The genetic Basis of Ab Structure Introduction The number of B and T cells with different antigenic specificities that can be generated in a single individual range from 1015-1018 i.e every person has the ability to generate 1015-1018 different immunoglobulins or T cell receptors "Ig or TCR". The number of genes in many species ranges from 20,000-25,000 genes. How do so few genes produce so many different antigen receptor molecules?

Susumu Tonegawa "Nobel prize winner" discovered that antibody genes can move and rearrange themselves within the genome of a differentiating B cell. The same applies for TCR. Many different V region genes can be linked up to a single constant "C" region gene. A prototypical gene coding for transmembrane "Non immunoglobulin" protein is shown in the following figure(fig.6.1)

Genes coding for proteins expressed at the cell surface have a leader sequence "L exon" at the 5` end. Leader sequence produces about ten hydrophobic amino acids "the signal peptide" at the NH2 end of the protein. The signal peptide directs the synthesis of the polypeptide chain to endoplasmic reticulum then it is cleaved off. The newly synthesized protein moves from the endoplasmic reticulum into the Golgi apparatus and then to the cell membrane.

V and C regions of Ig molecule are coded for by different genes. Ab genes can move and rearrange themselves within the genome of a differentiating cell. The process of rearrangement during differentiation brings together a set of genes that codes for the V and C regions. The set of rearranged genes is then transcribed and translated into a complete H or L chains. The generation of diversity in TCR is similar to Ig.

Genetic Events in the synthesis of Ig chains A) L chain genes Each  and  L chain polypeptide consists of two major domains "VL and CL". VL is 108 residue NH2-terminal. VL is coded by two separate gene segments: a variable segment (V) which codes for the NH2-terminal 95 residues. and a small joining segment (J) coding for 13 residues at the carboxy-terminal end of variable region.

One V gene and one J gene are brought together in the genome to create a gene unit that, together with the C region gene, codes for an entire Ig L chain. This unique gene rearrangement mechanism referred to as V(D)J recombination is used only by genes coding for Ig L and H chains and genes coding for TCRs. An enzyme known as V(D)J recombinase mediates the rearrangement of receptor genes in B and T cells. The products of two genes, RAG-1 and RAG-2 "recombination-activating genes" are critical in initiating recombination in lymphocyte precursor cells.

Both RAG-1 and RAG-2 proteins are required in the first stages of cutting Ig-and TCR-DNA. Mice lacking one of these genes "RAG knockout mice" are deficient in both B and T cells. While the V(D)J recombinase is found in all cells and is involved in the repair of DNA strands, RAG-1and RAG-2 gene products are expressed exclusively in lymphocytes.

 locus is found on chromosome 2.  chain synthesis  locus is found on chromosome 2. The arrangement of  genes in the germ line ((in any cell in the body)) is as follows: There are approximately 40 different V genes, each of which can code for the N-terminal 95 amino acids of a  variable region. these V genes are arranged linearly, each with its own L "leader" sequence and all separated by introns.   "L-sequence was omitted for simplicity"

A series of 5 J gene segments is found downstream (3`) of this region. Each J gene segment can encode the remaining 13 amino acid residues "96-108" of the  variable region. A long intron separates the C gene segment from the J gene segments. To make a  chain. An early cell in the B cell lineage selects one of the V genes from its DNA and physically joins it to one of the J segments "in Fig 6.2 V2 rearranges to J4". The V(D)J recombinase mediates the joining; it recognizes recombination recognition sequences that are located at the ends of the V and J gene segments.

Intervening DNA is looped, cut out and broken down. After rearrangement, primary RNA transcript is made, which is then spliced bringing V, J and C exons together in mature mRNA. At the ER, L sequence is cleaved off, and the mRNA is translated into the  polypeptide chain. The  chain moves into the lumen of the ER, where it can join with a newly synthesized H chain to form an Ig molecule.

