In the fully developed thymus, only ~60% of the double-negative thymocytes are immature T cells. The double-negative thymocyte pool (about 5% of all thymocytes) also includes two populations of more mature T cells that belong to minority lineages, including T cells expressing γ:δ T-cell receptors (see Section 8-16) and T cells bearing α:β T-cell receptors of very limited diversity (iNKT cells; see Section 6-19). In this and subsequent discussions, we reserve the term ‘double-negative thymocytes’ for the immature thymocytes that do not yet express a complete T-cell receptor molecule. These cells give rise to both γ:δ and α:β T cells (see Fig. 8.20), although most of them develop along the α:β pathway.
The α:β pathway is shown in more detail in Fig. 8.18. The double-negative stage can be further subdivided into four stages on the basis of expression of the adhesion molecule CD44, CD25 (the α chain of the IL-2 receptor), and Kit, the receptor for SCF (see Section 8-1). At first, double-negative thymocytes express Kit and CD44 but not CD25 and are called DN1 cells; in these cells, the genes encoding both chains of the T-cell receptor are in the germline configuration. As thymocytes mature, they begin to express CD25 on their surface and are called DN2 cells; later, expression of CD44 and Kit is reduced, and they are called DN3 cells.
Rearrangement of the T-cell receptor β-chain locus begins in DN2 cells with some Dβ to Jβ rearrangements and continues in DN3 cells with Vβ to DJβ rearrangement. Cells that fail to make a successful rearrangement of the β-chain locus remain at the DN3 (CD44lowCD25+) stage and soon die, whereas cells that make productive β-chain gene rearrangements and express the β-chain protein lose expression of CD25 once again and progress to the DN4 stage, in which they proliferate. The functional significance of the transient expression of CD25 is unclear: T cells develop normally in mice in which the IL-2 gene has been deleted by gene knockout (see Appendix I, Section A-35). By contrast, Kit is quite important for the development of the earliest double-negative thymocytes, in that mice lacking Kit have a much smaller number of double-negative T cells. In addition, continuous Notch signaling is important for progression through each stage of T-cell development. A second essential factor is IL-7, which is produced by the thymic stroma. In the absence of IL-7, IL-7 receptor α, γ-c, or the IL-7 receptor signaling protein Jak3, a severe block in T-cell development occurs in both mice and humans. In fact, the human primary immunodeficiency disease characterized by defects in T cells and NK cells, X-linked SCID (severe combined immunodeficiency disease), is caused by a genetic deficiency leading to the absence of γ-c protein expression.