title: Manipulation of devil facial tumour cells to enhance immunogenicity
Creators: name: Ong, CEB
Publisher: name: University of Tasmania
Year: 2022

description: Tasmanian devils (Sarcophilus harrisii), an icon of Tasmania, have been facing an ongoing threat for the past 25 years from devil facial tumour (DFT) disease caused by two independent transmissible cancers known as DFT1 and DFT2. Both tumour types are clonal cell lines, genetically distinct from host devils, and are transmitted as allografts through biting. Despite a functionally competent immune system that can mount cytotoxic and humoral responses against skin allografts, evidence of protective natural anti-DFT immunity in wild devils is rare, suggesting tumour circumvention of the immunological barriers to both cancer and allograft. The major histocompatibility complex class I (MHC-I) antigen presentation pathway plays a key role in adaptive immunity and is exploited in DFT cells to reduce tumour immunogenicity. DFT1 cells do not express MHC-I molecules on the cell surface. In DFT2, MHC-I molecules are expressed, but the most highly expressed allele commonly matches alleles in host devils, reducing the differences between host and tumour MHC for strong allogeneic responses. Monomorphic non-classical MHC-I molecules are also upregulated in DFT2 cells, likely inhibiting T cell and natural killer (NK) cell responses. In this thesis, the role of MHC expression on DFT cells in anti-DFT immune responses was investigated through in vitro assays using tissue samples from wild and captive devils. Stable expression of MHC-I and MHC-II molecules was induced in DFT cells through genetic modification of a variety of MHC regulators. Using RNA and protein expression studies such as RNA-sequencing, RT-PCR, flow cytometry, and western blot, the function and activities of selected genes in DFT cells were elucidated. The inflammatory cytokine interferon-gamma (IFNG) is a potent immune-modulatory molecule that can stimulate MHC-I expression on multiple cell types including DFT1 cells. However, IFNG is a pleiotropic cytokine with diverse downstream activities that encompass both anti-tumour and pro-tumour effects. Chapter 3 demonstrates the use of a conditional gene expression system known as the tetracycline (Tet)-Off system to modulate the expression of IFNG in transfected DFT1 cell lines. Using this system, the addition and removal of doxycycline, a tetracycline derivative, effectively controlled the expression of IFNG at the transcriptional level, permitting on demand upregulation of MHC-I on DFT1 cells. This allowed DFT1 cells to proliferate for research and vaccine purposes prior to inducing IFNG expression and the associated anti-proliferative, cytotoxic, and immune-inhibitory effects of IFNG. The adverse side effects of IFNG introduces a barrier for prolonged stimulation of DFT cells for constitutive MHC-I expression. NLRC5 is an MHC-I transcriptional co-activator involved in constitutive and IFNG-mediated MHC-I expression in humans and mice. The role of NLRC5 as an MHC regulator in devils was investigated in Chapter 4 through gene overexpression in DFT1 and DFT2 cells. Transcriptomic results suggested that NLRC5 plays a significant role in MHC-I regulation in devils, demonstrating the capacity to drive the expression of several components of the MHC-I machinery. Interestingly, NLRC5 did not upregulate the immune-inhibitory molecule PDL1 that was induced by IFNG. The role of MHC-I in anti-DFT immune response was explored using serum samples from devils with immunotherapy-induced or natural tumour regressions. The B2Mgene was knocked out from DFT1 cells using CRISPR technology to allow comparative analyses between antibody responses in the presence and absence of MHC-I on the cell surface of DFT1 cells. The diminished binding to B2M knockout compared to IFNG-treated and NLRC5 cell lines suggest that MHC-I is an important target in antibody responses to DFT1 cells. MHC-II expression is crucial for CD4\(^+\) T cell activation and is primarily confined to haematopoietic antigen-presenting cells. DFT1 and DFT2 cells do not typically express MHC-II, but the overexpression of Class II transactivator (CIITA) in both cell types revealed CIITA regulation of MHC-I and MHC-II pathways. Transcriptomic analysis in Chapter 5 illustrated the activity of CIITA in detail and the ability to induce MHC-I and MHC-II expression in DFT1 cells. In Chapter 6, the role of NLRC5- or CIITA-driven MHC expression in cell-mediated immunity was investigated by in vitro co-culture of devil PBMCs and DFT1 cell lines. Additionally, NLRC5- and CIITA-expressing DFT cell lines were modified to present co-stimulatory molecules CD80, CD86 or 41BBL to potentiate the immune response. The interaction between the tumour cell lines and PBMCs was analysed by flow cytometric-based assays that explored changes in PBMC and DFT1 cell viabilities, and upregulation of activation markers PD1 and IFNG on CD4\(^+\) and CD4\(^-\) cells following MHC and co-stimulatory molecule expressions on DFT1 cells. In Chapter 7, the Tet-Off regulatory system is revisited to develop an inducible suicide gene system for attenuation of DFT cells in a live tumour cell vaccine. Pro-apoptotic molecules BAX, BAK1,or BOK were conditionally overexpressed in transfected DFT1 cells, using doxycycline to regulate their expression. The efficacy of each suicide gene system was analysed by flow cytometry and luciferase assay. Overall, this thesis provided molecular insights into the regulation of MHC-I and MHC-II pathways in DFT cells and devils. Additionally, MHC molecules were shown to be important targets for anti-DFT immunity. The knowledge about MHC regulation, and the tools developed in this thesis, can help advance the development of immunogenic targets for immunisation against DFTD. Moreover, the use of gene expression systems in DFT cells provides an avenue of research for understanding gene function and aid in the development of vaccine and immunotherapeutic strategies for managing DFTD.