The aim of the project was to investigate the role of LCPTP (Leucocyte phosphotyrosine phosphatase) in T cell signalling and activation. The investigation was divided into three parts. In the first part, biochemical studies were used to identify possible LCPTP substrates/interacting proteins. Antibodies specific for LCPTP were used to characterise potential interactions of endogenous LCPTP protein in cells. Recombinant LCPTP proteins, including wildtype and a substrate-trap protein were also generated. These were used to show that LCPTP interacts with members of the MAP kinase family, specifically ERK1 and ERK2, suggesting that these proteins are major LCPTP substrates. In the second part, the effect of overexpressing LCPTP on T cell function was investigated. The Jurkat T cell line was transfected to produce stable cell lines expressing wildtype and mutant forms of LCPTP. LCPTP transfected cells were stimulated via the TCR and CD28 and monitored for cytokine production, surface marker expression and cell proliferation. It was shown that LCPTP inhibited IL-2 production and expression of the surface IL-2 receptor, CD25. LCPTP also protected cells from TCR induced cell death and promoted homotypic aggregation. Following on from the observations made in the Jurkat T cell line, the final part of the project was aimed at looking at the effects of LCPTP in primary T cells. LCPTP adenovirus constructs were made and used to infect both na?ve T cells as well as antigen differentiated Th1 and Th2 populations. Overexpressed LCPTP substrate-trap protein lead to increased levels of cellular phosphoERK. Increased levels of LCPTP resulted in changes in cytokine production in TCR stimulated cells relative to control cells. Cells overexpressing LCPTP had a higher basal level of proliferation. The results obtained support a role for LCPTP in regulating the early stages of T cell activation by changing MAP kinase activity.

The processing of antigens for presentation to CD8+ T-cells in association with MHC class I molecules involves many chaperones and accessory proteins in the ER. Once processed into a trimeric complex of beta-2-microglobulin (p2m), heavy chain and antigenic peptide the MHC class I molecule is complete and can traffic to the cell surface where it interacts with the T cell receptor of CD8+ T-cells. The main focus of this work is the use of MHC class I fusion proteins, which by means of a covalent linker form partially assembled class I molecules. Using a fusion protein in which an antigenic HLA-A2 binding peptide is linked to p2m (PB), it has been possible to show by immunoprecipitation that class I molecules can continue to interact with the peptide-loading complex after high affinity peptide has bound. This data is further supported by peptide release assays in which class I molecules are not released from the peptide-loading complex upon provision of high affinity peptide. The expression of fusion proteins in which p2in is linked to the heavy chain alleles HLA-B44 (p2m-B44) or HLA-A2 (p2m-A2), has highlighted an allelic difference in the requirement of MHC class I molecules for chaperones in the antigen processing pathway. Specifically it has been possible to show that while P2m-A2 is able to express in murine fibroblast K41 cells, p2m-B44 is not. This inability to express is not as a result of the absence of human tapasin, as previously reported, but because of some other unknown factor that is present in human cells but not in murine K41 cells. Interestingly the expression of p2m-B44 in K41 cells also disrupts native MHC class I expression implying that the construct may be occupying chaperone molecules in the ER and delaying processing of mouse class I molecules. Furthermore, in the same system it has been possible to show that the major function of calreticulin is not the recruitment of P2m to heavy chain, since the p2m-A2 fusion protein, like normal class I expressed 8-10 fold less well in calreticulin deficient cells than in 'wild type' cells.

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Through their functional diversification, T cells not only provide protection from a multitude of infectious agents but can act to both drive and constrain immune-mediated pathology. A locus encoding the transcription factor BACH2 is associated with diverse immune-mediated diseases including asthma, celiac disease, Crohn's disease, mUltiple sclerosis, generalized vitiligo and type 1 diabetes in humans. Despite this, a function for Bach2 in the maintenance of immune homeostasis had not been defined. We found that genetic ablation of Bach2 in mice resulted in the development of spontaneous lethal inflammation predominantly affecting the lungs and gut. Analysis of both thymic and induced Treg cell development revealed a cell intrinsic requirement for Bach2 in the formation of Treg cells. As a result, using bone marrow reconstitution experiments, we found that Bach2 was required for prevention of lethal inflammation in a manner that was FoxP3 and Treg cell dependent. In addition to CD4+ Treg cells, however, Bach2 was highly expressed in naive cells and lost upon effector differentiation. Strikingly, overexpression of Bach2 prevented differentiation into effector cells, suppressed cytokine production and resulted acquisition of memory-cell characteristics. Conversely, while naive Bach2- deficient CD8+ T cells formed effector responses following viral infection in vivo, there was a near-complete cell-autonomous defect in their ability to generate long-lived memory cells. Accordingly, deficiency of Bach2 resulted in impaired protective immunity following primary responses and Bach2-deficient cells exhibited impaired long-term anti-tumor functionality.

