Somatic Hypermutation - an overview (2023)

Somatic Hypermutation.

Centroblasts undergo somatic hypermutation of the immunoglobulin V region genes, which alters the antigen affinity of the antibody produced by the cell.^76,77 This process requires the activity of AID, which is induced in these cells. Somatic hypermutation results in marked intraclonal diversity of antibody-combining sites in a population of cells derived from only a few precursors. Studies of single centroblasts picked from the dark zone of germinal centers suggest that in the early stages, a germinal center may contain about 5 to 10 clones of centroblasts, which show only a moderate amount of immunoglobulin V region gene mutation; later, the number of clones diminishes to as few as three, and the degree of somatic mutation increases.⁵⁷ This process introduces somatic mutations in other genes expressed in the germinal center, such asBCL6 andPAX5, although at a lower frequency than is seen in the immunoglobulin genes.^78-80

1.1 Somatic Hypermutation

Somatic mutations of the V-region have been observed to accumulate between about 100 base pairs and about 2kb downstream of the promoter of the rearranged V(D)J gene, with the peak of mutation accumulation occurring within the V(D)J exon [7–10]. However, although the rest of the genome is normally protected from AID-induced mutations, low levels of SHM have been observed at non-antibody genes [11–17], and indeed, mutations at some of these genes have been associated with B-cell malignancies. Because the overexpression of AID leads to malignancies in T-cells and other tissues, but not in B-cells [18,19], it appears that B-cells have evolved to tolerate high mutation rates without eliciting a commensurate DNA damage response, cell cycle arrest, or apoptosis; but how this is accomplished remains an open question. We will address a number of possible explanations during this chapter.

Somatic Hypermutation

Immunoglobulin variants are generated among germinal center B cells by the process of somatic hypermutation, in which immunoglobulin genes accumulate apparently random point mutations within productively rearranged V, D, and J segments.⁸⁵² These variants undergo a selection process favoring B cells that bear sIg with high affinity for antigen. Such high-affinity immunoglobulin provides high levels of BCR signaling, favoring germinal center B-cell survival rather than a default pathway of apoptosis. Somatic hypermutation requires activation-induced cytidine deaminase (AID), which appears to be expressed only by germinal center B cells,⁸⁵³ and error-prone DNA polymerases.⁸⁵⁴ AID deaminates deoxycytidine residues in single-stranded DNA to deoxyuridines, which are processed by DNA replication, base excision, or mismatch repair to restore normal base pairing between the two DNA strands, resulting in somatic hypermutation. The effects of the mutator are focused on the variable region of immunoglobulin and its immediate flanking sequences. The peak of somatic mutation is approximately 10 to 12 days after immunization with a protein antigen. Somatic mutation is an important means for increasing antibody affinity, but it may also result in the acquisition of autoimmunity. Thus human B cells with newly acquired autoreactivity as a result of somatic mutation are not subject to receptor editing or other tolerance mechanisms.

Most neonatal and fetal immunoglobulin heavy chain gene variable regions appear not to have undergone somatic mutation,^806,808 consistent with the predominance of antigenically-naïve new emigrant B cells in the fetus and neonate. By contrast, somatic mutations are detectable in some neonatal B cells expressing IgG or IgA transcripts,⁸¹² and the mutational frequency per length of DNA is similar to that of adult B cells. In contrast to follicular B cells, in which somatic hypermutation appears to occur only in germinal centers, a subset of IgM⁺IgD⁺CD27⁺ B cells found in cord blood and in the fetal spleen demonstrate somatic hypermutation before the general appearance of germinal centers.⁸⁵⁵ In this context, extrafollicular somatic hypermutation appears to serve as a means to broaden the preimmune immunoglobulin repertoire of this B-cell subset. This early competency for somatic hypermutation by fetal B cells is supported by the similar ability of cord blood and adult peripheral blood B cells to increase expression of AID and error-prone polymerases in response to BCR engagement.⁸⁵⁴

