MHC gene copy number variation in Tasmanian devils: implications for the spread of a contagious cancer

Tasmanian devils face extinction owing to the emergence of a contagious cancer. Devil facial tumour disease (DFTD) is a clonal cancer spread owing to a lack of major histocompatibility complex (MHC) barriers in Tasmanian devil populations. We present a comprehensive screen of MHC diversity in devils and identify 25 MHC types and 53 novel sequences, but conclude that overall levels of MHC diversity at the sequence level are low. The majority of MHC Class I variation can be explained by allelic copy number variation with two to seven sequence variants identified per individual. MHC sequences are divided into two distinct groups based on sequence similarity. DFTD cells and most devils have sequences from both groups. Twenty per cent of individuals have a restricted MHC repertoire and contain only group I or only group II sequences. Counterintuitively, we postulate that the immune system of individuals with a restricted MHC repertoire may recognize foreign MHC antigens on the surface of the DFTD cell. The implication of these results for management of DFTD and this endangered species are discussed.     

Transmissible cancer in Tasmanian devils: localized lineage replacement and host population response

Tasmanian devil facial tumour disease (DFTD) is a clonally transmissible cancer threatening the Tasmanian devil (Sarcophilus harrisii) with extinction. Live cancer cells are the infectious agent, transmitted to new hosts when individuals bite each other. Over the 18 years since DFTD was first observed, distinct genetic and karyotypic sublineages have evolved. In this longitudinal study, we investigate the associations between tumour karyotype, epidemic patterns and host demographic response to the disease. Reduced host population effects and low DFTD infection rates were associated with high prevalence of tetraploid tumours. Subsequent replacement by a diploid variant of DFTD coincided with a rapid increase in disease prevalence, population decline and reduced mean age of the population. Our results suggest a role for tumour genetics in DFTD transmission dynamics and epidemic outcome. Future research, for this and other highly pathogenic emerging infectious diseases, should focus on understanding the evolution of host and pathogen genotypes, their effects on susceptibility and tolerance to infection, and their implications for designing novel genetic management strategies. This study provides evidence for a rapid localized lineage replacement occurring within a transmissible cancer epidemic and highlights the possibility that distinct DFTD genetic lineages may harbour traits that influence pathogen fitness.     

Allorecognition in the Tasmanian devil (Sarcophilus harrisii), an endangered marsupial species with limited genetic diversity

Tasmanian devils (Sarcophilus harrisii) are on the verge of extinction due to a transmissible cancer, devil facial tumour disease (DFTD). This tumour is an allograft that is transmitted between individuals without immune recognition of the tumour cells. The mechanism to explain this lack of immune recognition and acceptance is not well understood. It has been hypothesized that lack of genetic diversity at the Major Histocompatibility Complex (MHC) allowed the tumour cells to grow in genetically similar hosts without evoking an immune response to alloantigens. We conducted mixed lymphocyte reactions and skin grafts to measure functional MHC diversity in the Tasmanian devil population. The limited MHC diversity was sufficient to produce measurable mixed lymphocyte reactions. There was a wide range of responses, from low or no reaction to relatively strong responses. The highest responses occurred when lymphocytes from devils from the east of Tasmania were mixed with lymphocytes from devils from the west of Tasmania. All of the five successful skin allografts were rejected within 14 days after surgery, even though little or no MHC I and II mismatches were found. Extensive T-cell infiltration characterised the immune rejection. We conclude that Tasmanian devils are capable of allogeneic rejection. Consequently, a lack of functional allorecognition mechanisms in the devil population does not explain the transmission of a contagious cancer.     

Infectious disease and sickness behaviour: tumour progression affects interaction patterns and social network structure in wild Tasmanian devils

Infectious diseases, including transmissible cancers, can have a broad range of impacts on host behaviour, particularly in the latter stages of disease progression. However, the difficulty of early diagnoses makes the study of behavioural influences of disease in wild animals a challenging task. Tasmanian devils (Sarcophilus harrisii) are affected by a transmissible cancer, devil facial tumour disease (DFTD), in which tumours are externally visible as they progress. Using telemetry and mark–recapture datasets, we quantify the impacts of cancer progression on the behaviour of wild devils by assessing how interaction patterns within the social network of a population change with increasing tumour load. The progression of DFTD negatively influences devils'' likelihood of interaction within their network. Infected devils were more active within their network late in the mating season, a pattern with repercussions for DFTD transmission. Our study provides a rare opportunity to quantify and understand the behavioural feedbacks of disease in wildlife and how they may affect transmission and population dynamics in general.     

