No description available.

No description available.

No description available.

Vav proteins regulate receptor-mediated calcium mobilisation, a process for which the N-terminal calponin homology (CH) domain appears critical, yet the mechanisms by which this operate are unknown. We therefore attempted to characterise the role of this domain <i>in vivo</i> and identify mechanisms by which the CH domain regulates calcium mobilisation by generating mice expressing Vav1 with an N74A point mutation within the CH domain. Mice carrying the germline N74A mutation were generated and the presence of the mutation within the intact locus confirmed. Analysis of the expression of the mutant protein showed little to no protein was expressed in cells from the spleen or thymi. The defective expression of the mutant protein therefore precluded study of its role in lymphocyte development and function. We also aimed to study the effect of mutations within the Vav1 CH domain by retroviral complementation analysis. We successfully set up an optimised system for stem cell transduction and lymphocyte reconstitution in alymphoid recipient mice, but found limitations with this methodology when analysing relatively subtle changes in the proportions of lymphocyte subpopulations due to variations in reconstitution efficiency between mice. Like Vav proteins, members of the P13K family are also crucial for B cell survival and recent evidence suggests that survival mediated by the cytokine BAFF operates partly via Akt-dependent mechanisms. We therefore sought to determine the extent to which loss of the P13K subunit P110δ in B cells impacted on BAFF-mediated responses. We found that P110δ-deficient Be cells were impaired in BAFF-mediated survival and cell growth <i>in vitro</i>. These cells also showed an impaired ability to compete with wildtype B cells in competitive repopulation experiments. B cells that lack Vav proteins were also impaired in their ability to respond to BAFF-mediated survival and growth signals and showed reduced levels of the BAFF-receptor, although the significance of this remains to be tested.

It has been proposed that a self-renewing, “stem-cell” like stage of CD8⁺ T cell development exists in which a potential for proliferation and clonal expansion is maintained without effector differentiation. The possibility that the transcriptional repressor BCL6b, or costimulation via a receptor of the tumour necrosis factor superfamily of proteins, was responsible for allowing the expansion of CD8⁺ T cells without the development of effector properties was assessed.  This study demonstrates that BCL6b partially blocked the IL-2 induced increase in CD8⁺ T cell numbers and upregulation of the IL-2-regulated gene CD25 <i>in vitro</i>, but was unable to show an ability of this transcription of the IL-2-regulated gene CD25 <i>in vitro, </i>but was unable to show an ability of this transcription factor to prevent IL-2-mediated effector differentiation<i>. </i> Additionally, CD27 was identified as the costimulatory receptor which, in combination with T cell receptor (TCR) signals and the pro-survival cytokine IL-7, drives the IL-2-independent clonal expansion of CD8⁺ T cells. CD27 was shown to enhance TCR induced cells cycling and to maintain the expression of the IL-7 receptor. Importantly, cells stimulated via the TCR and CD27 did not acquire effector functions and were capable of subsequently expanding and differentiating in response to a viral challenge <i>in vivo.</i> Therefore, a pathway of CD8⁺ T cell clonal expansion that maintains the IL-7 receptor, is not associated with effector differentiation and preserves the potential of the cell for further clonal expansion and the development of effector functions has been defined, lending evidence to the existence of a “stem-cell” like population of CD8⁺ T cells.

Over-expression studies in cultured B cells showed that both TIS11b and TIS11d were able to block plasma cell differentiation <i>in vitro</i>. This action was specific as class switch recombination was not affected. TIS11b conditional knockout mice showed normal lymphocyte development and maturation and a normal humoral immune response. TIS11b conditional knockout mice lacked marginal zone B cells and had reduced numbers of peritoneal B1 cells. However they mounted a normal humoral response to immunisation with NP-KLH, with normal affinity maturation and a normal memory response. Single knockouts were inter-crossed to create double knockout (dKO) mice. Unexpectedly, close to 100% of dKO mice developed T-lymphoblastic leukaemia from three months of age. Pre-leukaemic mice showed arrested B cell development at the pro-B stage and significantly perturbed thymic development. This included the aberrant passage of thymocytes through the β-selection checkpoint in the absence of expression of a functional T cell receptor β chain. A genome-wide approach to identify deregulated TIS11b/d targets in pre-leukaemic mice showed elevated expression of the oncogenic transcription factor Notch1. Bioinformatic analysis revealed potentials binding sites for TIS11b and TIS11d within a highly conserved region of the Notch1 3’UTR. The ability of TIS11b and TIS11d to interact and suppress expression of Notch1 was demonstrated by electromobility shift assay (EMSA) and luciferase reporter assays. Furthermore, development and proliferation of T-ALL was Notch1 dependent, both <i>in vivo</i> and <i>in vitro.</i>

