![]() When B cells leave the bone marrow, they begin synthesizing IgM and IgD molecules embedded within the plasma membrane. IgM is the first immunoglobulin to be synthesized by B cells, which are inserted into the plasma membrane as a B cell receptor (BCR). Strong-affinity binders are selected for and proliferate and mature into antibody-secreting cells, whereas lower affinity clones are eliminated by apoptosis.ĭuring B cell development, cells switch between generating one class of antibody to another, a process known as class switch recombination (CSR). Hypermutated B cells undergo a selective process in germinal centers where B cells compete for various signals in an affinity-dependent manner, thereby outcompeting lower-affinity B cell clones. Mutations that enhance antigen-binding tend to be clustered in the CDR regions. Mutations in the framework regions of the variable domain tend to be selected against as they don’t enhance antigen-binding and alter the basic antibody structure. Some immunoglobulin mutants bind antigens better than others, while some mutants produce non-productive rearrangements. SHM facilitates the progressive increase in antibody affinity against the antigen known as affinity maturation (Figure 4). Double stranded DNA breaks are introduced into variable regions, eliciting DNA damage response pathways that facilitate error-prone repair and resulting in mutant antibodies. Point mutations by somatic hypermutation increase variation into the variable region and occur a million-fold more frequently than other genetic mutations. Somatic hypermutation (SHM)provides an additional level of antibody diversity after V(D)J recombination. The formed V(D)J exon is transcribed and translated into a functional HC or LC (Figure 3). This process known as junctional diversification introduces an additional level of antibody diversity in the variable region, specifically in the third complementarity-determining region (CDR3). Nucleotides are often lost or inserted at the joining sites during recombination resulting in a frameshift mutation. DNA ends are repaired by DNA repair enzymes, resulting in deletions or inversions of gene segments. Selected V, D, and J segments are rearranged and joined to form the V(D)J exon. This enzyme complex binds and cleaves the DNA at conserved sequences that flank each V, D, and J segment. Chromosomal DNA double stranded breaks are introduced by RAG recombinase. HCs contain 65 V segments that can join with any of the 6 J and 27 D segments to form roughly 11,000 possible variable HC regions.Ī process called site-specific recombination mediates V(D)J recombination. The genes for human λ LCs contain 30 V segments and 4 J segments resulting in 120 possible variable λ regions. For human κ LCs, 40 V segments can combine with any of the 5 J segments resulting in 200 possible combinations encoded by this pool. Recombination and joining of V, D, and J segments create a functional variable immunoglobulin region (Figure 3). Organization of gene segments in human heavy and light chain loci. The kappa light chain has the potential to be a major contributor toward generation of the antibody specificities of the rabbit pre-immune repertoire.Figure 2. Thus, in contrast to limited combinatorial diversity of its heavy chain, the rabbit can draw upon a diverse set of germline Igk-V genes. Some of the V(kappa)J(kappa) junctions had N and P nucleotide additions. The germline Igk-V genes display different lengths of the coding region 3' of Cys 88 ranging from 7 to 12 amino acids, resulting in CDR3 length heterogeneity among functional V(kappa)J(kappa) sequences ranging from 8 to 15 amino acids. The actual number of germline Igk-V genes is potentially greater than our conservative estimate of at least 39, 28 of which we found expressed as mRNA. Our analyses indicate that most of the sequences that we recovered from our libraries belong to a single family and some are extremely similar. To investigate the contribution of combinatorial diversity toward generation of the rabbit V(kappa) repertoire, we constructed five genomic libraries from rabbit kidney DNA and 1 cDNA library from the bone marrow of a 1-day-old rabbit using a series of polymerase chain reaction-based strategies. In rabbits, the contribution of the combinatorial mechanism to heavy chain diversity is minimal, as only a few Igh-V genes are rearranged and expressed. In mouse and human, generation of combinatorial diversity through use of different heavy and light chain variable region genes in immunoglobulin rearrangements can be a major contributor to the primary antibody repertoire.
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