The diversity of the B- and T-cell repertoires is created by random combinations of germline gene segments being joined together and by the addition or deletion of sequences at the junctions between these segments. Several genetic mechanisms contribute to this diversity (Fig. 1 and Table 1).
• Combinatorial diversity. Different combinations of gene segments united by V(D)J recombination produce different antigen receptors. The maximum possible number of combinations of these gene segments is the product of the numbers of V, J, and (if present) D gene segments at each antigen receptor locus. Therefore, the amount of combinatorial diversity that can be generated at each locus reflects the number of germline V, J, and D gene segments at that locus. After synthesis of antigen receptor proteins, combinatorial diversity is further enhanced by the juxtaposition of two different, randomly generated V regions (i.e., VH and VL in Ig molecules and Vα and Vβ in TCR molecules). Therefore, the total combinatorial diversity is theoretically the product of the combinatorial diversity of each of the two associating chains. The actual degree of combinatorial diversity in the expressed Ig and TCR repertoires in any individual is likely to be considerably less than the theoretical maximum. This is because not all combinations of gene segments are equally likely to occur and not all pairings of Ig heavy and light chains or TCR α and β chains may form functional antigen receptors. Importantly, because the numbers of V, D, and J segments in each locus are limited (see Table 1), the maxi mum possible numbers of combinations are on the order of 1 to 3 million (reflecting only combinatorial diversity). This is much less than the actual diversity of antigen receptors in mature lymphocytes.
• Junctional diversity. The largest contribution to the diversity of antigen receptors is made by the addition or removal of nucleotides at the junctions of the V and D, D and J, or V and J segments at the time these segments are joined. One way in which this can occur is if endonucleases remove nucleotides from the germline sequences at the ends of the recombining gene segments. In addition, new nucleotide sequences, not present in the germline, may be added at junctions (Fig. 2). As described earlier, coding segments (e.g., V and J gene segments) that are cleaved by RAG1 form hairpin loops. Each hairpin DNA is nicked (a single DNA strand is cleaved) asymmetrically by the enzyme ARTEMIS so that the hairpin is opened up and one DNA strand is then longer than its complementary strand. The shorter strand has to be extended with nucleotides complementary to the longer strand before the two coding segments can be ligated. The longer strand thus serves as a template for the addition of short lengths of new nucleotides called P nucleotides, and this process introduces new sequences at the V-D-J junctions. Another mechanism of junctional diversity is the random addition of up to 20 nontemplate encoded nucleotides called N nucleotides (see Fig. 2). N region diversification is more common in Ig heavy chains and in TCR β and γ chains than in Ig κ or λ chains. This addition of these new N nucleotides is mediated by the enzyme terminal deoxynucleotidyl transferase (TdT). In mice rendered deficient in TdT by gene knockout, the diversity of B- and T-cell repertoires is substantially less than in normal mice. The addition of P nucleotides and N nucleotides at the recombination sites may introduce frameshifts, theoretically generating termination codons in two of every three joining events (if the total number of added bases is not a multiple of three). These genes cannot produce functional proteins, but such inefficiency is the price that is paid for generating diversity.
Table1. Contributions of Different Mechanisms to the Generation of Diversity in Immunoglobulin (Ig) and T-Cell Receptor Genes
Fig1. Diversity of antigen receptor genes. The figure shows the two mechanisms that give rise to the diversity of antigen receptors—combinations of different V, D, and J gene segments and addition or removal of nucleotides at the joints. Although different V-D-J combinations and addition of N/P nucleotides are shown separately, to emphasize the different contributions of combinatorial and junctional diversity, both processes occur at the same time during rearrangement of gene segments. Not shown is removal of nucleotides, which also occurs simultaneously and contributes to more junctional diversity. The figure illustrates a few distinct antigen receptor genes produced by these mechanisms, but the total diversity is enormous (see Table1). D, Diversity; J, joining; V, variable.
Fig2. Junctional diversity. During the joining of different gene segments, addition or removal of nucleotides may lead to the generation of novel nucleotide and amino acid sequences at the junction. Nucleotides (P sequences) may be added to asymmetrically cleaved hairpins in a templated manner. Other nucleotides (N regions) may be added to the sites of V-D, V-J, or D-J junctions in a nontemplated manner by the action of the enzyme terminal deoxynucleotidyl transferase (TdT). These additions generate new sequences that are not present in the germline. RAG, Recombination-activating gene; RSS, recombination signal sequence; V, variable.
Because of junctional diversity, antibody and TCR molecules show the greatest variability at the junctions of V and C regions, which form the third hypervariable region, or CDR3. In fact, because of junctional diversity, the numbers of different amino acid sequences that are present in the CDR3 regions of Ig and TCR molecules are much greater than the numbers that can be encoded by germline gene segments. As expected, the CDR3 regions of Ig and TCR molecules are also the most important portions of these molecules for deter mining the specificity of antigen binding.
Although the theoretical limit to the number of Ig and TCR proteins that can be produced is enormous (see Table 1), the actual number of antigen receptors expressed on B or T cells in each individual at any one point in time is probably on the order of only 107 or 108.
A clinical application of our knowledge of junctional diversity is the determination of the clonality of lymphoid expansions that arise from B or T cells. This laboratory test is used to identify monoclonal tumors of lymphocytes and to distinguish tumors from polyclonal expansions. Because every lymphocyte clone expresses a unique antigen receptor CDR3 region, the sequence of nucleotides at the V(D)J recombination site serves as a specific marker for each clone. Thus, by determining the sequence of the junctional regions of Ig or TCR genes in different B- or T-cell expansions, one can establish whether these lesions arose from a single clone (indicating a neoplastic state) or independently from different clones (implying nonneoplastic proliferation of lymphocytes). The same method may be used to identify small numbers of tumor cells in the blood or tissues.
