The role of perforin in human health and disease

© 2011 Dr. Jenny ChiaCytotoxic 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