4.1 Diversity of Immune Cells by Activation-Induced Deaminase

Somatic hypermutation (SHM) and class switch recombination (CSR) are necessary for antibody diversification in antigen-specific memory B cells, and both mechanisms require activation-induced deaminase (AID) [164–166]. Because AID deaminates cytosine to generate uracil in DNA, a well-known BER substrate, involvement of BER in this pathway is being established [45,166]. The canonical BER reactions depicted in Fig. 17.1 do not likely occur during SHM and CSR. Instead, when uracil is generated in DNA by AID, it serves as a flag to recruit error-prone bypassing DNA polymerases for SHM and components of DNA DSB repair cooperate with some of the BER enzymes to lead to CSR [46,164]. Continued understanding of the mechanisms of SHM and CSR involving BER, other DNA-repair and -signaling pathways should illuminate the sophisticated crosstalk among the DNA-repair pathways.

Affinity maturation depends on somatic hypermutation and cell selection

The antibodies produced in a primary response to a TD antigen generally have a low average affinity. However, during the course of the response, the average affinity of the antibodies increases or matures. As antigen becomes limiting, the clones with the higher affinity will have a selective advantage. This process is calledaffinity maturation.

Although individual B cells do not usually change their overall specificity, the affinity of the antibody produced by a clone may be altered. Affinity maturation is achieved through the cellular events underlying B-cell development, described earlier: i.e. somatic hypermutation and antigen-driven selection and expansion of mutant clones expressing higher-affinity antibodies.

The mechanism by which affinity maturation occurs is thought to involve B-cell progeny binding to antigen held on FDCs in order to proliferate and to differentiate further. Unprocessed antigen in immune complexes is captured by the FDCs via their Fc and complement receptors and held there. As B cells encounter the antigen, there is competition for space on the surface of the FDC, leading to selection. When a B cell with higher affinity arises, it will stay there longer and be given a stronger signal. In addition, B cells with higher-affinity receptors will internalize more antigen and therefore they have a greater potential of presenting it to T cells and receiving T-cell help. Hence, B cells with higher-affinity antibodies have a selective survival advantage.

2 Molecular Mechanisms of SHM and CSR

SHM Occurs Also in Non-Ig Genes (from Storb, 2014)

SHM is initiated by AID deaminating cytosine (C) to uracil (U). This first phase is followed by a second phase (Neuberger etal., 2003) of error-prone repair of the uracils by corrupting the DNArepair mechanisms that outside of the SHM process faithfully revert the U to C: base excision repair (BER) and mismatch repair (MMR) (Longerich etal., 2006). How exactly this shift to error-prone mechanisms occurs is notknown, but the errors are due to the engagement of error-prone DNA lesion bypass DNA polymerases, such as pol eta (Sale etal., 2009). However, the system is not entirely fail-safe. Non-Ig genes are also mutated as bystanders; for example, the BCL6 gene is mutated in normal human memory B cells (Shen etal., 1998), and later was found mutated in germinal center-derived B cell lymphomas (Pasqualucci etal., 2001).

A large-scale DNA sequencing analysis of non-Ig genes in Peyer's patch B cells showed that many genes were mutated in AID-producing B cells from mice double deficient in DNA MMRand BER(Liu etal., 2008). The authors concluded that B cells were protected from excess mutagenesis by employing a separate mechanism for error-free repair in non-Ig genes. However, an in-depth review of SHM showed that Ig genes, the major biological targets of SHM, are also undergoing a high rate of error-free repair of AID-induced uracils (Storb etal., 2009). Thus, apparently error-free repair of SHM-associated uracils is universal. It represents an additional protection against genome-wide mutagenesis in B cells that express AID. It is likely that in cells that are wild type (wt) for DNA repair genes, Ig genes acquire more mutations than non-Ig genes because they are highly expressed and contain at leasttwo enhancers that are bound by various trans-acting factors that efficiently attract AID and are required for SHM. It appears that most, if not all, the non-Ig targets contain similar sequence motifs as those present in the enhancers of Ig genes (Duke etal., 2013).

