Affinity maturation is the process that leads to increased affinity of antibodies for a particular antigen as a T-dependent humoral response progresses, and it is the result of somatic mutation of Ig genes followed by selective survival of the B cells that produce the antibodies with the highest affinities. The process of affinity maturation generates antibodies with an increased ability to bind antigens and thus to more efficiently neutralize and eliminate microbes and their toxins (Fig. 1). Helper T cells and CD40:CD40L interactions are required for somatic mutation to be initiated, and, as a result, affinity maturation is observed only in antibody responses to T-dependent protein antigens.
Fig1. Affinity maturation. Early in the immune response, low affinity antibodies are produced. During the germinal center reaction, somatic mutation of immunoglobulin (Ig) V genes and selection of B cells with high-affinity antigen receptors result in the production of anti bodies with high affinity for antigen.
In proliferating germinal center B cells in the dark zone, rearranged Ig V genes undergo point mutations at an extremely high rate. This rate is estimated to be 1 in 103 V gene base pairs per cell division, which is approximately 1000 times higher than the spontaneous rate of mutation in other mammalian genes. For this reason, mutations in rearranged Ig V genes are called somatic hypermutation. The V genes of expressed heavy and light chains in each B cell contain a total of approximately 700 nucleotides. This implies that mutations will accumulate in expressed V regions at an average rate of almost one per cell division. Ig V gene mutations continue to occur in the progeny of individual B cells. As a result, a B-cell clone can accumulate more and more mutations during its life in the germinal center. It is estimated that as a consequence of somatic mutations, the nucleotide sequences of IgG antibodies derived from one clone of B cells can diverge as much as 5% from the original germline sequence. This usually translates to up to 10 amino acid substitutions. The mutations are clustered in the V regions, mostly in the antigen-binding complementarity-determining regions (CDRs) (Fig. 2), and the presence of mutations correlates with increasing affinities of the antibodies for the antigen that induced the response.
Fig2. Somatic mutations in immunoglobulin (Ig) V genes. Hybridomas were produced from the spleen cells of mice immunized 7 or 14 days previously with a hapten, oxazolone, coupled to a protein and from spleen cells obtained after secondary and tertiary immunizations with the same antigen. Hybridomas producing oxazolone-specific monoclonal antibodies were produced and the nucleotide sequences of the V genes encoding the Ig heavy and light chains were determined. Mutations in V genes increase with time after immunization and with repeated immunizations and are clustered in the complementarity-determining regions (CDRs). The location of CDR3 in the heavy chains is approximate. The affinities of the antibodies produced also tend to increase with more mutations, as indicated by the lower dissociation constants (Kd ) for hapten binding. (Modified from Berek C, Milstein C. Mutation drift and repertoire shift in maturation of the immune response. Immunol Rev. 1987;96:23–41.)
The enzyme AID, discussed earlier in the context of class switching, also plays an essential role in affinity maturation. The DNA deaminase activity of AID converts C residues to U residues at specific tetranucleotide (AGCT) hotspots that are found all over the genome but are targeted primarily in rear ranged V regions (or in switch regions, as discussed above). AID may recognize sequences in the location of the rearranged VDJ exon, which explains at least partially why rearranged V regions are highly susceptible to mutations. The mechanism by which these rearranged VDJ exons are specifically targeted by AID is still unclear. The Us that are generated from Cs may be changed to Ts when DNA replication occurs, thus generating a common type of C to T mutation, or the U may be excised by the enzyme uracil-DNA glycosylase (UNG), and the abasic site thus generated is repaired by an error-prone DNA repair process, eventually generating substitutions with any of the four DNA nucleotides at each site of AID-induced cytidine deamination. Two proteins, MSH2 and MSH6, involved normally in the process of DNA mismatch repair, are important participants in somatic hypermutation. MSH2 and MSH6 can recruit nucleases that remove not only “unnatural” uridine nucleotide but also adjacent nucleotides. This mutated stretch is then repaired by an error-prone DNA polymerase, thus extending mutations to residues beyond the C residues that are targeted by AID. It is unclear how two well-known DNA repair mechanisms, base excision repair (in which UNG is an important component) and mismatch repair, which are normally high-fidelity processes, recruit error-prone DNA polymerases in germinal center B cells in the context of somatic hypermutation.
