Even with conservative assumptions, the theoretical diversity of the pre-immune antibody repertoire exceeds 1016. Following antigen exposure, the processes of somatic hypermutation and in vivo affinity maturation drive additional (potentially exponential) diversification of the antibody repertoire.
There are three major sources of antibody diversity:
- Multiple inherited (germ-line) gene segments are used in different combinations to generate diversity. Recombination of human heavy chain variable (V), diversity (D), and joining (J) gene segments builds a functional VH chain; and recombination of light chain V and J genes (which are either kappa or lambda isotypes) builds a functional light chain. To build a functional human heavy chain variable region, there is a random assortment in which one of 39 functional V genes is coupled with one of 27 functional D genes and one of 6 J genes. The heavy chain can pair with either a kappa or lambda light chain. To build a functional human kappa light chain variable region, there is a random assortment in which one of 36 functional Vκ genes is coupled with one of 5 Jκ chains. To build a functional human lambda light chain variable region, there is a random assortment in which one of 33 functional Vλ genes is coupled with one of 5 Jλ chains. The pairing of a heavy chain with a light chain is also a source of diversity.
- Junctional diversity introduced via the gene rearrangement process adds diversity. The junctional position of heavy chain V-D-J and light chain V-J recombination, when translated, comprises their CDR3 domains, which is the main determiner of repertoire diversity. During the rearrangement process, a hairpin is formed that, when resolved via the activity of recombination-activating genes (RAG) and Terminal deoxynucleotidyl Transferase (TdT), can result in the addition or deletion of P- and N- nucleotides (respectively). This added junctional diversity in the heavy chain can result in >107 different variations, including CDR3 lengths ranging from 3-25 residues, arising from the same set of V, D, and J genes.
- After antigen exposure, the antibody genes undergo affinity maturation, generating new diversity from which antibodies with higher affinity to the targeted antigen are selected. This is accomplished via targeted somatic hypermutation by Activation-Induced Cytidine Deaminase (AID). The mutation rate of this programmed mutagenesis to the rearranged V(D)J region is ~10-3 base pairs per generation, a million-fold higher than the non-AID targeted genome of B cells. Those B cells whose variable regions have accumulated deleterious mutations and can no longer bind antigen die (negative selection). Those B cells whose variable regions have acquired mutations that result in improved antigen binding receive signals from CD4+ T cells that drive their proliferation and expansion, along with continued mutation. During the affinity maturation process, the average number of mutations in VH and VL are eight and five, respectively.
*Modified from Boyd et al. “High-Throughput DNA Sequencing Analysis of Antibody Repertoires”, Microbiology Spectrum 2014.
In applying the central dogma of molecular biology to antibodies, the genetic information encoded in the germline DNA undergoes somatic recombination to generate unique heavy and light chain mRNA sequences, which are translated as four polypeptide chains that assemble into a disulfide-bonded homodimer of disulfide-bonded heterodimers (heavy chain and light chain).