As I have marveled at several times before, antibodies have a remarkable repertoire that allows for the ability to bind to an incredible array of targets. This is both beneficial for immune defense and the protection of the host, as well as the ability to harness this capability in generating monoclonal antibodies. We have previously touched on the ability to generate a diverse repertoire, so I thought that I would explore the genetics and mechanics of how this is actually accomplished. For this review of antibody genetics, I will be focusing on mouse and human immunoglobulin genes. There are other species that have some fascinating genetic mechanisms associated with the development of antibody repertoires. I will leave that for another time.
II. Immunoglobulin Gene Structure
As discussed in the “Antibody Structure” blog, antibodies are generated by the production of heavy chain (HC) and light chain (LC) polypeptides that are synthesized from separate immunoglobulin (Ig) genes (Figure 1). The HC gene locus is on Chromosome (Chr) 14 in humans and 12 in mice, and the LC is produced either from the kappa (?) locus (Chr 2 in humans and Chr 6 in mice) or from the lambda (?) locus (Chr 22 in humans and Chr 16 in mice). The HC Ig gene is comprised of a variable (V) region, diversity (D) region, joining (J) region, and a constant (C) region. The C region from the HC determines the isotype of the antibody (i.e. IgM, IgG, IgA, IgE, or IgD). The V-, D-, and J-regions determine the antibody specificity. The LC Ig gene is comprised of V-, J-, and C-regions and the C region is characterised as ? or ? depending on the locus. Similar to the HC, the LC antibody specificity is derived from the V- and J-regions.
The combination of the VDJ regions and VJ regions is what generates the diverse population of antibodies (the process of how this happens will be described in the next section). Each individual mature B cell has a single, active HC Ig gene and a single, active LC Ig gene. As I have outlined above, there are three Ig loci (one HC and two LC) and two of each allele represented in the genome, but only one HC and one LC Ig gene will be active. This process is referred to as allelic exclusion. Once an Ig gene is determined to be functional, the other allele(s) will be shut down. So, once an HC gene is functional, the other allele (HC chromosomal region) will be shut down; in the case of the LC, once an LC gene becomes active the other three alleles are shut down. This process ensures that only one HC and one LC gene are active in an individual B cell.
Each of the HC V-,D-, J-, and C-regions and LC V-, J-, and C-regions are physically separated on their respective chromosomes (Figure 1). In fact, there are multiple copies of each of the regions, except for the LC ? C-region. In order for a functional HC gene to become active, the V-,D-, and J-regions need to be “joined” together to form a functional gene, in the LC the V- and J-regions need to be brought together. This means that the Ig genes,as the are found in the genome, are not functional; but need to be “activated’ in order to produce antibodies. As we will see below, the process is tightly regulated and some of the machinery necessary to accomplish this is only found in lymphocytes; therefore, antibodies can only be produced by B cells. The multiple combinations of VDJ and VJ segments is what generates the diversity of Ig genes and the antibody repertoire.
III. Immunoglobulin Gene Rearrangement
Now we are getting to the really important part: how the Ig gene segments rearrange to become active Ig genes and produce antibodies. The process is initiated in the bone marrow at the pro-B cell stage of differentiation (see the “B Cell Development” blog post for a review) when the D-region segment rearranges with a J-region segment (Figure 2). This is followed by V-region recombination to generate a V(D)J HC gene during the pre-B cell stage of differentiation. Here is one of the stumbling blocks in the process. The V(D)J rearrangement must generate a functional gene structure. In many cases, the process is faulty and results in a nonfunctional Ig gene structure. Both of the HC Ig alleles undergo rearrangement and therefore, if one allele leads to a nonfunctional Ig gene it is possible that the other allele will be functional and the process can move forward. If neither are functional, then that particular B cell will likely die due to its inability to complete the B cell differentiation process. However, if the HC V(D)J gene is determined to be functional through its association with the surrogate LC, the LC Ig gene rearrangement is initiated. The V-region segment recombines with a J-region segment (Figure 2) to generate a VJ LC Ig gene. The same is true for the LC as it was with the HC. If the rearrangement is faulty, then one of the other LC Ig alleles (remember there are three others for the LC) can compensate for the defective process. If any of the other alleles are unable to generate a functional LC Ig rearrangement, the cell will likely die. The process of allelic exclusion, mentioned earlier, will shut down the other HC or LC alleles once a functional rearrangement has occurred. Once we have a functional HC Ig gene and LC Ig gene, the cell becomes an immature B cell and will migrate from the bone marrow to the spleen for further maturation.