The role of the Major Histocompatibility Complex in the spread of contagious cancers

Major Histocompatibility Complex (MHC) genes play a key role in immune response to infectious diseases, immunosurveillance, and self/nonself recognition. Matching MHC alleles is critical for organ transplantation, while changes in the MHC profile of tumour cells allow effective evasion of the immune response. Two unique cancers have exploited these features to become transmissible. In this review I discuss the functional role of MHC molecules in the emergence and evolution of Devil Facial Tumour Disease (DFTD) and Canine Transmissible Venereal Tumour (CTVT). High levels of genetic diversity at MHC genes play a critical role in protecting populations of vertebrate species from contagious cancer. However, species that have undergone genetic bottlenecks and have lost diversity at MHC genes are at risk of transmissible tumours. Moreover, evolution and selection for tumour variants capable of evading the immune response allow contagious cancers to cross MHC barriers. Transmissible cancers are rare but they can provide unique insights into the genetics and immunology of tumours and organ transplants.     

The differentiation state of the Schwann cell progenitor drives phenotypic variation between two contagious cancers

Contagious cancers are a rare pathogenic phenomenon in which cancer cells gain the ability to spread between genetically distinct hosts. Nine examples have been identified across marine bivalves, dogs and Tasmanian devils, but the Tasmanian devil is the only mammalian species known to have given rise to two distinct lineages of contagious cancer, termed Devil Facial Tumour 1 (DFT1) and 2 (DFT2). Remarkably, DFT1 and DFT2 arose independently from the same cell type, a Schwann cell, and while their ultra-structural features are highly similar they exhibit variation in their mutational signatures and infection dynamics. As such, DFT1 and DFT2 provide a unique framework for investigating how a common progenitor cell can give rise to distinct contagious cancers. Using a proteomics approach, we show that DFT1 and DFT2 are derived from Schwann cells in different differentiation states, with DFT2 carrying a molecular signature of a less well differentiated Schwann cell. Under inflammatory signals DFT1 and DFT2 have different gene expression profiles, most notably involving Schwann cell markers of differentiation, reflecting the influence of their distinct origins. Further, DFT2 cells express immune cell markers typically expressed during nerve repair, consistent with an ability to manipulate their extracellular environment, facilitating the cell’s ability to transmit between individuals. The emergence of two contagious cancers in the Tasmanian devil suggests that the inherent plasticity of Schwann cells confers a vulnerability to the formation of contagious cancers.     

Genomic restructuring in the Tasmanian devil facial tumour: chromosome painting and gene mapping provide clues to evolution of a transmissible tumour

Devil facial tumour disease (DFTD) is a fatal, transmissible malignancy that threatens the world's largest marsupial carnivore, the Tasmanian devil, with extinction. First recognised in 1996, DFTD has had a catastrophic effect on wild devil numbers, and intense research efforts to understand and contain the disease have since demonstrated that the tumour is a clonal cell line transmitted by allograft. We used chromosome painting and gene mapping to deconstruct the DFTD karyotype and determine the chromosome and gene rearrangements involved in carcinogenesis. Chromosome painting on three different DFTD tumour strains determined the origins of marker chromosomes and provided a general overview of the rearrangement in DFTD karyotypes. Mapping of 105 BAC clones by fluorescence in situ hybridisation provided a finer level of resolution of genome rearrangements in DFTD strains. Our findings demonstrate that only limited regions of the genome, mainly chromosomes 1 and X, are rearranged in DFTD. Regions rearranged in DFTD are also highly rearranged between different marsupials. Differences between strains are limited, reflecting the unusually stable nature of DFTD. Finally, our detailed maps of both the devil and tumour karyotypes provide a physical framework for future genomic investigations into DFTD.     

