To find sequences involved in antigen/MHC recognition by the T cell receptor complex as well as in interactions within the T cell receptor complex and with accessory molecules. To study activation pathways, receptor internalization and apoptosis induced following stimulation of T cells via the T cell receptor complex and accessory molecules. To generate mutants and to express foreign genes in cultured cells as well as in transgenic mice to attain the previous objectives.
The structural determinants of antigen/MHC recognition for the TCRalpha/beta heterodimer of the T cell clone HA1.7, which is responsive to a complex of DR1 and influenza hemaglutinin peptide p14, have been characterized and a structural model for the ternary complex has been generated. A glutamic position in the complementary determining region 3 (CDR3) of TCRalpha has been shown to be determinant for the interaction with p14 through a lysine residue of the peptide.
The studies on how the TCR/CD3 complex recognizes antigen/MHC have been complemented with studies of the processes that result after TCR/CD3 stimulation: TCR/CD3 downregulation on the one hand, and the signaling mechanisms that lead to T cell activation and apoptosis on the other. Thus, T cells expressing two different TCR heterodimers have been generated. Stimulation of these cells with a superantigen specific for one of the TCRs, not only downmodulated and phosphorylated the directly stimulated TCR, but also the unstimulated one, suggesting that ligand binding to a TCR may induce its interaction with adjacent, untriggered TCRs. Experiments performed with isolated mutants and chimeras of CD3epsilon and CD3zeta suggest that these subunits could mediate internalization of complete as well as partial TCR/CD3 complexes. The internalization of CD3epsilon has been found to be mediated by the tyrosines contained in the Immunoreceptor Tyrosine-based Activation Motif (ITAM) which has been, on the other hand, structurally characterized by NMR spectroscopy. The signal for endoplasmic reticulum retention of CD3epsilon has been found to be functional and structurally related to internalization signals. In this regard, the signal that allows sorting of the internalized interleukin-2 receptor b chain from the other IL-2 receptor beta chains has also been characterized at the sequence and structural levels.
Because some tyrosine kinase inhibitors blocked superantigen-induced downregulation it is suggested that tyrosine kinases are involved in TCR downregulation, although the lack of involvement of CD45 and lck suggests that it is regulated differently to other signal transduction events.
In addition to the role of tyrosine phosphorylation in endocytosis, the role of tyrosine phosphorylation of several protein substrates in early T cell activation events has been studied. Crosslinking of the TCR/CD3 complex induces the rapid association of the protoonncogene vav and the tyrosine kinase ZAP70 to tubulin. Microtubule depolymerization inhibits the tyrosine phosphorylation of ZAP70 following TCR/CD3 stimulation, suggesting that its interaction with tubulin is functionally important, perhaps to mantain ZAP70 in close proximity to the TCR/CD3 complex. A functional analysis of the ZAP70 domains has shown that the kinase but not the SH2 domains are needed for its localization in the proximity of the plasma membrane suggesting that phosphorylation of a membrane-proximal substrate is involved in ZAP70's localization. The effects of TCR/CD3 activation on the upregulation of the TCR-beta enhancer have been studied, finding that the MAP kinase pathway components ras and raf and the tyrosine kinase src increase transcription of the TCR-beta enhancer via effects on the CBF/Ets factors. It has also been shown that Tpl-2 is a novel MAP kinase kinase kinase and that becomes tyrosine phosphorylated following TCR/CD3 activation. On the other hand, mice with null mutations in Ets-1 and CBFalphaA genes have finally been generated to study its importance for T cell development.
The signaling mechanisms leading to TCR/CD3 induced apoptosis have also been examined in Jurkat T cells by disecting the signals that result in CD95-ligand expression as well as by analyzing the signals elicited upon CD95 ligation. A point TCRbeta mutant has been generated that results in low CD3zeta association. This mutant is refractory to TCR/CD3 induced apoptosis, implying that CD3zeta is necessary for induction of CD95-ligand. On the other hand, CD95-ligand can also be induced by a PTK-independent, G-protein regulated mechanism in Jurkat cells transfected with the muscarinic receptor. CD95-ligand binds to CD95 and this transduces signals that result in apoptosis through a mechanism that has been characterized as PTK-dependent and modulated by PKC. Finally, to further study the apoptotic process, a monoclonal antibody has been developed that specifically recognizes cells in apoptosis.
MAJOR SCIENTIFIC BREAKTHROUGHS:
-Definition of TCR determinants of specificity for DR1/p14 in TCRa/b.
-NMR structure of the CD3e ITAM motif.
-Functional crosstalk between TCRs upon activation.
-Separate sorting of the subunit components of the IL-2 receptor upon endocytosis.
-ets-1 and CBFaA knockout mice.
-Role of vav as mediator of very early T cell activation events.
-Role of Tpl-2 as a novel MAP kinase kinase kinase.
-Characterization of signaling mechanisms leading to apoptosis upon TCR activation.
-Characterization of a monoclonal antibody specific for apoptotic T cells.
Funding SchemeCSC - Cost-sharing contracts
WC2A 3PX London
NW7 1AA London