Organization and Rearrangement of H chain Genes

Figure 6.4 shows the organization of genes coding for the H chain. In contrast to the V region of L chain which is constructed from two gene segments, the variable region of H chain is constructed from three gene segments "VH, DH, JH". Diversity "D" segment is the extra segment. The D and J segments code for amino acid sequences in the third hypervariable, or complementarily determining region "CDR3" of H chain. Human H chain locus includes 50 VH genes, 20 DH gene segments and 6 JH gene segments.

There are multiple genes coding for the C region of Ig in the germ line. The C genes, each flanked by introns, are separated from VH genes by a large intron. The C genes closest to the V region genes are  and  which are first transcribed during B cell development. H chain synthesis uses the same mechanism described for L chains "the use of V(D)J recombinase to mediate the joining of different gene segments. In the early life of a particular B cell, two rearrangements of germ life DNA must occur: The first brings one D segment alongside one J segment. The second brings one V segment next to DJ unit "V2 D2 J5 in fig. 6.4", fixing the Ag specificity of the H chain.

The rearranged DNA is then transcribed along with the closest C region genes  and . This primary transcript can be spliced in two different ways "alternative splicing" to yield either a VDJ- or VDJ- mRNA. These two messages may then be translated in RE to yield either  or  polypeptide. In this way, an individual B cell may express both  and  with identical antigenic specificity.

Alternative splicing of H chain primary transcript also generates membrane and secreted forms of a heavy chain polypeptide. Two additional exons are found at 3` end of each CH gene” not shown in fig. 6.4” these code for transmembrane form of the molecule and the C-terminal end of the secreted form of the molecule. Both exons are transcribed into the primary transcript, but one or other of the transcribed exons is spliced out. This results in mRNAs membrane form or the secreted form of the H chain polypeptide.

Regulation of Ig Gene Expression Each B cell uses only one set of VDJ genes and one type of light chain. As a result, a single B cell produces an Ig of only one antigenic specificity. Ig chains are coded for by only one set of genes, either from the maternal or the paternal chromosome, e.g. H chain may be coded for by genes on the paternal chromosome and the L chain " or " by genes on the maternal chromosome. This phenomenon of using genes from only one parental chromosome is known as allelic exclusion. If the process of rearrangement fails in one chromosome, the second parental chromosome will continue rearrangement. Gene exclusion ensures that every B cell and the Ab it synthesizes are monospecific e.g. specific for one epitope.

Class or Isotype Switching One B cell makes Ab of a single specificity that is fixed by the nature of VJ "L chain" and VDJ "H chain" rearrangements. These rearrangements occur in the absence of Ag in the early stages of B cell differentiation. B cells can synthesize IgM and IgD with the same antigenic specificity. An individual B cell have the ability to switch to make a different class of Ab... such as IgG, IgE, IgA. This phenomenon is known as class or isotypic switching. Class switching changes the effector function of the B cell but does not change the cell's antigenic specificity.

Class switching occur in Ag-stimulated mature B cells synthesizing IgM and IgD, and involves further DNA arrangement, juxtaposing the rearranged VDJ genes with a different heavy - chain C region gene. In addition to Ag, class switching is dependent on the presence of cytokines released by T cells. Cytokines induce further rearrangement of B cell DNA and produce switching to other Ig classes depending on the switches occurring in the DNA coding for its H chain. Figure 6.5 shows the mechanism by which mature B cells undergo class switching.

At the 5` end of every H chain C region "CH" gene apart from C delta , is a stretch of repeating base sequences called a switch "S" region. This S region permits any of the CH genes "other than C delta" to associate with the VDJ unit, in the figure, only the CH genes γ1, γ3, and 2, are shown, but other CH genes may also be used. Under the stimulating influence of Ag & T cell-derived cytokines, a B cell with a VDJ unit linked to Cu and C delta further rearranges its DNA to link the VDJ to an S region in front of another CH region gene " γ1 in fig 6.5".

After a primary RNA transcript is made from the rearranged DNA, the introns are spliced out to give a mRNA coding for the IgG1 H chain, the intervening C region DNA is removed. At this stage, the cell loses its ability to revert to making a class of Ab whose C region gene has been deleted "IgM , IgD, or IgG3 in this example".

