Hannah V. Siddle

Transmission of a fatal clonal tumor by biting occurs due to depleted MHC diversity in a threatened carnivorous marsupial

A fatal transmissible tumor spread between individuals by biting has emerged in the Tasmanian devil (Sarcophilus harrisii), a carnivorous marsupial. Here we provide genetic evidence establishing that the tumor is clonal and therefore foreign to host devils. Thus, the disease is highly unusual because it is not just a tumor but also a tissue graft, passed between individuals without invoking an immune response. The MHC plays a key role in immune responses to both tumors and grafts. The most common mechanism of immune evasion by tumors is down-regulation of classical cell surface MHC molecules. Here we show that this mode of immune escape does not occur. However, because the tumor is a graft, it should still be recognized and rejected by the hosts immune system due to foreign cell surface antigens. Mixed lymphocyte responses showed a lack of alloreactivity between lymphocytes of different individuals in the affected population, indicating a paucity of MHC diversity. This result was verified by genotyping, providing a conclusive link between a loss of MHC diversity and spread of a disease through a wild population. This novel disease arose as a direct result of loss of genetic diversity and the aggressive behavior of the host species. The neoplastic clone continues to spread although the population, and, without active disease control by removal of affected animals and the isolation of disease-free animals, the Tasmanian devil faces extinction.

Reconstructing an Ancestral Mammalian Immune Supercomplex from a Marsupial Major Histocompatibility Complex

The first sequenced marsupial genome promises to reveal unparalleled insights into mammalian evolution. We have used theMonodelphis domestica (gray short-tailed opossum) sequence to construct the first map of a marsupial major histocompatibility complex (MHC). The MHC is the most gene-dense region of the mammalian genome and is critical to immunity and reproductive success. The marsupial MHC bridges the phylogenetic gap between the complex MHC of eutherian mammals and the minimal essential MHC of birds. Here we show that the opossum MHC is gene dense and complex, as in humans, but shares more organizational features with non-mammals. The Class I genes have amplified within the Class II region, resulting in a unique Class I/II region. We present a model of the organization of the MHC in ancestral mammals and its elaboration during mammalian evolution. The opossum genome, together with other extant genomes, reveals the existence of an ancestral “immune supercomplex” that contained genes of both types of natural killer receptors together with antigen processing genes and MHC genes.

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.

Reversible epigenetic down-regulation of MHC molecules by devil facial tumour disease illustrates immune escape by a contagious cancer

Contagious cancers that pass between individuals as an infectious cell line are highly unusual pathogens. Devil facial tumor disease (DFTD) is one such contagious cancer that emerged 16 y ago and is driving the Tasmanian devil to extinction. As both a pathogen and an allograft, DFTD cells should be rejected by the host–immune response, yet DFTD causes 100% mortality among infected devils with no apparent rejection of tumor cells. Why DFTD cells are not rejected has been a question of considerable confusion. Here, we show that DFTD cells do not express cell surface MHC molecules in vitro or in vivo, due to down-regulation of genes essential to the antigen-processing pathway, such as β2-microglobulin and transporters associated with antigen processing. Loss of gene expression is not due to structural mutations, but to regulatory changes including epigenetic deacetylation of histones. Consequently, MHC class I molecules can be restored to the surface of DFTD cells in vitro by using recombinant devil IFN-γ, which is associated with up-regulation of the MHC class II transactivator, a key transcription factor with deacetylase activity. Further, expression of MHC class I molecules by DFTD cells can occur in vivo during lymphocyte infiltration. These results explain why T cells do not target DFTD cells. We propose that MHC-positive or epigenetically modified DFTD cells may provide a vaccine to DFTD. In addition, we suggest that down-regulation of MHC molecules using regulatory mechanisms allows evolvability of transmissible cancers and could affect the evolutionary trajectory of DFTD.

A second transmissible cancer in Tasmanian devils

Significance Transmissible cancers are somatic cell lineages that are spread between individuals via the transfer of living cancer cells. Only three transmissible cancers have been reported in nature, suggesting that such diseases emerge rarely. One of the known transmissible cancers affects Tasmanian devils, and is threatening this species with extinction. Here we report the discovery of a second transmissible cancer in Tasmanian devils. This cancer causes facial tumors that are grossly indistinguishable from those caused by the first-described transmissible cancer in this species; however, tumors derived from this second clone are genetically distinct. These findings indicate that Tasmanian devils have spawned at least two different transmissible cancers, and suggest that transmissible cancers may arise more frequently in nature than previously considered. Clonally transmissible cancers are somatic cell lineages that are spread between individuals via the transfer of living cancer cells. There are only three known naturally occurring transmissible cancers, and these affect dogs, soft-shell clams, and Tasmanian devils, respectively. The Tasmanian devil transmissible facial cancer was first observed in 1996, and is threatening its host species with extinction. Until now, this disease has been consistently associated with a single aneuploid cancer cell lineage that we refer to as DFT1. Here we describe a second transmissible cancer, DFT2, in five devils located in southern Tasmania in 2014 and 2015. DFT2 causes facial tumors that are grossly indistinguishable but histologically distinct from those caused by DFT1. DFT2 bears no detectable cytogenetic similarity to DFT1 and carries a Y chromosome, which contrasts with the female origin of DFT1. DFT2 shows different alleles to both its hosts and DFT1 at microsatellite, structural variant, and major histocompatibility complex (MHC) loci, confirming that it is a second cancer that can be transmitted between devils as an allogeneic, MHC-discordant graft. These findings indicate that Tasmanian devils have spawned at least two distinct transmissible cancer lineages and suggest that transmissible cancers may arise more frequently in nature than previously considered. The discovery of DFT2 presents important challenges for the conservation of Tasmanian devils and raises the possibility that this species is particularly prone to the emergence of transmissible cancers. More generally, our findings highlight the potential for cancer cells to depart from their hosts and become dangerous transmissible pathogens.

