Mechanism of somatic hypermutation
The process of enhanced immunoglobulin gene mutation occurs exclusively during an immune response. It involves actively proliferating B cells (B centroblasts) located in germinal centers. Hypermutation ceases at the mature plasma cell stage.
The main initiator is the enzyme activation-induced cytidine deaminase, encoded by the AID gene (Activation-induced cytidine deaminase). The enzyme deaminates cytidine in single-stranded DNA, converting it to deoxyuridine. Initially, this causes a cytosine-to-thymine (C→T) substitution, after which multiple somatic mutations arise as transitions.
The frequency of genetic changes during this process increases by three to four orders of magnitude (from $10^{-6}$ to $10^{-2}$). Mutations are localized strictly in the V domain, most intensively affecting the CDR3 segment, where up to 3% of bases are altered with each division. Most mutations reduce the antibody's ability to bind antigen, but due to the vast number of variants, clones with increased affinity emerge. The survival of such cells is called affinity maturation.
Switching constant genes (isotypes)
Isotype switching provides the molecular basis for changing antibody classes. The IGH gene cluster contains a set of constant (C) genes. In humans on chromosome 14q, they are arranged in an evolutionarily fixed order: from C$\mu$ and C$\delta$ to C$\alpha$2.
Triggering the switch requires a T-dependent immune response, contact with T cells via CD40 and CD154 molecules, and the action of cytokines. Upstream of each C gene (except C$\delta$) lies an S-region (from switch), consisting of GAGCT or GGGGGT repeats.
Stages of recombination:
- Polymerization of S-regions and chromatin reorganization.
- Bringing selected S-regions into proximity.
- Formation of a DNA loop enclosing all intervening C genes.
- Double-strand DNA breaks mediated by endonucleases at the edges of the loop.
- Excision of the loop and immediate ligation of the free ends.
As a result, the new C gene joins the original variable V gene. Antibody specificity does not change because the V gene remains the same; only the effector function changes.
Influence of cytokines and formation of secreted forms
The direction of isotype switching is regulated by cytokines produced by T helper cells:
- IL-4 directs switching toward IgE synthesis (and IgG1 in mice as well).
- IFN$\gamma$ induces the transition to IgG3 production.
- TGF$\beta$ promotes IgA production.
In addition to isotype switching, the B cell switches synthesis from membrane receptors to secreted antibodies. At the 3' end of C genes, there are an SC segment (encoding the soluble part) and two MC segments (encoding the hydrophobic membrane anchor).
In naive cells, both regions are transcribed, but during splicing, the SC fragment is removed, and the receptor is anchored in the membrane. Upon differentiation into a plasma cell, transcription termination (poly-A signal) occurs early—right after the SC segment. As a result, the MC regions are not transcribed, and the cell synthesizes only soluble antibodies.
Briefly: answers to topical questions
What is the exact order of the human heavy chain constant domain (IGH) genes on chromosome 14q?
The exact order of human heavy chain constant domain genes on chromosome 14q includes the VDJ segment followed by C genes and a pseudogene.
Order of arrangement in the IGH cluster: VDJ — C$\mu$ — C$\delta$ — C$\gamma$3 — C$\gamma$1 — $\psi$C$\epsilon$ — C$\alpha$1 — C$\gamma$2 — C$\gamma$4 — C$\epsilon$ — C$\alpha$2.
The presence of two homologous gene sets is the result of evolutionary duplication. A pseudogene homologous to the C$\epsilon$ gene is located between the C$\gamma$1 and C$\alpha$1 genes. The arrangement and exact set of C genes are species-specific.
Which primary immunodeficiency is caused by genetic defects in the AID enzyme or the CD154 molecule?
A genetic defect in the CD154 molecule leads to the development of a primary immunodeficiency: hyper-IgM syndrome.
- Hyper-IgM syndrome arises due to a mutation in the TNFSF5 gene (Xq chromosome) and impaired expression of the CD154 molecule or its receptor, CD40.
This blocks normal intercellular interaction between T cells and macrophages, dendritic cells, and B lymphocytes. As a result, the patient has impaired C-gene switching. There is no information in the provided materials regarding primary immunodeficiency caused by an AID enzyme defect.
To remember the cytokine-isotype association: IL-4 ("four" sounds like "E" in Russian mnemonics) switches to IgE. TGF-beta ("beta" and "A") directs synthesis toward IgA.
Class and exam questions
Does hypermutation occur in plasma cells?
No, this process is detected only in activated proliferating B centroblasts. It does not occur in differentiated plasma cells.
Does antibody specificity change during isotype switching?
Antigen specificity does not change. The variable (V) gene remains the same; only the constant (C) gene, which determines the effector properties of the molecule, is replaced.
Why do naive B cells express two isotypes at once—IgM and IgD?
This is achieved through alternative splicing of the primary mRNA transcript. Depending on where introns are excised, either the $\mu$ or $\delta$ chain is formed.
Is antibody class switching reversible?
As a rule, this is an irreversible process, because the DNA loop containing the intermediate constant genes is physically excised from the cell genome and lost.
What else to study on this topic
- Mechanisms of alternative splicing during simultaneous expression of IgM and IgD
- Structure and functions of CD40 and CD154 molecules in the T-dependent immune response
- Evolutionary gene duplication in the human IGH cluster on chromosome 14q
- Molecular structure of S-regions and their susceptibility to endonucleases
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