In the thymus, bone marrow-derived stem cells undergo a programmed series of differentiation and selection steps that results in the generation of mature functional CD4+ and CD8+ cells that are exported to the periphery. The earliest stage of thymocyte development is the so-called double-negative (DN) cell that lacks expression of both the CD3/TcR complex and the CD4 and CD8 coreceptors. DN cells then express the Rag1 and Rag2 genes, rearrange the TRB genes (CD3/TcR+) and express CD4 and CD8 coreceptors to become double-positive (DP) cells. DP cells rearrange the TRA genes and undergo both positive and negative selection through interaction with major histocompatibility complex (MHC) molecules on thymic epithelium and dendritic cells to become SP CD4+ helper or CD8+ cytotoxic T cells.
The GATA3 zinc finger transcription factor is positioned at the very earliest stages of the T cell lineage. GATA3 also influences Th2 differentiation, thus playing a role in multiple stages of T cell development.
Lymphoid enhancer factor 1 (LEF1) and T cell factor 1 (TCF1) are closely related members of the "HMG box" family of transcription factors that regulate the TRA enhancer in vitro. Another HMG family member, Sox4, is also critical in the transition from the DN to the DP stage. Although TCF1/LEF1 regulate the expression of the TcRα genes, downregulation of TcRα cannot explain the block since TcRα expression in not required for the DN to DP transition. TCF/LEF proteins are downstream targets of the Wnt/Wingless signaling pathway shown to control development in Drosophila and Xenopus. TCF/LEF proteins mediate Wnt/Wingless signalling by direct interaction with and recruitment of Armadillo/β -catenin as a transcriptional coactivator. However, TCF factors can also act as repressors in Drosophila, Xenopus, and Caenorhabditis elegans. The interaction of a single transcription factor with both activator and repressor proteins would achieve appropriate expression of target genes in cell-fate decisions.
Two signalling pathways in addition to Wnt appear to be critical as negative rather than positive regulators for the generation of T cells. The constitutive activation of the p38 MAP kinase pathway in the thymus blocks the differentiation of DN into DP thymocytes. Conditional ablation of the Csk kinase, which inhibits Scr family kinases (Lck, Fyn) by phosphorylating C-terminal tyrosine residues, completely overcomes the DN to DP block and allows CD4+ thymocyte development in both TcRβ -deficient and MHC class II-deficient-mice .
Interestingly, CD8+ cells are not generated in the absence of Csk. An intronic silencer has been reported to control the tissue-specific expression of CD4 in T helper cells. There are at least two factors binding to the silencer: HES1, a bHLH protein that is a homolog of the Drosophila Hairy/Enhancer of split and a homeodomain protein, termed SAF. Mutation of the HES1- and SAF-binding sites affects function, suggesting for HES1, that the Notch signalling pathway is important in thymic development.
The LKLF transcription factor is critical both in controlling the DP to SP transition in the thymus and in maintaining T cells quiescence in the periphery.
At the DN stage, the T cell lineage splits to generate two distinct types of T cells, distinguished by the type of T cell receptor they bear TcRα β and TcRγδ. Occupancy of the CBF, Myb, and GATA sites in the δ enhancer in DN cells decreases at the DP stage at the time when the Eα enhancer is active.