Toll-like receptors in the endangered Tasmanian devil and devil facial tumour disease

Devil facial tumour disease (DFTD) describes two genetically distinct transmissible cancers threatening the survival of the world's largest carnivorous marsupial, the Tasmanian devil (Sarcophilus harrisii). Denoted DFT1 and DFT2, these aggressive cancers successfully evade immune detection to be transferred as clonal allografts via biting behaviours of the devil. A prophylactic DFTD vaccine will protect Tasmanian devils from transmission of DFT1 and DFT2. As with other vaccines, its success will depend upon the activation of effective immune responses. In human cancer studies, ligands of toll-like receptors (TLRs) are incorporated into immunotherapies and vaccines to provide potent immune stimulation. Conserved TLR genes have been identified in the genome of the Tasmanian devil, and as such TLR ligands could be effective vaccine adjuvants in DFTD. Analysis of TLR signalling in the Tasmanian devil is required to determine whether this is the case, as TLR function has not previously been investigated in any marsupial species. In this thesis, the role of TLRs in immune activation in the Tasmanian devil has been assessed using gene expression assays, in vitro mononuclear cell (MNC) stimulations and in vivo immunisation trials. Furthermore, the response of DFTD tumour cells to TLR ligands in vitro has also been investigated, as tumour cells frequently express TLRs with a range of anti- and pro-tumoural functions. These analyses revealed that Tasmanian devil MNCs express genes encoding homologues of human TLRs 2, 3, 4, 5, 6, 7, 8, 9, 10 and 13. Stimulation of these cells with ligands of TLRs increased expression of inflammatory cytokines including interleukin-1˜í¬± and interleukin-6, indicating that TLR signalling is functional in the immune system of the Tasmanian devil. In particular, a combination of the TLR ligands poly-ICLC (TLR3) and imiquimod (TLR7) significantly increased the expression of inflammatory cytokines and production of interferon-˜í‚â• from devil MNCs, suggesting that these ligands may be effective adjuvants in a DFTD vaccine. To confirm this finding, immunisation trials were performed in Tasmanian devils using both model and DFTD antigens. These immunisations revealed that the combination of poly-ICLC and imiquimod activates potent antigen-specific immune responses in Tasmanian devils that are both more rapid and long-lived than responses activated by previously used DFTD vaccine adjuvants. Importantly, this finding reduces the current DFTD immunisation protocol in Tasmanian devils from four to two immunisations. This shorter immunisation protocol will allow for more feasible delivery of DFTD vaccines to affected wild Tasmanian devil populations. Analysis of the response of DFTD cells to in vitro stimulation with TLR ligands revealed a variety of anti- and pro-tumoural functions. In particular, stimulation of the gram-positive bacterial sensor TLR2/6 increased the expression of both angiogenic and immunosuppressive cytokines from DFT1 cells in culture, highlighting a novel mechanism of immune evasion in DFTD. In comparison, other TLR ligands including poly-IC (TLR3) suppressed DFTD cell proliferation, while imiquimod (TLR7) was demonstrated to be a potent inducer of apoptosis in DFTD cell lines. RNA sequencing and proteomic mass spectrometry analysis of the mechanisms by which imiquimod deregulated DFTD survival demonstrated activation and subsequent overload of oxidative and ER stress responses via TLR7-independent pathways. These findings highlight the potential of stress responses as therapeutic targets in DFTD, and provide the first whole transcriptome and proteome analysis of imiquimod action in any mammalian tumour cell line. This thesis has expanded the current knowledge of the Tasmanian devil's immune system by verifying that devils have functional TLRs that respond to a range of prototypic TLR ligands. This is the first description of functional TLRs in any marsupial species, contributing to our understanding of marsupial immunology and the evolution of TLR signalling. This thesis has also identified the TLR ligands poly-ICLC and imiquimod as potent immunostimulatory agents in the Tasmanian devil, and demonstrated that in combination these ligands induce rapid and long-lived antigen-specific responses to DFTD immunisation. These ligands also exhibit antitumour effects in DFTD, suggesting that they may have additional uses as immunomodulatory agents for therapeutic application. This study provides the first analysis of candidate DFTD vaccine adjuvants in Tasmanian devils. Future DFTD vaccines will be formulated with poly-ICLC and imiquimod as adjuvants for improved vaccine efficacy and DFTD protection.