Infection of the fittest: devil facial tumour disease has greatest effect on individuals with highest reproductive output

Emerging infectious diseases rarely affect all members of a population equally and determining how individuals’ susceptibility to infection is related to other components of their fitness is critical to understanding disease impacts at a population level and for predicting evolutionary trajectories. We introduce a novel state‐space model framework to investigate survival and fecundity of Tasmanian devils (Sarcophilus harrisii) affected by a transmissible cancer, devil facial tumour disease. We show that those devils that become host to tumours have otherwise greater fitness, with higher survival and fecundity rates prior to disease‐induced death than non‐host individuals that do not become infected, although high tumour loads lead to high mortality. Our finding that individuals with the greatest reproductive value are those most affected by the cancer demonstrates the need to quantify both survival and fecundity in context of disease progression for understanding the impact of disease on wildlife populations.     

Regulation of the Hippo-YAP Pathway by G-Protein-Coupled Receptor Signaling

The Tasmanian devil (Sarcophilus harrisii), the largest marsupial carnivore, is endangered due to a transmissible facial cancer spread by direct transfer of living cancer cells through biting. Here we describe the sequencing, assembly, and annotation of the Tasmanian devil genome and whole-genome sequences for two geographically distant subclones of the cancer. Genomic analysis suggests that the cancer first arose from a female Tasmanian devil and that the clone has subsequently genetically diverged during its spread across Tasmania. The devil cancer genome contains more than 17,000 somatic base substitution mutations and bears the imprint of a distinct mutational process. Genotyping of somatic mutations in 104 geographically and temporally distributed Tasmanian devil tumors reveals the pattern of evolution and spread of this parasitic clonal lineage, with evidence of a selective sweep in one geographical area and persistence of parallel lineages in other populations.     

Low major histocompatibility complex diversity in the Tasmanian devil predates European settlement and may explain susceptibility to disease epidemics

The Tasmanian devil (Sarcophilus harrisii) is at risk of extinction owing to the emergence of a contagious cancer known as devil facial tumour disease (DFTD). The emergence and spread of DFTD has been linked to low genetic diversity in the major histocompatibility complex (MHC). We examined MHC diversity in historical and ancient devils to determine whether loss of diversity is recent or predates European settlement in Australia. Our results reveal no additional diversity in historical Tasmanian samples. Mainland devils had common modern variants plus six new variants that are highly similar to existing alleles. We conclude that low MHC diversity has been a feature of devil populations since at least the Mid-Holocene and could explain their tumultuous history of population crashes.     

Extreme Telomere Length Dimorphism in the Tasmanian Devil and Related Marsupials Suggests Parental Control of Telomere Length

Telomeres, specialised structures that protect chromosome ends, play a critical role in preserving chromosome integrity. Telomere dynamics in the Tasmanian devil (Sarcophilus harrisii) are of particular interest in light of the emergence of devil facial tumour disease (DFTD), a transmissible malignancy that causes rapid mortality and threatens the species with extinction. We used fluorescent in situ hybridisation to investigate telomere length in DFTD cells, in healthy Tasmanian devils and in four closely related marsupial species. Here we report that animals in the Order Dasyuromorphia have chromosomes characterised by striking telomere length dimorphism between homologues. Findings in sex chromosomes suggest that telomere length dimorphism may be regulated by events in the parental germlines. Long telomeres on the Y chromosome imply that telomere lengthening occurs during spermatogenesis, whereas telomere diminution occurs during oogenesis. Although found in several somatic cell tissue types, telomere length dimorphism was not found in DFTD cancer cells, which are characterised by uniformly short telomeres. This is, to our knowledge, the first report of naturally occurring telomere length dimorphism in any species and suggests a novel strategy of telomere length control. Comparative studies in five distantly related marsupials and a monotreme indicate that telomere dimorphism evolved at least 50 million years ago.     

