The formation of functional genes that encode B and T lymphocyte antigen receptors is initiated by somatic recombination of gene segments that code for the variable regions of the receptors, and diversity is generated during this process.

Inherited Antigen Receptor

 Genes Hematopoietic stem cells in the bone marrow and early lymphoid progenitors contain Ig and TCR genes in their inherited, or germline, configuration. In this configuration, Ig heavy-chain and light-chain loci and the TCR α chain and β chain loci each contain multiple variable region (V) gene segments, numbering about 30 to 45, and one or a few constant region (C) genes (Fig. 1). Between the V and C gene segments are groups of several short coding sequences called diversity (D) and joining (J) gene segments. (All antigen receptor gene loci contain V, J, and C gene segments, but only the Ig heavy chain and TCR β chain loci also contain D gene segments.) These separated gene segments cannot code for functional antigen receptor proteins, so they have to be brought together as lymphocytes mature. 

Fig1. Germline organization of antigen receptor gene loci. In the germline, inherited antigen receptor gene loci contain coding segments (exons, shown as colored blocks of various sizes) that are separated by segments that are not expressed (introns, shown as gray sections). Each immunoglobulin (Ig) heavy-chain constant (C) region and T cell receptor (TCR) C region consists of multiple exons, which are not shown, that encode the domains of the C regions; the organization of the Cμ exons in the Ig heavy-chain locus is shown as an example. The diagrams illustrate the antigen receptor gene loci in humans; the basic organization is the same in all species, although the precise order and number of gene segments may vary. The numbers of V, D, and J gene segments are estimates of functional gene segments (those that can code for proteins). The sizes of the segments and the distances between them are not drawn to scale. D, Diversity; J, joining; L, leader sequence (a small stretch of nucleotides that encodes a peptide that guides proteins through the endoplasmic reticulum and is cleaved from the mature proteins); V, variable.

Somatic Recombination and Expression of Antigen Receptor Genes

The commitment of a lymphocyte progenitor to become a B lymphocyte is associated with the recombination of randomly selected gene segments in the Ig heavy-chain locus—first one D gene segment with one J segment to form a fused DJ complex, followed by the rearrangement of a V segment to the fused DJ complex (Fig. 2).

Thus, the committed but still-developing B cell now has a recombined VDJ exon in the heavy-chain locus. This gene is transcribed, and in the primary RNA transcript, the VDJ exon is spliced to the C-region exons of the μ chain, the most 5′ C region, to form a complete μ messenger RNA (mRNA). The μ mRNA is translated to produce the μ heavy chain, which is the first Ig protein synthesized during B cell maturation.

Essentially the same sequence of DNA recombination and RNA splicing leads to production of a light chain in B cells, except that the light-chain loci lack D segments, so a V region exon recombines directly with a J segment. The rearrangement of TCR α chain and β chain genes in T lymphocytes is similar to that of Ig L and H chains, respectively. 

Fig2. Recombination and expression of immunoglobulin (Ig) genes. The expression of an Ig heavy chain involves two gene recombination events (D-J joining, followed by joining of a V region to the DJ complex, with deletion of intervening gene segments). The recombined gene is transcribed, and the VDJ complex is spliced onto the C region exons of the first heavy-chain RNA (which is μ), to give rise to the μ messenger RNA (mRNA). The mRNA is translated to produce the μ heavy-chain protein. The recombination of other antigen receptor genes—that is, the Ig light chain and the T cell receptor (TCR) α and β chains—follows essentially the same sequence, except that in loci lacking D segments (Ig light chains and TCR α), a V gene recombines directly with a J gene segment.

Mechanisms of V(D)J Recombination

T he somatic recombination of V and J, or of V, D, and J, gene segments is mediated by a lymphoid-spe cific enzyme, the VDJ recombinase, and additional enzymes, most of which are not lymphocyte specific and are involved in repair of double-stranded DNA breaks introduced by the recombinase. The VDJ recombinase is composed of the recombination-activating gene 1 and 2 (RAG-1 and RAG-2) proteins. It recognizes DNA sequences that flank all antigen receptor V, D, and J gene segments. As a result of this recognition, the recombinase brings two Ig or TCR gene segments close together and cleaves the DNA at specific sites. The DNA breaks are then repaired by ligases, producing a full-length recombined VJ or VDJ exon without the intervening DNA segments (see Fig. 2). The VDJ recombinase is expressed only in immature B and T lymphocytes. Although the same enzyme can mediate recombination of all Ig and TCR genes, intact Ig heavy-chain and light-chain genes are rearranged and expressed only in B cells, and TCR α and β genes are rearranged and expressed only in T cells. The lineage specificity of receptor gene rearrangement appears to be linked to the expression of lineage-specific transcription factors. In B cells, B lineage-specific transcription factors “open” the Ig gene locus at the chromatin level but not the TCR locus, whereas in developing T cells, transcriptional regulators help open the TCR locus but not the Ig locus. The “open” loci are the ones that are accessible to the recombinase. 

