Vibhuti P. Dave

The zinc finger transcription factor Th-POK regulates CD4 versus CD8 T-cell lineage commitment

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/Krüppel-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.

CD3δ deficiency arrests development of the αβ but not the γδ T cell 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.

CD3delta couples T-cell receptor signalling to ERK activation and thymocyte positive selection.

Thymocytes from mice lacking the CD3δ 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, CD3δ -/- 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 CD3δ-/- 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 CD3δ maps to its extracellular or transmembrane domains, or both, as expression of a tail-less CD3δ rescues both ERK activation and positive selection in CD3δ-/- mice. Furthermore, the defect correlates with severely impaired tyrosine phosphorylation of the linker protein LAT, and of the CD3ζ chain that is localized to membrane lipid rafts upon TCR engagement. Our data indicate that the blockade of positive selection of CD3δ-/- thymocytes may derive from defective tyrosine phosphorylation of CD3ζ in lipid rafts, resulting in impaired activation of the LAT/Ras/ERK pathway.

Distinctive CD3 Heterodimeric Ectodomain Topologies Maximize Antigen-Triggered Activation of αβ T Cell Receptors

The αβ TCR has recently been suggested to function as an anisotropic mechanosensor during immune surveillance, converting mechanical energy into a biochemical signal upon specific peptide/MHC ligation of the αβ clonotype. The heterodimeric CD3εγ and CD3εδ subunits, each composed of two Ig-like ectodomains, form unique side-to-side hydrophobic interfaces involving their paired G-strands, rigid connectors to their respective transmembrane segments. Those dimers are laterally disposed relative to the αβ heterodimer within the TCR complex. In this paper, using structure-guided mutational analysis, we investigate the functional consequences of a striking asymmetry in CD3γ and CD3δ G-strand geometries impacting ectodomain shape. The uniquely kinked conformation of the CD3γ G-strand is crucial for maximizing Ag-triggered TCR activation and surface TCR assembly/expression, offering a geometry to accommodate juxtaposition of CD3γ and TCR β ectodomains and foster quaternary change that cannot be replaced by the isologous CD3δ subunit’s extracellular region. TCRβ and CD3 subunit protein sequence analyses among Gnathostomata species show that the Cβ FG loop and CD3γ subunit coevolved, consistent with this notion. Furthermore, restoration of T cell activation and development in CD3γ−/− mouse T lineage cells by interspecies replacement can be rationalized from structural insights on the topology of chimeric mouse/human CD3εδ dimers. Most importantly, our findings imply that CD3γ and CD3δ evolved from a common precursor gene to optimize peptide/MHC-triggered αβ TCR activation.

Critical and Multiple Roles for the CD3ε Intracytoplasmic Tail in Double Negative to Double Positive Thymocyte Differentiation

The preTCR is associated with signal-transducing CD3γ, δ, ε, and ζ polypeptides. It is generally agreed that CD3 chains play redundant roles in the receptor-mediated signal transduction. In the present study, we show that the intracytoplasmic (IC) domain of CD3ε is essential for early thymocyte maturation. We demonstrate that the IC domain-deleted CD3ε fails to restore the double negative (DN) to double positive (DP) thymocyte development in CD3ε-deficient mice. Additional experiments show that the membrane proximal basic amino acid rich sequence in the IC domain of CD3ε is sufficient for the DN to DP differentiation, whereas the proline rich sequence is required for efficient proliferation. This is probably due to impaired ligand independent recruitment of Nck to the proline rich sequence motif of CD3ε within the context of the preTCR. The data presented in this study elucidates mechanistic basis for the preTCR-induced proliferation of the DN thymocytes and have identified distinct roles for individual motifs of CD3ε in the preTCR-mediated differentiation and proliferation. These data provide the first genetic and phenotypic evidence for requirement of the IC domain of a CD3 chain in thymocyte development.