Repeated stimulation by T cell–dependent protein antigens (in the light zone, as described below) leads to the migration of B cells into the dark zone and an increasing number of mutations in the Ig genes of antigen-specific germinal center B cells. Some of these mutations are likely to be useful because they will generate high-affinity antibodies. However, many of the mutations may result in a decline or even a loss of antigen binding. Therefore, the next and crucial step in the process of affinity maturation is the selection of the most useful, high-affinity B cells, a type of Darwinian natural selection that ensures survival of the best B cells (fittest in terms of antigen binding), and this occurs in the light zone. In the dark zone, some B cells undergo extensive DNA damage and apoptosis.
B cells that bind antigens in germinal centers with high affinity are selected to survive (Fig.3). The early response to antigen results in the production of antibodies, some of which form complexes with residual antigen and may activate complement. FDCs express receptors for the Fc portions of antibodies and for products of complement activation, including C3b and C3d. These receptors bind and display antigens that are complexed with antibodies and complement products. Meanwhile, germinal center B cells that have undergone somatic mutation migrate into the FDC-rich light zone of the germinal center. These B cells die by apoptosis, unless they are rescued by recognition of antigen. Only B cells with high-affinity receptors for the antigen are able to bind the antigen when it is present at low concentrations, and these B cells survive preferentially because of several mechanisms. First, antigen recognition induces the expression of the transcription factor c-MYC, which in turn induces expression of antiapoptotic proteins of the BCL-2 family. Second, high-affinity B cells will preferentially endocytose and present the antigen and interact with the limited numbers of Tfh cells in the germinal center. These helper T cells may signal via CD40L to promote the survival of the B cells with which they interact.
Fig3. B-cell selection in germinal centers. Somatic mutation of V genes in germinal center B cells generates antibodies with different affinities for antigen. Binding of the B cells to antigen displayed on follicular dendritic cells (FDCs) is necessary to rescue the B cells from programmed cell death. B cells may also present antigen to germinal center T follicular helper (Tfh) cells, which promote B-cell survival. The B cells with the highest affinity for antigen thus have a selective advantage for survival as the amount of available antigen decreases during an immune response. This leads to an average increase in the affinity of antibodies for antigen as the humoral immune response progresses. Ig, Immunoglobulin.
As more antibody is produced, more of the antigen is eliminated and less is available in the germinal centers. Therefore, the B cells that will be able to specifically bind this antigen and to be rescued from death need to express antigen receptors with higher and higher affinity for the antigen. As a result, as the antibody response to an antigen progresses, the B cells that are selected to survive in germinal centers produce Ig of increasing affinity for the antigen. This selection process results in affinity maturation of the antibody response, and is a rationale for boosters (repeated immunization) to increase the efficacy of vaccines. Because somatic mutation also generates many B cells that do not express high-affinity receptors for antigen and cannot therefore be selected to survive, the germinal centers are sites of tremendous apoptosis.
The DNA breaks associated with somatic hypermutation and class switching predispose to chromosomal translocations of various oncogenes into Ig gene loci, producing tumors of B cells (lymphomas). This may explain why many lymphomas develop from germinal center B cells. Germinal centers may also, in a few diseases driven by high-affinity autoantibodies, contribute to pathogenesis if somatic mutation and selection by a foreign antigen drives a B-cell clone in the germinal center to become more and more strongly self-reactive as well. However, tight selection mechanisms in the germinal center most often “focus” B cells on the foreign antigen driving the response and away from self reactivity. In addition, self-reactive B cells may be controlled by a specialized regulatory T cells, known as T follicular regulatory cells, that express CXCR5 and enter the follicle.