Generation of Ig and TCR Diversity

 Diversity of antigen receptors is produced by the use of different combinations of V, D, and J gene segments in different clones of lymphocytes (called combinatorial diversity) and even more by changes in nucleotide sequences introduced at the junctions of the recombining V, D, and J gene segments (called junctional diversity; Fig. 3). Combinatorial diversity is limited by the number of available V, D, and J gene segments, but junctional diversity is almost unlimited. Junctional diversity is produced by three mechanisms, which generate more sequences than are present in the germline genes:

• Exonucleases may remove nucleotides from V, D, and J gene segments at the sites of recombination.

 • A lymphocyte-specific enzyme called terminal deoxyribonucleotidyl transferase (TdT) catalyzes the random addition of nucleotides that are not part of germline genes to the junctions between V and D segments and D and J segments, forming so-called N regions.

 • During an intermediate stage in the process of V(D)J recombination, the two broken strands of the DNA at each end of the cut DNA form hairpin loops. As a first step in the repair process, the loops are asymmetrically cut, forming overhanging DNA sequences. These overhangs have to be filled in with new nucleotides, which are called P-nucleotides, introducing even more variability at the sites of recombination.

As a result of these mechanisms, the nucleotide sequence at the site of V(D)J recombination in antibody or TCR genes in one clone of lymphocytes differs from the sequence at the V(D)J site of antibody or TCR molecules made by every other clone. These junctional sequences and the D and J segments encode the amino acids of the CDR3 loop, mentioned earlier as the most variable of the CDRs and the most important for anti gen recognition. Thus, junctional diversity maximizes the variability in the antigen-recognizing portions of antibodies and TCRs. In the process of creating junctional diversity, many genes may be produced with out-of-frame sequences that cannot code for proteins and are therefore useless. This is the price the immune system pays for generating tremendous diversity. The risk of producing nonfunctional genes also is why the process of lymphocyte maturation contains checkpoints at which only cells with useful receptors are selected to survive.

The uniqueness of CDR3 sequences in every lymphocyte clone can be exploited to distinguish neoplastic and reactive proliferations of B and T lymphocytes. In tumors arising from these cells, all the cells of the tumor will have the same CDR3 (because they all arose from a single B or T cell clone), but in proliferations that are reactions to external stimuli, many CDR3 sequences will be present. The same principle can be used to define the magnitude of an immune response—measuring the number of CDR3 sequences present in a population before and during a response is an indicator of the amount of proliferative expansion of a B or T cell clone. 

Fig3. Mechanisms of diversity in antigen receptors. Diversity in immunoglobulins and T cell receptors is produced by random combinations of V, D, and J gene segments, which is limited by the numbers of these segments and by removal and addition of nucleotides at the V-J or V-D-J junctions, which is almost unlimited. The numbers of gene segments refer to the average numbers of functional genes (which are known to be expressed as RNA or protein) in humans. Junctional diversity maximizes the variations in the CDR3 regions of the antigen receptor proteins, because CDR3 includes the junctions at the site of V-J and V-D-J recombination. The diversity is further enhanced by the juxtaposition of the V regions of the two types of chains in Ig or TCRs to form the complete antigen binding sites, and thus the total diversity is theoretically the product of the total diversity of each of the juxtaposed V regions. The estimated contributions of these mechanisms to the total possible numbers of distinct B and T cell antigen receptors are shown. Although the upper limit on the number of immunoglobulin (Ig) and TCR proteins that may be expressed is extremely large, each individual contains on the order of only 107–109 clones of B cells and T cells with distinct specificities and receptors; in other words, only a fraction of the potential repertoire may actually be expressed. (Modified from Davis MM, Bjorkman PJ: T-cell antigen receptor genes and T-cell recognition, Nature 334:395–402, 1988.)