2 Physiologic Role of Activation-Induced Cytidine Deaminase

SHM and CSR are unique genetic events specifically observed in B cells. SHM is defined as the introduction of nucleotide alterations at the V regions of heavy and light chain genes with a high frequency, thereby enabling the selection of B cells producing high-affinity antibodies [5–7]. In contrast, CSR exchanges the heavy chain constant domain (C_H) for one of a set of downstream C_H exons, allowing B cells to produce different antibody isotypes (IgG, IgA, or IgE) with distinct physiologic functions, but without changing antigen specificity [8,9]. Although these two events occur independently [10,11], they are similar in that they basically take place in the germinal center of antigen-stimulated B cells [12–14]. These B cell–specific genetic modification events are initiated by the same molecule, activation-induced cytidine deaminase (AID) [15,16]. AID is a member of the apolipoprotein B mRNA-editing enzyme catalytic polypeptide (APOBEC) family comprising nucleotide-editing enzymes that insert nucleotide alterations into target DNA or RNA sequences through cytidine deamination [17]. AID is expressed only at the germinal center of activated B cells under physiologic conditions [15,18]. No hypermutations or gene-switching events at the Ig loci are found in patients with congenital AID deficiency [16,19]. Furthermore, the fact that AID-deficient B cells fail to undergo SHM and CSR confirms that AID is essential for both of these events [16,20,21]. AID was first considered to be an RNA-editing enzyme that edits target mRNA sequences to initiate either SHM or CSR [15,16,22]. Recent studies, however, support a role of AID in the deamination of DNA sequences [23–34].

Somatic mutation

COMPARISON OF CSR WITH SHM

CSR and SHM were previously thought to be mediated by different mechanisms, and the requirement of AID for both events surprised many scientists in the field. However, when we carefully compare the two events, they share several features that are critical to the molecular mechanisms of SHM and CSR. The targets of CSR and SHM must be transcribed before the reaction takes place. The efficiency of these genetic alterations is correlated with the level of transcription. The targets of both events do not have specific primary sequences, yet defined DNA regions are altered. Not only SHM but also CSR is associated with mutations. Most important, they both require AID activity.

CSR has important features mechanistically distinct from SHM. Not only do the products of CSR and SHM differ but also the initiation of the two events is dissimilar. CSR requires two DSBs, one each in two different S regions. By contrast, SHM is more likely to be initiated by a single nick on one strand of the target DNA (Faili et al., 2002b). During CSR, two separate DNA ends, which originally can be located 100 kb apart, have to be held in a close proximity. Without proteins that can hold the two recombining ends close together, it is probably impossible to join the DNA ends. It is therefore reasonable to assume that CSR depends on a protein complex that differs from a SHM complex. The CSR complex, which we refer to as “recombinasome,” may contain AID or the putative protein encoded by the mRNA it edits, MMR proteins, base excision repair enzymes, error-prone DNA polymerases, and NHEJ proteins. Although SHM may not require a complex of proteins involved in DNA synapsis, the “mutasome” may contain the DNA nicking enzyme, MMR, base excision repair enzymes, and error-prone DNA polymerases. Additional components in these complexes may be required to target them to the correct DNA segments.

During evolution, SHM appeared first in cartilaginous and bony fish, whereas these fish do not appear to have CSR (Litman et al., 1999; Flajnik, 2002). Amphibians, which evolved later, were the first to carry out both CSR and SHM. Another indication of the distinct functions of AID for CSR and SHM is found in hyper-IgM type II patients with mutations in AID who have significant levels of SHM, but almost no CSR (Revy et al., 2000). The results can be explained most easily by the possibility that AID interacts with different protein molecules when inducing SHM and CSR. Perhaps, in these patients, proteins required for CSR are unable to associate with the mutated AID, whereas proteins required for SHM interact normally. In addition, CSR and SHM are independently regulated. For example, LPS stimulation of splenic B cells efficiently induces CSR but very rarely SHM. Thus, although a single protein, AID, mediates both CSR and SHM, the two processes evolved stepwise and are regulated differently, although it is possible that in germinal center cells they occur concurrently. Altogether, the data suggest that CSR and SHM are likely to have different molecular mechanisms downstream of AID.