Natural killer cell mediated cytotoxic responses in the Tasmanian devil

The Tasmanian devil (Sarcophilus harrisii), the world's largest marsupial carnivore, is under threat of extinction following the emergence of an infectious cancer. Devil facial tumour disease (DFTD) is spread between Tasmanian devils during biting. The disease is consistently fatal and devils succumb without developing a protective immune response. The aim of this study was to determine if Tasmanian devils were capable of forming cytotoxic antitumour responses and develop antibodies against DFTD cells and foreign tumour cells. The two Tasmanian devils immunised with irradiated DFTD cells did not form cytotoxic or humoral responses against DFTD cells, even after multiple immunisations. However, following immunisation with xenogenic K562 cells, devils did produce cytotoxic responses and antibodies against this foreign tumour cell line. The cytotoxicity appeared to occur through the activity of natural killer (NK) cells in an antibody dependent manner. Classical NK cell responses, such as innate killing of DFTD and foreign cancer cells, were not observed. Cells with an NK-like phenotype comprised approximately 4 percent of peripheral blood mononuclear cells. The results of this study suggest that Tasmanian devils have NK cells with functional cytotoxic pathways. Although devil NK cells do not directly recognise DFTD cancer cells, the development of antibody dependent cell-mediated cytotoxicity presents a potential pathway to induce cytotoxic responses against the disease. These findings have positive implications for future DFTD vaccine research.     

Telomere dynamics and homeostasis in a transmissible cancer

Background

Devil Facial Tumour Disease (DFTD) is a unique clonal cancer that threatens the world''s largest carnivorous marsupial, the Tasmanian devil (Sarcophilus harrisii) with extinction. This transmissible cancer is passed between individual devils by cell implantation during social interactions. The tumour arose in a Schwann cell of a single devil over 15 years ago and since then has expanded clonally, without showing signs of replicative senescence; in stark contrast to a somatic cell that displays a finite capacity for replication, known as the “Hayflick limit”.

Methodology/Principal Findings

In the present study we investigate the role of telomere length, measured as Telomere Copy Number (TCN), and telomerase and shelterin gene expression, as well as telomerase activity in maintaining hyperproliferation of Devil Facial Tumour (DFT) cells. Our results show that DFT cells have short telomeres. DFTD TCN does not differ between geographic regions or between strains. However, TCN has increased over time. Unlimited cell proliferation is likely to have been achieved through the observed up-regulation of the catalytic subunit of telomerase (TERT) and concomitant activation of telomerase. Up-regulation of the central component of shelterin, the TRF1-intercating nuclear factor 2 (TINF2) provides DFT a mechanism for telomere length homeostasis. The higher expression of both TERT and TINF2 may also protect DFT cells from genomic instability and enhance tumour proliferation.

Conclusions/Significance

DFT cells appear to monitor and regulate the length of individual telomeres: i.e. shorter telomeres are elongated by up-regulation of telomerase-related genes; longer telomeres are protected from further elongation by members of the shelterin complex, which may explain the lack of spatial and strain variation in DFT telomere copy number. The observed longitudinal increase in gene expression in DFT tissue samples and telomerase activity in DFT cell lines might indicate a selection for more stable tumours with higher proliferative potential.     

Characterization of major histocompatibility complex class I and class II genes from the Tasmanian devil (<Emphasis Type="Italic">Sarcophilus harrisii</Emphasis>)

The Tasmanian devil (Sarcophilus harrisii) is currently threatened by an emerging wildlife disease, devil facial tumour disease. The disease is decreasing devil numbers dramatically and may lead to the extinction of the species. At present, nothing is known about the immune genes or basic immunology of the devil. In this study, we report the construction of the first genetic library for the Tasmanian devil, a spleen cDNA library, and the isolation of full-length MHC Class I and Class II genes. We describe six unique Class II beta chain sequences from at least three loci, which belong to the marsupial Class II DA gene family. We have isolated 13 unique devil Class I sequences, representing at least seven Class I loci, two of which are most likely non-classical genes. The MHC Class I sequences from the devil have little heterogeneity, indicating recent divergence. The MHC genes described here are most likely involved in antigen presentation and are an important first step for studying MHC diversity and immune response in the devil.     

Towards a Case Definition for Devil Facial Tumour Disease: What Is It?

In the mid 1990s an emerging disease characterised by the development of proliferative lesions around the face of Tasmanian devils (Sarcophilus harrisii) was observed. A multi-disciplinary approach was adopted to define the condition. Histopathological and transmission electron microscopic examination combined with immunohistochemistry help define Devil Facial Tumour Disease (DFTD) as a neoplastic condition of cells of neuroendocrine origin. Cytogenetic analysis of neoplastic tissue revealed it to be markedly different from normal devil tissue and having a consistent karyotype across all tumours examined. Combined with evidence for Major histocompatability (MHC) gene analysis there is significant evidence to confirm the tumour is a transmissible neoplasm.     