The protein tyrosine phosphatase SHP2 regulates phosphotyrosine mediated signalling cascades by dephosphorylating specific phosphotyrosine residues on its protein substrates. It is regulated by tandem N terminal SH2 domains and tyrosine phosphorylation sites in the C terminus. Although the mechanism and targets of SHP2 have been extensively studied in the context of receptor tyrosine kinase and cytokine mediated signalling systems, its <I>in vivo</I> substrates and role in T cell antigen receptor (TCR) mediated signalling remain uncertain. The TCR complex possesses no intrinsic tyrosine kinase activity, yet its engagement by MHC-peptide complexes of stimulatory antibodies leads to an orchestrated sequence of signalling events initiated by the protein tyrosine kinases Lck and ZAP70 and leading ultimately to activation of the T cell, anergy, or death depending on the context and character of the engagement. It has previously been observed in this and other laboratories that SHP2 is recruited to the plasma membrane in association with an adaptor protein following TCR engagement, leading to the hypothesis that recruitment of SHP2 to the plasma membrane enables it to act on specific membrane associated substrates, and therefore play a role in determining the outcome of TCR engagement. In order to test this hypothesis, it was attempted to determine the identity of the adaptor protein to which SHP2 binds when recruited to the plasma membrane. A catalytically inactive (Cysteine to Serine) mutant form of SHP2 was also expressed in the Jurkat T cell line, a widely used model system for investigating TCR signalling. The effect of SHP2 on elements of the IL2 promoter, on signalling pathways distal to TCR engagement and on membrane associated phosphotyrosine containing protein complexes was investigated. Catalytically inactive SHP2 was found to inhibit the activity of the NFκB complex in Jurkat T cells, and to modulate the activity of its upstream activator, IκBα, implicating SHP2 in modulation of IL2 transcription following TCR engagement. Proteins which are targets of SHP2 regulated modulation of tyrosine phosphorylation at the plasma membrane following TCR engagement were also identified.

The adaptive immune system relies upon the presentation of antigens to T cells on major histocompatibility complex molecules (MHC) to regulate the immune response. The expression of MHC class II (MHCII) molecules is tightly controlled and generally confined to the professional antigen presenting cells and those stimulated with interferon gamma. This regulation is achieved by 3 specific transcription factors that bind to the 5’ X-box of all MHCII genes called RFX5, RFXANK and RFXAP, and a signal non DNA binding transcriptional transactivator, CIITA, which interacts with the RFX genes and the ubiquitously expressed cellular transcriptional machinery and its regulators. CIITA was analysed and shown to be unsuitable for bacterial expression. In addition computational analysis allowed the identification of two distinct domains the C-terminal containing RNI leucine rich repeats in the C-terminus of the protein. This could offer a possible mechanism for nuclear localization. RFXAP was bacterially expressed and biophysical characterization and crystal trials were undertaken. With the support of computational evidence this data supported RFXAP being an unstructured protein which only folded into an active from in the presence of is binding partners. RFXANK was bacterially expressed and analysed using biophysical techniques as well as crystallography. The computational identification of unannotated ankyrin repeats in the N-terminus of the protein has lead to the hypothesis that RFXANK is an non DNA binding adaptor vital for the assembly of the enhancesome complex. RFX5 was computationally analysed but no significant observations were made.

No description available.

No description available.