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URI [dataset] University of Southampton Faculty of Environmental and Life Sciences School of Biological Sciences Investigating Cellular Origins to Identify Peptide Vaccine Targets in two Independent Transmissible Tumours Circulating in the Tasmanian Devil (Sarcophilus harrisii) DOI: by Rachel Siân Owen ORCID ID: 0000‐0001‐6441‐2213 Thesis for the degree of Doctor of Philosophy September 2019 University of Southampton Abstract Faculty of Environmental and Life Sciences School of Biological Sciences Thesis for the degree of Doctor of Philosophy Investigating Cellular Origins to Identify Peptide Vaccine Targets in two Independent Transmissible Tumours Circulating in the Tasmanian Devil (Sarcophilus harrisii) by Rachel Siân Owen The Tasmanian devil (Sarcophilus harrisii) is under threat from two independent lineages of contagious cancer; Devil Facial Tumour 1 (DFT1) and Devil Facial Tumour 2 (DFT2), which are spread as allografts by biting and cause large tumours around the oral cavity of the animals. DFT1 is a Schwann cell derived tumour first identified in 1996 which persists in most wild devil populations, carrying a near 100% mortality and causing extreme population declines. DFT2 was identified in 2014 and is confined to a peninsula in the South-east of Tasmania, where its impact on devil populations has not yet been established. Despite indistinguishable gross phenotypes, evidence indicates that the tumours emerged independently in different animals. While DFT1 affected population declines have stabilised, DFT2 may be evolving to become a more widespread tumour, and further population declines threaten the future of the species. This project is part of a larger study to identify candidate vaccine targets in DFT1 and DFT2. DFT1 evades immune detection by downregulating Major Histocompatibility Complex (MHC) class I, but expression can be restored with interferon gamma (IFNγ) and the presence of MHC class I on DFT1 cells increases their immunogenicity. In contrast, DFT2 cells maintain expression of MHC class I. We postulate that a vaccine against MHC class I restricted peptides could induce specific immune responses against both tumours. To identify potential tumour specific vaccine targets it is necessary to characterise and compare the peptides bound by MHC class I on tumour cells to those presented on healthy progenitor cells and other devil tissues. Currently, nothing is known about the binding properties of Tasmanian devil MHC class I molecules and the progenitor cells of DFT1 and DFT2 lack specific definition. In this thesis I aim to identify the cellular origins of DFT2 and define the peptide binding motif of a biologically significant MHC class I allele in order to identify specific vaccine targets. Using a proteomics approach, I have demonstrated that DFT2 expresses proteins and genes specific to myelinating glial cells, indicating a similar origin to DFT1. However, DFT2 expresses lower levels of myelin associated proteins than DFT1 and shows an enrichment for developmental glial markers. These data indicate that DFT2 has emerged from an immature myelinating glial cell, whilst DFT1 emerged from a myelinating or pro-myelinating Schwann cell. Additionally, using immunohistochemical techniques I have demonstrated that the MHC class I expression profile of Schwann cells is potentially immune evasive, making these cells prone to transmissible malignant transformation. I also show that like DFT1, DFT2 upregulates MHC class I in response to IFNγ, but demonstrates a transcriptional response that is indicative of an oligodendroglial origin, and a distinct immunosuppressive gene expression profile which is largely unaffected by IFNγ. I have generated a DFT2 cell line overexpressing a recombinant MHC class I allele which is present in the genomes of DFT1 and DFT2 and common in the wider devil population. This is, to our knowledge, the first stable transfection of DFT2 cells and these cell lines and experimental pipelines can be used for further study of the antigen presentation pathway in the Tasmanian devil and other non-model species. These data have indicated unusual properties of MHC class I peptide binding in the Tasmanian devil which were previously unknown. Perhaps most importantly, the data generated in this thesis indicates that despite their independent origins, DFT1 and DFT2 may share neoantigens, thus these tumours may respond to a single vaccination strategy, a finding with significant scientific and economic interest for the DFT vaccination programme. Table of Contents Table of Contents Table of Contents ............................................................................................................ i Table of Tables ............................................................................................................... ix Table of Figures .............................................................................................................. xi List of Accompanying Materials ..................................................................................... xv Research Thesis: Declaration of Authorship ................................................................. xvii Acknowledgements ...................................................................................................... xix Definitions and Abbreviations ...................................................................................... xxi Chapter 1 Review of the Literature .............................................................................. 1 1.1 Cancer and the immune system ................................................................................. 1 1.1.1 Contagious cancers ............................................................................................ 1 1.1.2 CD8+ T‐cell mediated tumour immunosurveillance and graft rejection ........... 4 1.2 Major Histocompatibility Complex Class I ................................................................. 8 1.2.1 Classical MHC class I ........................................................................................... 9 1.2.2 Non‐classical MHC class I ................................................................................. 10 1.2.3 The structure of MHC class I ............................................................................ 11 1.2.4 The antigen presentation pathway .................................................................. 13 1.2.5 MHC class I peptide binding ............................................................................. 15 1.2.6 MHC class I and T‐cell receptor interaction ..................................................... 17 1.2.7 Tumour associated and tumour specific antigens ........................................... 19 1.2.8 Cancer vaccines ................................................................................................ 21 1.3 Cancer progenitors ................................................................................................... 22 1.3.1 Schwann cells ................................................................................................... 23 1.3.2 Schwann cell development and differentiation ............................................... 24 1.3.3 Schwann cells in culture ................................................................................... 27 1.3.4 Oligodendrocytes ............................................................................................. 27 1.3.5 MHC expression by Schwann cells and oligodendrocytes ............................... 28 1.4 Devil facial tumour disease ...................................................................................... 29 1.4.1 The Tasmanian devil ......................................................................................... 29 1.4.2 The emergence of DFT1 ................................................................................... 30 i Table of Contents 1.4.3 The origins of DFT1 .......................................................................................... 32 1.4.4 Immune responses to DFT1 ............................................................................. 34 1.4.5 The discovery and origins of DFT2 ................................................................... 38 1.4.6 Immune responses to DFT2 ............................................................................. 40 1.4.7 Vaccination strategies for DFTs ....................................................................... 42 1.4.8 MHC class I in Tasmanian devils ...................................................................... 50 1.5 Aims