the pre-T-cell receptor at the DN3 stage of thymocyte development induces signals that cause the phosphorylation and degradation of RAG-2, thus halting β-chain gene rearrangement and ensuring allelic exclusion at the β locus. These signals also induce the DN4 stage, in which rapid cell proliferation occurs, and eventually the co-receptor proteins CD4 and CD8 are expressed. The pre-T-cell receptor signals constitutively via the cytoplasmic protein kinase Lck, an Src-family tyrosine kinase (see Fig. 7.12), but seems not to require a ligand on the thymic epithelium. Lck subsequently associates with the co-receptor proteins. In mice genetically deficient in Lck, T-cell development is arrested before the CD4+CD8+ double-positive stage, and no α-chain gene rearrangements can be made.
The role of the expressed β chain in suppressing further β-locus rearrangement can be demonstrated in mice containing a rearranged TCRβ transgene: these mice express the transgenic β chain on virtually 100% of their T cells, and rearrangement of their endogenous β-chain genes is strongly suppressed. The importance of pTα has been shown in mice deficient in pTα, in which there is a hundredfold decrease in α:β T cells and an absence of allelic exclusion at the β locus.
During the proliferation of DN4 cells triggered by expression of the pre-T-cell receptor, the RAG-1 and RAG-2 genes are repressed (see Fig. 8.18). Hence, no rearrangement of the α-chain locus occurs until the proliferative phase ends, at which time RAG-1 and RAG-2 are transcribed again, and the functional RAG-1:RAG-2 complex accumulates. This ensures that each cell in which a β-chain gene has been successfully rearranged gives rise to many CD4+CD8+ thymocytes. Once the cells stop dividing, each of them can independently rearrange its α-chain genes, so that a single functional β chain can be associated with many different α chains in the progeny cells. During the period of α-chain gene rearrangement, α:β T-cell receptors are first expressed and selection by self peptide:self MHC complexes on the thymus cells can begin.
The progression of thymocytes from the double-negative to the double-positive and finally to the single-positive stage is accompanied by a distinct pattern of expression of proteins involved in DNA rearrangement, signaling, and T-cell-specific gene expression (see Fig. 8.18). TdT, the enzyme responsible for the insertion of N-nucleotides, is expressed throughout T-cell receptor gene rearrangement; N-nucleotides are found at the junctions of all rearranged α and β genes. Lck and another tyrosine kinase, ZAP-70, are both expressed from an early stage in thymocyte development. As well as its key role in signaling from the pre-T-cell receptor, Lck is also important for γ:δ T-cell development. In contrast, gene knockout studies (see Appendix I, Section A-35) show that ZAP-70, although expressed from the double-negative stage onward, is not essential for pre-T-cell receptor signaling, as double-negative thymocytes also express the related Syk kinase, which is capable of fulfilling this role. Instead, ZAP-70 is required later, to promote the development of single-positive thymocytes from double-positive thymocytes; at this stage, Syk is no longer expressed. Fyn, an Src-family kinase similar to Lck, is expressed at increasing levels from the double-positive stage onward. It is not essential for the development of α:β thymocytes as long as Lck is present, but is required for the development of iNKT cells (see Section 8-26).
8-18 T-cell α-chain genes undergo successive rearrangements until positive selection or cell death intervenes.