Non-selective T cell depletion reduces the incidence of severe graft-versus-host disease after allogeneic haematopoietic stem cell transplantation but the cost is delayed and disordered antigen-specific immune reconstitution and increased infection. A method of selective depletion of alloreactive donor T cells expressing the activation marker CD69 after co-culture with recipient stimulator cells in a mixed lymphocyte reaction has previously been shown to reduce alloreactivity, whilst retaining third party responses in vitro and in a mismatched murine model led to donor T cell engraftment with a virtual absence of GvHD and significantly increased survival. We have further developed this technique by comparing two methods of potentiating allostimulation in the HLA-matched setting. Cytokine pre-treated recipient stimulator cells led to successful allostimulation of a minority of HLA-matched responder cells whereas the strategy of pre-treatment of recipient stimulator cells with OKT3 led to effective allostimulation in all pairs tested and led to more efficient selective abrogation of alloresponses after depletion of responder alloreactive cells. The retention of donor antiviral T cell frequencies were compared after selective HLA matched allodepletion using both techniques of potentiating allostimulation. Using both techniques the majority of CMV-specific T cells (quantified by HLA Class I tetramer assay and IFN-y ELISpot) and EBV-specific T cells (quantified by IFN-gamma ELISpot) were retained in the selectively allodepleted T cell pool. Preservation of antiviral CTLs in selectively albdepleted stem cell grafts would lead to improved antiviral immunity post transplant. The phenotypic characteristics of the alloreactive and non-alloreactive T cells within the donor pool were examined. 0069"* alloreactive T cells were found to consist of both naive and memory T cells and to exhibit significant skewing of TCR Vp sub-family distribution in both the HLA-mismatched and HLA-matched setting. The technique of selective allodepletion based on CD69 expression was found to retain functional CD4+CD25+ T regulatory cells The retention of immunosuppressive CD4+CD25+ T regulatory cells could lead to more ordered immune reconstitution and further suppress alloreactive responses post transplant. Direct stimulation of donor T cells with CMV peptide led to up regulation of CD69 on CMV-specific T cells and these cells exhibited TCR Vp sub-family overuse consistent with previously published data. Homology of TCR Vp sub-family overuse in HLA A*0201+ donor T cells following CMV peptide stimulation and HLA-matched allostimulation was demonstrated in some individuals suggesting supporting the existence of donor T cells possessing TCRs with affinity for CMV and minor histocompatability antigens. Sequential selective allodepletion and CMV antigen stimulation of donor T cells might lead to production of CMV-specific non-alloreactive donor T cell pools suitable for use as adoptive immunotherapy post-allogeneic haematopoietic stem cell transplantation. The techniques for allostimulation and selective allodepletion at a clinical scale and under sterile conditions have been developed in order to test the safety and the efficacy of this technique in a clinical pilot study of HLA-matched sibling donor allogeneic haematopoietic stem cell transplantation in adults with acute myeloid leukaemia.

This PhD thesis aims to examine the growth of T cells in response to cytokines of the common gamma chain (yc) family, in particular interleukin (IL)-2 and IL- 15. Cell growth is commonly used to describe cells in exponential division however the growth of a cell is defined by its size and volume, which is directly related to the rate of cell metabolism and protein synthesis. Naive T cells are small and circulate around the body maintaining a minimal rate of metabolic activity and protein synthesis. During an immune response naive T cells undergo rapid proliferation and differentiate into effector cells. These cells produce and secrete large amounts of cytokines and effector molecules allowing them to mediate their immune function. The differentiation of antigen activated T cells to mature effectors takes several days and is regulated by cytokines. These cytokines need to maintain high rates of cell metabolism for a prolonged period. Thus the cytokines that regulate proliferation and differentiation of antigen activated T cells need to sustain cell growth. Data presented in this thesis shows differential roles for gamma chain cytokines, specifically IL-2 and IL-15 in the regulation of protein synthesis and uptake of amino acids, whilst maintaining equal mitogenic capacity. This thesis highlights the possible uncoupling of the rate of cellular division and protein synthesis induced by cytokines and defines a unique role for common gamma chain cytokines in regulation of protein synthesis and ultimately the regulation of cellular function.

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