Development of immature T-cell precursors (thymocytes) to either the CD4 helper or CD8 killer T-cell lineages correlates precisely with their T-cell receptor specificity for major histocompatibility complex class II or class I molecules, respectively, indicating that the process is carefully regulated. Although intensively studied owing to its importance in determining the composition of the mature T-cell compartment and as a general model of binary lineage decisions, the underlying molecular pathways remain obscure. We have previously reported a spontaneous mouse mutant (HD (helper deficient) mice) in which lineage commitment is specifically perturbed without affecting positive selection. Here we show that a point mutation in the zinc finger transcription factor Th-POK (T-helper-inducing POZ/Krü ppel-like factor) is responsible for redirection of class-II-restricted thymocytes to the CD8 lineage in HD mice. Furthermore, we demonstrate that constitutive expression of this factor during thymic development leads to redirection of class-I-restricted thymocytes to the CD4 lineage, indicating that Th-POK is a master regulator of lineage commitment.Developing ab T cells progress through three major stages in the thymus, defined by differential expression of the CD4 and CD8 coreceptor molecules; that is, CD4 2 CD8 2 (double negative), CD4 þ CD8 þ (double positive) and CD4 þ CD8 2 or CD4 2 CD8 þ (single positive). The double-positive to single-positive transition depends on productive rearrangement of both a-and b-subunits of the T-cell receptor (TCR) and engagement of the complete ab TCR by intrathymic ligands (positive selection). Simultaneously, thymocytes diverge into the functionally distinct T-helper and T-killer lineages, defined by expression of CD4 and CD8, respectively. Mature T cells show an almost perfect correlation between CD4 or CD8 expression and their TCR specificity towards class II or class I major histocompatibility complex (MHC) molecules, respectively. Alternative instructive and stochastic/selective models have been proposed to explain this marked correlation (for recent reviews see refs 1, 2). Current thinking favours a quantitative version of the instructive model, whereby lineage choice is determined by the relative strength or duration of TCR engagement 3-9 ; however, the intracellular pathways that are involved remain unknown.Progress in the field has been hindered because lineage commitment is so intimately tied to the process of positive selection that it is difficult to study in isolation. Hence, no specific pathways have been identified that are required for lineage commitment but not positive selection. Recently, we identified a spontaneous recessive mutation in mice, the HD mutation, which appeared to identify a genetic locus specifically required for lineage commitment 10 . Notably, this mutation caused redirection of all class-II-restricted thymocytes to the CD8 lineage 11 . The existence of such a mutation demonstrated a mechanistic distinction between the pathways governing lineage ...

The CD3 complex found associated with the T cell receptor (TCR) is essential for signal transduction following TCR engagement. During T cell development, TCR‐mediated signalling promotes the transition from one developmental stage to the next and controls whether a thymocyte undergoes positive or negative selection. The roles of particular CD3 components in these events remain unclear. Indeed, it is unknown whether they have specialized or overlapping roles. However, the multiplicity of CD3 components and their evolutionary conservation suggest that they serve distinct functions. Here the developmental requirement for the CD3δ chain is analyzed by generating a mouse line specifically lacking this component (δ−/− mice). Strikingly, CD3δ is shown to be differentially required during development. In particular, CD3δ is not needed for steps in development mediated by pre‐TCR or γδTCR, but is required for further development of thymocytes expressing αβTCR. Absence of CD3δ specifically blocks the thymic selection processes that mediate the transition from the double‐positive to single‐positive stages of development.

Thymocytes from mice lacking the CD3delta chain of the T-cell receptor (TCR), unlike those of other CD3-deficient mice, progress from a CD4- CD8- double-negative to a CD4+ CD8+ double-positive stage. However, CD3delta-/- double-positive cells fail to undergo positive selection, by which double-positive cells differentiate into more mature thymocytes. Positive selection is also impaired in mice expressing inactive components of the Ras/mitogen activated protein (MAP) kinase signalling pathway. Here we show that CD3delta-/- thymocytes are defective in the induction of extracellular signal-regulated protein kinase (ERK) MAP kinases upon TCR engagement, whereas activation of other MAP kinases is unaffected. The requirement for CD3delta maps to its extracellular or transmembrane domains, or both, as expression of a tail-less CD3delta rescues both ERK activation and positive selection in CD3delta-/- mice. Furthermore, the defect correlates with severely impaired tyrosine phosphorylation of the linker protein LAT, and of the CD3zeta chain that is localized to membrane lipid rafts upon TCR engagement. Our data indicate that the blockade of positive selection of CD3delta-/- thymocytes may derive from defective tyrosine phosphorylation of CD3zeta in lipid rafts, resulting in impaired activation of the LAT/Ras/ERK pathway.

Developing alphabeta T cells diverge into the CD4 and CD8 lineages as they mature in the thymus. It is unclear whether lineage commitment is mechanistically distinct from the process that selects for the survival of T cells with useful T cell receptor (TCR) specificities (positive selection). In HD mice, which lack mature CD4+ T cells, major histocompatibility complex (MHC) class II-restricted T cells are redirected to the CD8 lineage independent of MHC class I expression. However, neither TCR-mediated signaling nor positive selection is impaired. Thus, the HD mutation provides genetic evidence that lineage commitment may be mechanistically distinct from positive selection.

Maturation of immature CD4−CD8− (DN) thymocytes to the CD4+CD8+ (DP) stage of development is driven by signals transduced through a pre–T cell receptor (TCR) complex, whose hallmark is a novel subunit termed pre-Tα (pTα). However, the precise role of pre-TCRs in mediating the DN to DP transition remains unclear. Moreover, progress in understanding pre-TCR function has been hampered thus far because previous attempts to demonstrate expression of pTα-containing pre-TCRs on the surface of normal thymocytes have been unsuccessful. In this report, we demonstrate for the first time that pTα-containing pre-TCR complexes are expressed at low levels on the surface of primary thymocytes and that these pre-TCR complexes comprise a disulfide-linked pTα–TCR-β heterodimer associated not only with CD3-γ and -ε, as previously reported, but also with ζ and δ. Interestingly, while CD3-δ is associated with the pre-TCR complex, it is not required for pre-TCR function, as evidenced by the generation of normal numbers of DP thymocytes in CD3-δ–deficient mice. The fact that any of the signaling components of the pre-TCR are dispensable for pre-TCR function is indeed surprising, given that few pre-TCR complexes are actually expressed on the surface of primary thymocytes in vivo. Thus, pre-TCRs do not require the full array of TCR-associated signaling subunits (γ, δ, ε, and ζ), possibly because pTα itself possesses signaling capabilities.