Pathology - Theses
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ItemPerforin biochemistry: function and dysfunction( 2016)Natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), collectively referred to as cytotoxic lymphocytes (CLs), are responsible for clearing virus infected and cancerous cells. The predominant manner by which CLs do this is through the delivery of the pore forming protein, perforin, and pro-apoptotic granzymes that synergise to induce apoptosis in a conjugated target cell. Humans who inherit bi-allelic inactivating perforin mutations develop the immunoregulatory disease familial haemophagocytic lymphohistiocytosis (FHL) and/or haematological malignancies, demonstrating the critical importance of expressing functional perforin for the maintenance of immune homeostasis and tumour immune surveillance. While most disease associated perforin mutations are rare, 8-9% of the Caucasian population are carriers of polymorphism A91V (rs35947132, 272C>T). It has been suggested that >50% of individuals homozygous for the A91V allele develop FHL and/or cancer and that individuals heterozygous for A91V have an increased susceptibility to ALL. Despite its frequency and disease association, it remained unknown whether heterozygous inheritance of the A91V allele impairs human CL cytotoxicity and, more broadly, whether perforin is rate limiting in CL cytotoxicity. Here, it has been demonstrated that NK cells from healthy humans heterozygous for the A91V allele show an almost 50% reduction in cytotoxicity compared to individuals homozygous for WT perforin. This reduction in function was due to A91V perforin protein being misfolded within human primary NK cells. Moreover, it was also observed that heterozygous perforin knockout mouse CTLs showed an ~50% reduction in cytotoxicity. Taken together, these data demonstrate that perforin is indeed rate limiting for CLs cytotoxicity and therefore, individuals heterozygous for defective perforin alleles have impaired CL function. Although FHL predominantly presents shortly after birth, a subset of patients present at an age greater than three years, owing to the expression of misfolded perforin variants. Previous studies have shown that when transiently expressed in CTLs, perforin variants associated with late onset disease failed to traffic within CTLs and the cells remained non-functional. Therefore, it was unknown how patient CTLs expressing these variants could avoid FHL in infancy, and maintain a level of immune homeostasis for many years, or even decades. Here, it is shown that perforin variants associated with late onset disease can fold correctly and traffic within CTLs, and thus provide a significant level of cytotoxic function. However, this function was found to be lost if CTLs were cultured at an increased temperature (39 ̊C). Taken together, these data suggest that the CTLs of late onset FHL patients may have sufficient cytotoxicity to delay FHL onset in infancy. However, prolonged fever and, potentially, a more rapid exhaustion of the limited pool of correctly folded perforin mutants may result in the loss of CTL function, leading to FHL and cancer later in life. Prior to its secretion from the CL, the evolutionarily conserved C-terminal residues of perforin are proteolytically cleaved. The functional significance of C- terminal processing has remained controversial. Here it is shown that perforin enriched from human NK cell with an intact and glycosylated C-terminus was not cytotoxic. However, removal of the C-terminal glycan from the protein was found to completely restore function. As full-length deglycosylated perforin has wild type activity, these data suggest that C-terminal cleavage of perforin is permissive for cytotoxic function due to removal of an inhibitory N-linked glycan moiety at the C-terminus of the protein. These findings position the protease(s) responsible for perforin cleavage as critical to CL function. In summary, the studies described in this thesis have added to the understanding to how perforin mutations affect CL cytotoxicity and described a critical final step in perforin maturation. Together, these advancements in perforin biology may contribute to the treatment of disease arising from perforin deficiency and also define new factors critical for CL function and human health.
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ItemThe role of perforin in human health and disease( 2011)Cytotoxic lymphocytes (CL) are key effectors of the immune system responsible for the elimination of intracellular pathogen-infected and transformed cancerous cells. This occurs predominantly through the perforin-dependent granule exocytosis pathway, where perforin pores formed in the target cell membrane allow the delivery of pro-apoptotic serine proteases, granzymes, into the cytoplasm of the target cell and the initiation of apoptosis. However, despite a critical role of perforin in CL biology, the physiological role of perforin in human health and disease states is not fully understood. In humans, perforin deficiency is most commonly associated with an autosomal recessive disorder, Type 2 Familial Haemophagocytic Lymphohistiocytosis (FHL), the manifestations of which suggest a role for perforin in maintaining immune homeostasis. However, unlike studies in perforin deficient mice, no evidence has yet linked defective perforin cytotoxicity with cancer susceptibility in humans. Here, a putative role for perforin in the immune surveillance of human cancers was investigated. Although most patients with bi-allelic perforin mutations develop FHL in early infancy, some individuals have been reported to develop an atypical delayed late-onset of disease. Perforin function was examined in these individuals, using surrogate assays developed in rat basophilic leukaemia cells and mouse cytotoxic T lymphocytes (CTLs). Accordingly, it was found that perforin dysfunction was associated with an increased susceptibility to haematological malignancies, providing the most compelling evidence to date for a role for perforin and thus CLs in human cancer immune surveillance. To account for the delayed age of disease onset in these patients, it was postulated that partial CL cytotoxicity protected from overwhelming FHL in early childhood. Novel assays were developed in both mouse and human natural killer (NK) cells to compare the effects of early- and late-onset disease-associated perforin mutations on CL cytotoxicity. Whilst perforin alleles associated with early-onset FHL were completely detrimental to NK cell cytotoxicity, perforin alleles associated with atypical late-onset disease retained partial activity in NK cells. By contrast, almost all these mutations were completely detrimental to CTL cytotoxicity. Hence, it appears that residual NK cell cytotoxicity, rather than CTL cytotoxicity, is responsible for protecting against overwhelming FHL in early infancy, supporting a predominant role for the loss of NK cell cytotoxicity in the pathogenesis of FHL. Furthermore, these studies revealed that partial perforin deficiency is caused by protein misfolding, providing a rationale for the development of specific drug therapies aimed at stabilising perforin structure. Finally, the case studies of two patients with clinical suspicion of FHL were presented. Sequence analysis of the patients’ perforin genes was performed in order to confirm diagnosis, resulting in the identification of two novel perforin mutations. The molecular basis of perforin dysfunction was analysed in each case using the functional assays generated in previous chapters, in combination with structural information extrapolated from the recently resolved crystal structure of mouse perforin. These studies revealed valuable insight into the role of perforin and CLs in disease pathology, including the discovery of the gain of glycosylation as a pathogenic mechanism common to perforin mutations associated with early-onset disease. In summary, the body of work presented in this thesis highlights the absolute importance of functional perforin in maintaining human health. It has also widened the range of available diagnostic assays and provided possible novel approaches for managing diseases associated with perforin deficiency.