Whole-Brown-_thesis.pdf (5.46 MB)
Manipulating the immune response of Tasmanian Devils to target Devil Facial Tumour Disease
thesisposted on 2023-05-26, 03:02 authored by Brown, GK
The Tasmanian devil (Sarcophilus harrisii) is a marsupial carnivore confined to the island of Tasmania, where it is the top predator in the natural ecosystem. However, the Tasmanian devil is in danger of extinction due to the emergence of a contagious cancer. Devil Facial Tumour Disease (DFTD) is transmitted between devils by biting, a common behaviour which occurs during feeding and mating. The disease was first identified in 1996 and has since spread through over 60% of the natural distribution of the Tasmanian devil. Once contracted, the disease is invariably fatal, and has reduced Tasmanian devil population numbers by over 80%. Epidemiology estimates that the Tasmanian devil may become extinct in the wild within 20 years. Considering the importance of this animal in the natural ecosystem, its extinction in the wild must be prevented. One of the few options to achieve this is to develop an immunological intervention, a vaccine or immunotherapy capable of targeting this deadly tumour. This thesis aimed to characterise the anti-tumour immune response of the Tasmanian devil and to identify target pathways for immunological intervention. This thesis details the first characterisation of specific anti-tumour responses in Tasmanian devils. An analysis of DFTD infected Tasmanian devils found no evidence for a natural anti-tumour response against the tumour cells. The integrity of specific anti-tumour immunity was analysed using xenogeneic tumour cell immunisation with human K562 tumour cells to induce strong responses. Cytotoxicity responses were measured using chromium release assays and antibody production was analysed using flow cytometry. This result suggested that, with a sufficiently immunogenic preparation, specific anti-tumour responses could also be induced against DFTD. However, trials using six different killed DFTD cell preparations failed to induce consistent immune responses, with only two of fourteen devils showing evidence for a response against whole cells. During this project, investigations performed between our laboratory and collaborators verified a lack of MHC I expression on DFTD tumours. In the absence of this protein, cytotoxic T lymphocytes would not target the tumours, providing an explanation for the poor response in the vaccine trials. However, the K562 tumour cells used in the xenogeneic immunisations also lacked surface MHC I, but they were successfully recognised by the Tasmanian devil's immune system. This thesis also analysed the mechanisms of this anti-tumour response against K562 cells. Evidence was provided for the presence of functional natural killer (NK)-like cells in Tasmanian devils, which could consistently kill K562 cells by antibody dependent cell mediated cytotoxicity (ADCC). However, although Tasmanian devils can form strong ADCC responses against MHC I negative cells, antibody responses against DFTD are generally poor, and the results of initial experiments testing ADCC killing of DFTD cells were not promising. Some immunotherapy strategies used in humans can induce MHC I independent killing of tumour cells using non-specific stimulation with cytokines, activating antibodies or mitogens. This thesis reports the discovery of a technique which can consistently activate Tasmanian devil lymphocytes to target DFTD cells. Stimulation with the mitogen Con A resulted in the generation of cytotoxic cells which had the capacity to kill up to 80% of DFTD cells in vitro. Activation could also be achieved using the cytokine-rich supernatant from Con A cultures, recombinant Tasmanian devil IL-2 and the toll-like receptor agonist Poly I:C. This promising result provided a basis for immunotherapy of DFTD, and this thesis also reports the use of these mitogen-activated killer cells in the first successful treatment of DFTD in a Tasmanian devil. The work presented in this thesis provided the first evidence for functional anti-tumour responses in Tasmanian devils, and that it is possible to induce cytotoxic responses against DFTD cells in Tasmanian devils. The identification of NK-like cells and a technique to consistently activate these and other lymphocytes to kill DFTD cells was a major advance which provided a basis for an immunotherapy. The results of this preliminary trial were extremely promising and should direct the development of vaccine and immunotherapy strategies for the disease in the future.
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