The a chain, like the immunoglobulin L chain, is encoded by V, J, and C gene segments. The p chain, like the immunoglobulin H chain, is encoded by V, D, J, and C gene segments. Rearrangement of the TCR a- and p-chain gene segments results in VJ joining for the a chain and VDJ joining for the p chain (Figure 9-6).
After transcription of the rearranged TCR genes, RNA processing, and translation, the a and p chains are expressed as a disulfide-linked heterodimer on the membrane of the T cell. Unlike immunoglobulins, which can be membrane bound or secreted, the ap heterodimer is expressed only in a membrane-bound form; thus, no differential RNA processing is required to produce membrane and secreted forms. Each TCR constant region includes a connecting sequence, a transmembrane sequence, and a cytoplasmic sequence.
The germ-line DNA encoding the TCR a and p chain constant regions is much simpler than the immunoglobulin heavy-chain germ-line DNA, which has multiple C gene segments encoding distinct isotypes with different effector functions. TCR a-chain DNA has only a single C gene segment; the p-chain DNA has two C gene segments, but their protein products differ by only a few amino acids and have no known functional differences.
MECHANISM OF TCR DNA REARRANGEMENTS
The mechanisms by which TCR germ-line DNA is rearranged to form functional receptor genes appear to be
LVal LVaw L Vgl L Vg» D81D82 J81J82 C8 L Vs5 Ja1Ja2Jan Ca
Rearranged a-chain DNA
Protein product aß heterodimer
Rearranged P-chain DNA
Example of gene rearrangements that yield a functional gene encoding the ap T-cell receptor. The a-chain DNA, analogous to immunoglobulin light-chain DNA, undergoes a variable-region Va-Ja joining. The P-chain DNA, analogous to immunoglobulin heavy-chain DNA, undergoes two variable-region joinings: first Dp to Jp and then Vp to DpJp. Transcription of the rearranged genes yields primary transcripts, which are processed to give mRNAs encoding the a and p chains of the membrane-
bound TCR. The leader sequence is cleaved from the nascent polypeptide chain and is not present in the finished protein. As no secreted TCR is produced, differential processing of the primary transcripts does not occur. Although the P-chain DNA contains two C genes, the gene products of these two C genes exhibit no known functional differences. The C genes are composed of several exons and introns, which are not individually shown here (see Figure 9-7).
similar to the mechanisms of Ig-gene rearrangements. For example, conserved heptamer and nonamer recombination signal sequences (RSSs), containing either 12-bp (one-turn) or 23-bp (two-turn) spacer sequences, have been identified flanking each V, D, and J gene segment in TCR germ-line DNA (see Figure 5-6). All of the TCR-gene rearrangements follow the one-turn/two-turn joining rule observed for the Ig genes, so recombination can occur only between the two different types of RSSs.
Like the pre-B cell, the pre-T cell expresses the recombination-activating genes (RAG-1 and RAG-2). The RAG-1/2 recombinase enzyme recognizes the heptamer and non-amer recognition signals and catalyzes V-J and V-D-J joining during TCR-gene rearrangement by the same deletional or inversional mechanisms that occur in the Ig genes (see Figure 5-7). As described in Chapter 5 for the immunoglobulin genes, RAG-1/2 introduces a nick on one DNA strand between the coding and signal sequences. The recombinase then catalyzes a transesterification reaction that results in the formation of a hairpin at the coding sequence and a flush 5' phosphorylated double-strand break at the signal sequence. Circular excision products thought to be generated by looping-out and deletion during TCR-gene rearrangement have been identified in thy-mocytes (see Figure 5-8).
Studies with SCID mice, which lack functional T and B cells, provide evidence for the similarity in the mechanisms of Ig-gene and TCR-gene rearrangements. As explained in Chapter 19, SCID mice have a defect in a gene required for the repair of double-stranded DNA breaks. As a result of this defect, D and J gene segments are not joined during rearrangement of either Ig or TCR DNA (see Figure 5-10). This finding suggests that the same double-stranded break-repair enzymes are involved in V-D-J rearrangements in B cells and in T cells.
Although B cells and T cells use very similar mechanisms for variable-region gene rearrangements, the Ig genes are not normally rearranged in T cells and the TCR genes are not rearranged in B cells. Presumably, the recombinase enzyme system is regulated in each cell lineage, so that only rearrangement of the correct receptor DNA occurs. Rearrangement of the gene segments in both T and B cell creates a DNA sequence unique to that cell and its progeny. The large number of possible configurations of the rearranged genes makes this new sequence a marker that is specific for the cell clone. These unique DNA sequences have been used to aid in diagnoses and in treatment of lymphoid leukemias and lymphomas, cancers that involve clonal proliferation of T or B cells (see Clinical Focus on page 208).
As mentioned above, the 8 genes are located within the agene complex and are deleted by a-chain rearrangements. This event provides an irrevocable mode of exclusion for the 8 genes located on the same chromosome as the rearranging a genes. Allelic exclusion of genes for the TCR a and p chains occurs as well, but exceptions have been observed.
The organization of the p-chain gene segments into two clusters means that, if a nonproductive rearrangement occurs, the thymocyte can attempt a second rearrangement. This increases the likelihood of a productive rearrangement for the p chain. Once a productive rearrangement occurs for one p-chain allele, the rearrangement of the other p allele is inhibited.
Exceptions to allelic exclusion are most often seen for the TCR a-chain genes. For example, analyses of T-cell clones that express a functional ap T-cell receptor revealed a number of clones with productive rearrangements of both a-chain alleles. Furthermore, when an immature T-cell lymphoma that expressed a particular ap T-cell receptor was subcloned, several subclones were obtained that expressed the same p-chain allele but an a-chain allele different from the one expressed by the original parent clone. Studies with transgenic mice also indicate that allelic exclusion is less stringent for TCR a-chain genes than for p-chain genes. Mice that carry a productively rearranged ap -TCR transgene do not rearrange and express the endogenous p-chain genes. However, the endogenous a-chain genes sometimes are expressed at various levels in place of the already rearranged a-chain transgene.
Since allelic exclusion is not complete for the TCR a chain, there are rare occasions when more than one a chain is expressed on the membrane of a given T cell. The obvious question is how do the rare T cells that express two ap T-cell receptors maintain a single antigen-binding specificity? One proposal suggests that when a T cell expresses two different ap T-cell receptors, only one is likely to be self-MHC restricted and therefore functional.
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