Epitope specificity of immunoglobulin molecules produced by B cells and of T-cell receptors is determined before they encounter antigen. Moreover, several possible epitope-binding specificities greatly exceeds several genes within the human genome. This presents a paradox: How does the immune system generate a diverse array of antigen-specific molecules from a limited number of genes? The immune system’s genetic solution is both fascinating and elegant.
Properties of Lymphocyte Antigen Receptors
Domains located at the amino terminus of immunoglobulin heavy and light chains (variable or VH and VL regions) produced by different B cells are highly variable in amino acid sequence. In contrast, other regions, termed C or constant regions, are limited in variability for immunoglobulins of the same isotype produced by different B cells. Light chains have a single constant domain (CL, also designated as C? for kappa chains or C? for lambda chains), and heavy chains contain multiple constant regions CH1, CH2, CH3, and for some CH4 domains. Heavy or light chain DNA gene segments for both variable and constant regions are rearranged, transcribed into RNA, and translated into a single heavy or light chain polypeptide.
Individuals codominantly inherit maternal and paternal sets of alleles for light chain and heavy chain loci. A single B cell or plasma cell may express only the kappa (V?C?) or the lambda (V?C?) light chain alleles, either maternal or paternal, to the exclusion of all others (Fig. 8.1). Likewise, a single B cell may express only the maternal or paternal VHCH heavy chain alleles but not both. The restriction of VLCL and VHCH expression to a single member of each of the involved chromosome pairs is termed allelic exclusion. However, the combined contributions of all of the B cells mean that both maternal and paternal allotypes (allelic forms) are expressed within any particular individual.
Figure 8.1. Allelic exclusion.
Immunoglobulin light and heavy gene clusters are located on different chromosome pairs, each pair having a maternally derived and a paternally derived chromosome. Each B cell and its progeny use only one parental chromosome to encode its light (#2 or #22) and heavy (#14) chains. A given B cell uses these same variable region gene clusters throughout its lifetime for the immunoglobulins it produces to the exclusion of all others.
The same principles apply to the genes encoding the ?? and ?? T-cell receptors, which also include light chains (? or ?) and heavy chains (? or ?). Each chain consists of a variable region at its amino terminus and a constant region at its carboxy terminus. The variable regions of the chains are determined by the rearrangement of the DNA encoding them and the production of an mRNA transcript, including both the variable and constant regions. Splicing of the mRNA that is then translated into the polypeptide chains unites the variable and constant regions.
Immunologists estimated that each person has the ability to produce a range of individual antigen-specific receptors capable of binding as many as 1015 different epitopes. DNA chromosomal rearrangement is responsible for a significant portion of epitope-specific diversity among T and B cell receptors. Rearrangement occurs at both the DNA and RNA levels by the deletion of nucleotides, followed by reannealing, to bring together gene segments that were previously separated (Fig. 8.2).
Inversion of certain DNA sequences, notably within V?, leads to rearranged nucleotide sequences of the same length as the original (see Fig. 8.2). Additional variation comes from junctional diversity (Fig. 8.3) as “exposed” ends of gene segments (V, D, and J genes) undergoing rearrangement are modified through the addition or removal of nucleotides by deoxynucleotidyl transferase (TdT) before the genes are linked together. Thus, even if the same two genes were to be linked together, the nucleotide sequence at their junctions may be different, and the amino acid sequences encoded would differ.
Figure 8.2. Chromosome rearrangement.
Segments of DNA encoding a series of genes are rearranged by the deletion of intervening DNA. Joining of the remaining segments (a process called annealing) then brings together genes that were originally separated on the chromosome. In addition, DNA rearrangement is sometimes accomplished by the inversion of DNA segments, changing the linear sequence of genes without the removal of intervening DNA.
Figure 8.3. Junctional diversity.
Terminal deoxynucleotidyl transferase (TdT) can add or remove exposed ends of DNA before annealing, producing additional variation in nucleotide sequence.
As we noted earlier, T cells express distinct, epitope-specific cell-surface receptors (TCRs) that are heterodimers composed of either ?? or ?? light–heavy chain pairs. Each polypeptide contains a single variable region domain and a single constant region domain. T cells express and display T-cell receptor (TCR) complexes composed of an ?? or a ?? TCR (but never both) heterodimer pair, associated CD3 (?, ?, and ? and a CD247 ?-chain homodimer), and CD4 or CD8 molecules. Associated transmembrane molecules, such as CD4 or CD8, stabilize the interaction of the TCR with a specific peptide-MHC (pMHC) combination.