Class switching is a mechanism unique to Ig H chains of B cells Class switching is a mechanism unique to Ig H chains of B cells. It allows an Ab with a single antigenic specificity to associate with a variety of different constant region chains and thus have different effector functions. e.g. an Ab with a VDJ unit specific for a bacterial Ag may be linked to C γ to produce an Ig G molecule, this Ig G Ab interacts with cells such as MØ that express receptors for Fcγ. Alternatively the same VDJ unit may be linked to CЄ to produce IgE. IgE interacts with cells such as mast cells that express receptors for FcЄ.

Cytokines play a key role in CH gene selection during isotype switching. e.g. in the presence of IFN- γ, B cell can rearrange its VDJ to Cγ2 H chain, and the cell switches to IgG2 synthesis. In the presence of IL-4, a B cell can rearrange its VDJ Cγ4 or CЄ, and the cell switches to IgG4 or IgE synthesis respectively. Each cytokine is thought to loosen the structure of DNA double helix at only certain points along the Ig locus, allowing an enzyme known as a "switch recombinase" to recognize DNA coding for specific C regions.

other mechanisms for generating diversity Presence of multiple V genes in the germ line represent the minimum number of different Abs that can be produced. VJ and VDJ Combinatorial Association they contribute to the structure of the variable region. 40 Vk × 5 Jk = 200 k chains formed 40 V × 4 J = 160  chains 50 V × 20 D × 6 J = 6000 H chains different.

The additional diversity generated is termed N region diversity. 3. Random Assortment of H and L chanins If any H chain can associate with any K or  chain, a total of 1.2 × 106 different K-containing Ig molecules "200 × 6000" and 0.96×106(160×6000 -containing molecules can be generated from just 165 different genes "All H, K, ". 4. Junctional and Insertional Diversity The absence of precision in joining during DNA rearrangement leads to deletions or changes of amino acids "junctional diversity" that affect Fab region as it occurs in parts of the hypervariable region. Small sets of nucleotides may be inserted "insertional diversity" at the V-D and D-J junctions. It is mediated by the enzyme terminal deoxynucleotidyltransferase "TdT". The additional diversity generated is termed N region diversity.

5. Somatic Hypermutation Mutations that occur in V genes of H and/or L chains increase the variety of Abs produced by B cells. Ab of low affinity is produced in primary response to Ag. As the sequence matches that of germ line DNA. As the response matures, and after secondary stimulation by Ag, the affinity of Abs increases and the sequences diverge from those on the germ line DNA.

This divergence results from point mutation in the VDJ recombined unit of Ab V genes which results in changes in amino acids. This phenomenon is referred to as somatic hypermutation because it occurs at a rate at least 10,000-fold higher than the normal rate of mutation. Somatic hypermutation results in increased affinity of Abs in secondary response. 6. Somtic gene conversion. Figure 6.6with its footnote.

7. Receptor editing A cell in the B cell lineage can undergo a second rearrangement of its L chain variable gene segments, after it has formed a recombined VJ unit. This process is known as receptor editing. In fig. 6.3 unrearranged elements V1 and J5 can be used in a second rearrangement. Thus the diversity is increased. Receptor editing can occur when a B cell with a receptor specific for a self Ag interacts with that self Ag. One outcome of this interaction is that the V and J genes of the B cell can undergo a second rearrangement which may generate a VJ recognition unit specific for a foreign rather than a self Ag thus diversity is increased to foreign Ags.  

N.B All these mechanisms contribute to the formation of a huge library. Or repertoire of B lymphocytes that contain all the specificities required to deal with the multitude of diverse epitopes that antibodies could encounter. The number of total Ig specificities that can be generated in an individual are on the order of 1015 which is increased even more by somatic hypermutation.

Role of Activation-induced Cytidine deaminase(AID) in Generating Antibody Diversity   AID plays a key role in initiating class switch recombination, somatic Hypermutation, and gene conversion, the three major pathways that generate diversity of antibodies. AID removes cytidine groups from the DNA in activated B cells to form uridine, this generates U:G base pairs in DNA, the mismatched base pairs are then removed by one of several different enzymes. This in turn , results In changes in the DNA , primarily in Ig V-region genes.