 4.14 Mutations in regions of five IgM and five IgG monoclonal phosphorylcholine antibodies generated during an antipneumococcal response in a single mouse are compared with the primary structure of the T15 germline sequence. A line indicates identity with the T15 prototype and an orange circle a single amino acid difference. Mutations have only occurred in the IgG molecules and are seen in both hypervariable and framework segments. (After Gearhart P.J. (1982) Immunology Today 3, 107.) Although in some other studies somatic hypermutation has been seen in IgM antibodies, the amount of mutation usually greatly increases following class switching.

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