Others, such as those in the CD3 complex, participate in signal transduction events after TCR-ligand engagement (Fig. 8.4). The short cytoplasmic tail of the TCR lacks signaling sequence or immunoreceptor tyrosine activation motifs (ITAMs). The CD3 and CD247 molecules supply these. Unlike antibodies, TCRs cannot bind free epitopes. They can bind only enzymatically cleaved fragments of larger polypeptides that are presented as pMHC complexes.
Figure 8.4. T-cell receptors (TCRs).
T cells express either ?? or ?? TCR heterodimers. The CD3 complex associates with the TCR to transduce a signal to the interior of the cell when the TCR engages a peptide-MHC complex.
A. Gene clusters encoding T-cell receptors
Gene clusters encoding ? and ? chains of the TCR on chromosome 14 are arranged such that the entire ? chain gene cluster (D?, J?, and C?) lies within the a gene cluster between the D? and J? chain genes (Fig. 8.5). Genes encoding ? and ? chains are located in separate clusters on chromosome 7.
Figure 8.5. Gene clusters encoding TCR chains.
Gene clusters encoding TCR light chains are located on chromosomes 14 (? chain) and 7 (? chain), and heavy chains on chromosomes 7 (? chain) and 14 (? chain).
The first level of TCR diversity comes from DNA recombination to produce the variable regions. The selection of V, D, and J genes for rearrangement appears to be random from cell to cell: 2475 ? chain sequences (45 V? × 55 J?) × 1200 ? chain sequences (50 V? × 2 D? × 12 J?). Random association of ? and ? chains yields nearly 3 million different epitope-binding sites for ?? chains.
For ?? TCRs, 25 ? chain sequences (5 V? × 5 J?) × 24 ? chain sequences (2 V? × 3 D? × 4 J?) yields 600 ?? TCR epitope-binding site possibilities. Junctional diversity, a process mediated by TdT (see Fig. 8.3), contributes a second level of diversity by the insertion or deletion of up to 20 nucleotides at the time of recombination. Thus, the total number of possible TCR specificities increases by many orders of magnitude.
B. Variable regions: Rearrangement of V, D, and J genes
TCRs are generated by recombination enzymes or recombinases (e.g., Rag-1 and Rag-2) that mediate genetic rearrangement and recombination, processes similar to those seen for immunoglobulin (see sections 8.V.B and 8.V.C). Each T cell produces ?? or ?? TCR heterodimers, never both. Gene rearrangement begins by the excision and deletion of DNA between V? and J? (light, Fig. 8.5) or V? and D? (heavy) chain genes.
Because the entirety of the ? chain genetic material lies within the ? light chain sequence (between V? and J?), initiation of recombination by a chain genes deletes ? chain DNA. Conversely, initiation of recombination by ? chain (between V?, D?, and D?) DNA precludes a light chain DNA recombination. For the details of this process, see Figure 8.6.
Figure 8.6. Rearrangement to form an ?? TCR.
For the ? chain, DNA is deleted to join randomly selected V and J genes. An mRNA transcript is then produced containing united VJ genes and a constant gene. This transcript is then spliced to unite the VJ and C genes to form mRNA that can be directly translated to a polypeptide-containing conjoined VJC segments. A similar process occurs for the ? chain with the addition of D genes to form a VDJ variable region. ?? TCRs are synthesized in a similar manner.
C. Uniting variable and constant regions
TCR constant region genes (already rearranged into VJ or VDJ units) are united with their respective light chain VJ (C? or C?) by transcription into mRNA, followed by splicing to delete intervening mRNA. The united VJC (C? or C?) or VDJC (C? or C?) transcripts are then translated into proteins that are the joint product of the rearranged genes.
D. Random combinations of light and heavy chains
Genetic rearrangement and junctional diversity randomly create TCR chains that vary among, but not within, individual T cells. Each developing T cell randomly produces a unique light–heavy chain (?? or ??) combination with unique specificity. The theoretical number of possible combinations produced within the body may be estimated to be the product of several possible light chains and several possible heavy chains (Fig. 8.7).
Immunologists estimate that 1 to 5 million epitope-binding combinations are possible for TCRs. However, unlike immunoglobulin genes, TCRs do not undergo subsequent changes equivalent to the isotype switching and somatic hypermutation that occur in—immunoglobulins to further increase their diversity (see Sections 8.V.E and 8.V.G).
Figure 8.7. Formation of TCR peptide-MHC binding regions.