Sir Peter MacCallum Department of Oncology - Theses
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ItemA cell-based functional assay that accurately links genotype to phenotype in Familial HLH( 2023-04)Cytotoxic lymphocytes protect humans against viral pathogens and cancer by killing infected and transformed cells, through perforin-mediated mechanism. Mutations in perforin (PRF1) itself or in the secretory machinery responsible for its release (UNC13D, STX11, and STXBP2) are catastrophic, and lead to fatal immune dysregulation, an autosomal recessive disease called familial haemophagocytic lymphohistiocytosis (FHL). Traditionally, FHL has been associated with infant patients. However, it is now apparent that many patients remain disease-free for years, and then present with highly variable and often unexpected symptoms. They remain undiagnosed for a long time and, instead of receiving curative stem cell transplantation, they are treated symptomatically leading to high risk of severe neurological impairment, organ failure and/or death. While the pathogenicity of frame-shift/nonsense mutations is rarely in doubt, the effect of missense mutations on protein function can vary enormously. Yet, over the last two decades, the pathogenicity of missense mutations was almost invariably assumed, and invasive stem cell transplantation was considered without confirmed pathogenicity of mutations. Sadly, transplantation without genetically proven FHL results in a 20% increased mortality compared to patients with proven FHL. Therefore, early and accurate diagnosis of the disease is essential to determine the most appropriate treatment option. Due to the diversity of genetic causes of FHL, there was no test available to directly assess the effect of mutations on cytotoxic lymphocyte function, leading to delayed/erroneous diagnoses. To overcome this diagnostic problem, we have developed a simple, rapid, and robust method that takes advantage of the functional equivalence of the human and mouse orthologues of PRF1, UNC13D, STX11 and STXBP2 proteins. By knocking out endogenous mouse genes in CD8+ T cells and simultaneously expressing their mutated human orthologues, we can accurately assess the effect of mutations on cell function. The wide dynamic range of this novel system allowed us to understand the basis of otherwise cryptic cases of FHL/HLH and, in some instances, to demonstrate that previously reported mutations are unlikely to cause FHL. In addition to diagnosing patients, this unique approach will be paramount for assessing the prognosis of asymptomatic siblings and to guide genetic counselling advice for prospective parents.
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ItemInvestigating the mechanism of cytotoxic lymphocyte resistance to perforin( 2020)Cytotoxic lymphocytes are highly efficient killer cells of the immune system. They destroy cognate target cells by secreting highly toxic effector molecules, the pore-forming protein perforin and pro-apoptotic serine proteases granzymes, into the confines of the immune synapse. Despite both the lymphocyte and target cell plasma membrane being equally exposed to the perforin and granzymes, the lymphocytes invariably survive that encounter as they remain resistant to perforin pores. This project investigates the mechanisms behind this unique phenomenon.