Low MHC class II diversity in the Tasmanian devil (Sarcophilus harrisii)

The largest remaining carnivorous marsupial, the Tasmanian devil (Sarcophilus harrisii), is currently under threat of extinction due to a fatal contagious cancer-devil facial tumour disease. Low major histocompatibility complex (MHC) class I diversity is believed to have contributed to the transmission of the tumour allograft through devil populations. Here, we report low MHC class II variability in this species, with DA β chain genes (Saha-DAB1, 2 and 3) exhibiting very limited diversity and the sole α chain gene (Saha-DAA) monomorphic. Three, six and three alleles were found at Saha-DAB1, 2 and 3, respectively, with a predominant allele found at each locus. Heterozygosity at these three loci is low in the eastern population and modestly higher in northwestern individuals. The results are indicative of a selective sweep likely due to an infectious disease resulting in the fixation of selectively favoured alleles and depletion of genetic diversity at devil class II loci. Several attempts were made to isolate the other marsupial classical class II gene family, namely, DB, resulting in only one DBB pseudogene being found. These findings further support the view that this species has a compromised capacity to respond to pathogen evolution, emerging infectious diseases and environmental changes.     

Modifying the tumour microenvironment and reverting tumour cells: New strategies for treating malignant tumours

The tumour microenvironment (TME) plays a pivotal role in tumour fate determination. The TME acts together with the genetic material of tumour cells to determine their initiation, metastasis and drug resistance. Stromal cells in the TME promote the growth and metastasis of tumour cells by secreting soluble molecules or exosomes. The abnormal microenvironment reduces immune surveillance and tumour killing. The TME causes low anti‐tumour drug penetration and reactivity and high drug resistance. Tumour angiogenesis and microenvironmental hypoxia limit the drug concentration within the TME and enhance the stemness of tumour cells. Therefore, modifying the TME to effectively attack tumour cells could represent a comprehensive and effective anti‐tumour strategy. Normal cells, such as stem cells and immune cells, can penetrate and disrupt the abnormal TME. Reconstruction of the TME with healthy cells is an exciting new direction for tumour treatment. We will elaborate on the mechanism of the TME to support tumours and the current cell therapies for targeting tumours and the TME—such as immune cell therapies, haematopoietic stem cell (HSC) transplantation therapies, mesenchymal stem cell (MSC) transfer and embryonic stem cell‐based microenvironment therapies—to provide novel ideas for producing breakthroughs in tumour therapy strategies.     

Lack of genetic diversity across diverse immune genes in an endangered mammal,the Tasmanian devil (Sarcophilus harrisii)

The Tasmanian devil (Sarcophilus harrisii) is threatened with extinction due to the spread of devil facial tumour disease. Polymorphisms in immune genes can provide adaptive potential to resist diseases. Previous studies in diversity at immune loci in wild species have almost exclusively focused on genes of the major histocompatibility complex (MHC); however, these genes only account for a fraction of immune gene diversity. Devils lack diversity at functionally important immunity loci, including MHC and Toll‐like receptor genes. Whether there are polymorphisms at devil immune genes outside these two families is unknown. Here, we identify polymorphisms in a wide range of key immune genes, and develop assays to type single nucleotide polymorphisms (SNPs) within a subset of these genes. A total of 167 immune genes were examined, including cytokines, chemokines and natural killer cell receptors. Using genome‐level data from ten devils, SNPs within coding regions, introns and 10 kb flanking genes of interest were identified. We found low polymorphism across 167 immune genes examined bioinformatically using whole‐genome data. From this data, we developed long amplicon assays to target nine genes. These amplicons were sequenced in 29–220 devils and found to contain 78 SNPs, including eight SNPS within exons. Despite the extreme paucity of genetic diversity within these genes, signatures of balancing selection were exhibited by one chemokine gene, suggesting that remaining diversity may hold adaptive potential. The low functional diversity may leave devils highly vulnerable to infectious disease, and therefore, monitoring and preserving remaining diversity will be critical for the long‐term management of this species. Examining genetic variation in diverse immune genes should be a priority for threatened wildlife species. This study can act as a model for broad‐scale immunogenetic diversity analysis in threatened species.