The process occurs through a series of gene rearrangements generated by recombination. The recombination occurs at specific sites mediated by the RAG1 (recombination-activating gene 1) and RAG2 genes which are uniquely expressed in B cells and T cells (which have a similar rearrangement for the generation of T cell receptors). While the RAG genes initiate the site specific recombination, components of the non-homologous end joining machinery are required for the repair and ligation of the DNA double strand breaks produced by RAG1 and RAG2. Each step of the HC V(D)J and LC VJ rearrangement is tightly controlled, as well as tested for function. Most of the B cells that begin differentiation do not make it out of the bone marrow, indicating that there is high failure rate of generating functional HC and LC genes.
IV. Antibody Diversity
Now that we have a basic understanding of the Ig gene rearrangement process, we can consider how antibody repertoires are generated. During a successful rearrangement, a single V(D)J and single VJ combination will be generated in each B cell. As this process is random in the selection of each component, the final specific HC and LC will be varied in each individual B cell. The number of possible combinations is determined by the number of possible HC and LC pairings that can occur. It is estimated that there are approximately 40 human HC V-regions (110 for mice), 24 HC D-regions (10 in mice), 6 HC J-regions (4 in mice), 82 LC V-regions ( 98 for mice), and 10 LC J-regions (7 for mice). If the HC and LC combinations are calculated, there are in the neighborhood of 2 million antibodies that can be generated by Ig gene rearrangement. The survival of the B cell through this process is dependent on the successful rearrangement of the HC and LC Ig genes, so only B cells with “functional” antibodies are produced.
While 2 million different Ig gene combinations is a substantial antibody repertoire, it is nowhere near the 1013 possible antibodies that are predicted to be the full repertoire that are found in both human and mouse immune systems. There are two mechanisms that bridge this gap. The first occurs during the HC D-J recombination event, which generates variation at the D-J junction. The recombination process is initiated by the RAG genes at specific sites which generates a unique double strand break at the 3’ of the D-region and the 5’ of the J-region, which yields a “loop” at the end of each break. The repair and joining of these ends can lead to: 1) deletion of some of the nucleotides at the junction, 2) the addition of non-germline (or N-region) nucleotides at 3’ end of the D-region, and/ or 3) the generation of palindromic (or P-region) nucleotides that arise from the opening of the loop at the 5’ end of the J-region. Each can contribute to a substantial increase in diversity of the germline HC D- and J-region segments.
The second mechanism that contributes significantly to the antibody repertoire is the process of somatic hypermutation (SHM). SHM is a result of errors or mutations introduced into the variable region during replication of the rearranged Ig genes. Activation induced cytidine deaminase and DNA polymerase “errors” introduce nucleotide changes into the LC and HC genes which modify the germline DNA sequence of the variable regions (we do not see changes to the constant region). These modifications may lead to amino acid sequence changes and alterations in the binding capacity or target specificity (both increased and decreased). SHM is initiated in the germinal center during a process called affinity maturation (I will discuss this in more detail in an upcoming blog). During the expansion of an antigen specific B cell, SHM will lead to modification of the HC and/ or LC sequence. With the antigen present, those that have the highest affinity will capture antigen to a greater capacity, which will support their survival. Those that cannot compete, will not thrive and may not survive. Therefore, the higher affinity B cell clones will be selected and mature to produce antibodies with increased specificity and higher affinity to the antigen.
The combination of the variation in selection of the V(D)J and VJ regions, recombination variation at the D-J junction, as well as SHM generate the incredible diversity of antibodies that allow for the protection of the host. In addition we can induce responses to antigens through immunization and vaccination, both for the protection of populations (vaccination) or for the development of polyclonal or monoclonal antibodies (immunization) for use as research tools, reagents for diagnostics, and even as therapeutics. . The fact that the rearrangement process is random, each individual will have a slightly different repertoire, even if they are genetically identical, as would be the case for inbred mouse strains (and monozygotic twins). In addition, the differences between individuals, and inbred strains, would yield even greater differences in the overall repertoire. Therefore, considering the role genetics play in establishing diversity of the antibody repertoire it is important to keep this in mind when initiating monoclonal antibody projects.