Generation of Immune Diversity

(c); constant regions; from Nature, Vol 430 July 8, 2004

The ability of the immune system to recognize antigens depends on antibodies generated by B cells and on antigen receptors expressed by T cells. These antigen receptors, antibodies or T-cell receptors, are generated by a gene-rearrangement process during lymphocyte development. The antigen-binding or variable (V) region is formed by the rearrangement of V, D (diversity) and J (joining) gene segments for one chain (antibody heavy chain) of the receptor, and of V and J segments for the other chain (antibody light chain). The enzymes RAG1 and RAG2 stitch these segments together. The RAG enzymes rearrange genes by recognizing recombination signal sequences (RSS) that flank the variable (V), diversity (D) and joining (J) gene segments at heavy-chain and light-chain loci.

The multigenic nature and imprecise joining of the V, D and J segments result in each individual having a tremendous repertoire of antigen receptors.

Organization and Structure of the Germ-line Ig genes

The light chain of an amino acid has an amino terminal half of about 110 amino acids which varies among the different proteins. This region is sometimes called the variable region. The carboxyl terminal half of the molecule, called the constant (C) region has 2 basic amino acid sequences which are designated "kappa" and "lambda." In humans, 60% of the L chains are kappa, and 40 are lamda. The heavy chain also contains a variable amino acid terminal as well as a constant region.

DNA rearrangement of Ig and TCR Genes

Coordination of the series of DNA rearrangement events required to assemble Ig and TCR genes from component V, D and J segments presents a highly complex regulatory event. There are seven structurally unique antigen receptor loci, some spanning 3 or 4 megabases, each composed of multiple V, J. and sometimes D segments, along with nonrearranging constant (C) gene segments. Rearrangement is cell lineage restricted, so that Ig loci are fully rearranged only in B cells, and TCR genes are completely assembled only in T cells.

Rearrangement occurs in a specific order. Ig heavy chain (IgH) genes are joined before the assembly of the Ig light chain (к and ג).  With the IgH and TCRβ loci, D to J segment joining occurs first, followed by fusion of a V segment to the newly assembled DJ element. Because the same recombinase machinery, including the two proteins that specifically cut the DNA (RAG1 and RAG2), is responsible for V(D)J recombination at all loci, regulation must take place at the level of the recombination sites.

The к and ג  light chains and the heavy chains are encoded by separate multigene families situated on different chromosomes. Each of these multigene families contains a series of coding sequences, called gene segments, separated by noncoding regions. During cell maturation, these gene segments are rearranged and brought together to form functional immunoglobulin genes.  The к and ג  light chain families contain the V, J, and C gene segments. The heavy chain family contains V, D, J and C gene segments. Each V gene segment is preceded at its 5' end by a short signal leader peptide that guides the heavy or light chain through the ER. The conservation of important biological effector functions of the antibody molecule is maintained by the limited number of heavy-chain constant region genes. In both humans and mice, the C_H gene segments are arranged sequentially in the order mu (for IgM), IgD...

Each V_H and V_L gene segment has a promoter located just upstream from the leader sequence. The promoter contains a highly conserved AT rich sequence called the TATA box, to which RNA polymerase II binds. After binding to the TATA box, RNA polymerase starts transcribing the DNA from the initiation site about 25-35 bp downstream of the TATA box. The rate of transcription is almost negligible in unrearranged germ line DNA but increases following rearrangement due to enhancer sequences which are brought closer together.

Variable-Region Gene Rearrangements

Functional genes that encode Ig light and heavy chains are assembled by recombinational events at the DNA level. Variable region gene rearrangement occur in an ordered sequence during B cell maturation in the bone marrow. The heavy chain (shown above) variable region genes rearrange first, then the light chain variable region genes. Generation of the heavy chain requires 2 separate rearrangement events within the variable region. First, DH gene segment joins to JH segment. The resulting DHJH segment then joins a VH segment to generate a VHDHJH unit that encodes the entire variable region. Once heavy chain gene rearrangement is accomplished, RNA polymerase binds to the promoter sequence and transcribes the entire heavy chain gene, including the introns. Initially, both C mu and C episolon are transcribed. Differential polyadenylation and RNA splicing remove the introns and process the primary transcript to generate either the C mu or C episolon chains.

Following antigenic stimulation of a B cell, the heavy chain DNA can undergo additional rearrangement in which the V_HD_HJ_H unit can combine with any C_H gene segment in a process called "class switching."  The mechanism involves DNA flanking sequences called switch regions located upstream from each CH segment (except C episolon). Various cytokines secreted by activated T_H cells have been shown to induce B cells to class switch to a particular isotope.

Long after the coding regions have been assembled, when B cells are stimulated by antigen and T_H cells to generate memory cells, there is usually a progressive increase in the affinity of antibodies produced against the immunizing antigen. This is somatic mutation and probably involves some form of error prone DNA repair process targetted to the rearranged V region coding sequence. Somatic mutation does not play a significant role in the generation of TCR diversity.

Mechanism of Variable-Region Gene Rearrangements

Functional immunoglobulin genes are generated during B cell maturation by a process whereby the gene segments are rearranged and brought together. Joining is mediated by an enzyme complex called the V(D)J recombinase which are encoded by RAG proteins which introduce double strand breaks at flanking DNA sequences. During lymphocyte development, the RAG enyzmes rearrange genes by recognizing recombination signal sequences (RSS) that flank the variable (V), diversity (D) and joining (J) gene segments at heavy-chain and light chain loci (C, constant regions). These segments are stitched together by enzymes, and the functional gene encodes the binding sites that can recognize foreign antigen.

Rearranged VJ gene segments encode the variable region of the light chains. Thus a functional ג variable region gene contains 2 coding segments (or exons), a 5' V segment and a 3' J segment which are separated by a noncoding DNA sequence (or introns) in unrearranged germ-line DNA. Rearranged VDJ gene segments encode the variable region of the heavy chain. The C gene segment encodes the constant regions of the light or heavy chains.

In most cases of site specific recombination, DNA joining is precise. But during the joining of antibody (and T cell receptor) gene segments, a variable number of nucleotides are often lost from the ends of the recombining gene segments and one or more randomly chosen nucleotides may also be inserted. This random loss and gain of nucleotides at joining sties is called junctional diversification and it adds to the diversity of V region coding sequences created by recombination. In many cases junctional diversification results in a shift in the reading frame that produces a nonfunctional gene. Thus many developing B cells never make a functional antibody molecule and consequently die in the bone marrow. B cells making functional antibody molecules that bind strongly to self antigens in the bone marrow are stimulated to reexpress the RAG proteins and undergo a second round of V(D)J rearrangements thereby changing the specificity of the cell surface antibody they make in a process referred to as receptor editing.

Control of Accessibility

Impact of Aleterations in Chromatin Structure

A broad range of genetic and biochemical evidence suggests that much of the developmental regulation of V(D)J joining is mediated by specific alterations in chromatin structure that render regions with a locus accessible or inaccessbile to the recombinase machinery. A strong correlation between histone acetylation (or factors that increase acetylation) and a recombinationally accessible chromatin state has been shown in vivo, including the mapping of acetylated histone H3 and H4 to recombinationally accessible gene segments at a series of loci. In vitro, RAG proteins cannot cleave unmodified nucleosomal DNA, but acetylation and hSWI/SNF remodeling render nucleosomal DNA accessible for cleavage.

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