The role of serpins and granzymes in cancer and immunity

Abstract

This thesis describes the results of our research regarding the expression and function of granzymes and serpins in immune cells and in tumours. Granzymes are cytotoxic proteases that are expressed by cytotoxic lymphocytes. The family of human granzymes consists of five members: GrA, GrB, GrH, GrK, and GrM. Upon perforin-dependent entry into a target cell they can all induce cell death by cleaving specific intracellular substrates. Serpins, on the other hand, can inhibit the proteolytic activity of cognate proteases and thereby fulfil a regulatory function in many processes. The novel findings regarding granzymes and serpins can be divided into three themes. First, the expression and regulation of two granzymes by specific immune cells has been investigated. This is of importance to determine which cell populations can initiate cell death by one particular granzyme. We showed that human mast cells produce and release GrB upon activation. These results propose an additional function for mast cells, whereas they preclude the use of GrB as an exclusive marker for cytotoxic lymphocytes. Using a novel specific antibody, we determined that NK cells, NKT cells, gamma-delta T cells, and CD8+ T cells express GrM. This suggests that GrM plays a role in both the innate and the adaptive immune response. Second, the mechanism underlying GrM-induced cell death has been investigated as well as a possibility for tumour cells to evade GrM-induced cell death. Novel substrates of GrM have been identified using a proteomics approach, including the cytoskeleton proteins alpha-tubulin and ezrin. Cleavage of these proteins disturbs their function and likely contributes to GrM-induced cell death. Biochemical analysis revealed that SERPINB4 inhibits the proteolytic activity of GrM. SERPINB4 traps GrM into a typical serpin-target protease SDS-stable complex and impairs hydrolysis of GrM-substrates. Since SERPINB4 is highly expressed by certain tumours, these results suggest that SERPINB4 expression constitutes a novel mechanism by which tumour cells evade GrM-induced cell death. Third, the expression of two serpins by specific cell types and corresponding tumours has been studied. In addition, the use of these serpins as tumour marker has been analysed. Normal squamous epithelium highly expresses SERPINB13, whereas this is strongly down-regulated in head and neck squamous cell carcinomas (HNSCC). Moreover, down-regulation of SERPINB13 protein expression by HNSCC associates with a poor clinical outcome of the patients. SERPINB8 is expressed by normal neuroendocrine cells as well as neuroendocrine pancreas tumours. Since expression by the latter one is highly sensitive and specific, SERPINB8 can be used as an additional diagnostic marker for neuroendocrine tumours of the pancreas. These novel insights may lead to improvement in the diagnosis and treatment of patients with immunological disorders, viral infections, and/or cancer.