The Immune Response of the Tasmanian Devil (Sarcophilus harrisii) and Devil Facial Tumour Disease

One of the most remarkable aspects of Devil Facial Tumour Disease (DFTD) is its infectious nature, and for successful transmission it must avoid detection by the devil’s immune system. For this to occur, the devil either is severely immunosuppressed or factors produced by the tumor contribute to its avoidance of immune detection. An analysis of the devil’s immune system revealed the presence of normal-looking lymphoid organs and lymphoid cells. At a functional level the lymphocytes proliferated in response to mitogen stimulation. Subcutaneous injection of a cellular antigen produced a strong antibody response, providing compelling evidence that the devil has a competent immune system. Tumor cell analysis demonstrated that the tumor expresses the genes of the major histocompatibility complex; however, there was a limited diversity. Therefore, the most likely explanation for devil-to-devil transmission of DFTD is that the tumor is not recognized by the devil as “non-self” because of the limited genetic diversity. With its consistent morphology and relatively stable genome, this tumor would provide a reasonable target for a vaccine approach, provided the immune system can be coaxed into recognizing the tumor as “non-self.”

Characterization of major histocompatibility complex class I and class II genes from the Tasmanian devil (Sarcophilus harrisii)

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 β 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.

MHC-linked and un-linked class I genes in the wallaby

BackgroundMHC class I antigens are encoded by a rapidly evolving gene family comprising classical and non-classical genes that are found in all vertebrates and involved in diverse immune functions. However, there is a fundamental difference between the organization of class I genes in mammals and non-mammals. Non-mammals have a single classical gene responsible for antigen presentation, which is linked to the antigen processing genes, including TAP. This organization allows co-evolution of advantageous class Ia/TAP haplotypes. In contrast, mammals have multiple classical genes within the MHC, which are separated from the antigen processing genes by class III genes. It has been hypothesized that separation of classical class I genes from antigen processing genes in mammals allowed them to duplicate. We investigated this hypothesis by characterizing the class I genes of the tammar wallaby, a model marsupial that has a novel MHC organization, with class I genes located within the MHC and 10 other chromosomal locations.ResultsSequence analysis of 14 BACs containing 15 class I genes revealed that nine class I genes, including one to three classical class I, are not linked to the MHC but are scattered throughout the genome. Kangaroo Endogenous Retroviruses (KERVs) were identified flanking the MHC un-linked class I. The wallaby MHC contains four non-classical class I, interspersed with antigen processing genes. Clear orthologs of non-classical class I are conserved in distant marsupial lineages.ConclusionWe demonstrate that classical class I genes are not linked to antigen processing genes in the wallaby and provide evidence that retroviral elements were involved in their movement. The presence of retroviral elements most likely facilitated the formation of recombination hotspots and subsequent diversification of class I genes. The classical class I have moved away from antigen processing genes in eutherian mammals and the wallaby independently, but both lineages appear to have benefited from this loss of linkage by increasing the number of classical genes, perhaps enabling response to a wider range of pathogens. The discovery of non-classical orthologs between distantly related marsupial species is unusual for the rapidly evolving class I genes and may indicate an important marsupial specific function.

Antigen-presenting genes and genomic copy number variations in the Tasmanian devil MHC

BackgroundThe Tasmanian devil (Sarcophilus harrisii) is currently under threat of extinction due to an unusual fatal contagious cancer called Devil Facial Tumour Disease (DFTD). DFTD is caused by a clonal tumour cell line that is transmitted between unrelated individuals as an allograft without triggering immune rejection due to low levels of Major Histocompatibility Complex (MHC) diversity in Tasmanian devils.ResultsHere we report the characterization of the genomic regions encompassing MHC Class I and Class II genes in the Tasmanian devil. Four genomic regions approximately 960 kb in length were assembled and annotated using BAC contigs and physically mapped to devil Chromosome 4q. 34 genes and pseudogenes were identified, including five Class I and four Class II loci. Interestingly, when two haplotypes from two individuals were compared, three genomic copy number variants with sizes ranging from 1.6 to 17 kb were observed within the classical Class I gene region. One deletion is particularly important as it turns a Class Ia gene into a pseudogene in one of the haplotypes. This deletion explains the previously observed variation in the Class I allelic number between individuals. The frequency of this deletion is highest in the northwestern devil population and lowest in southeastern areas.ConclusionsThe third sequenced marsupial MHC provides insights into the evolution of this dynamic genomic region among the diverse marsupial species. The two sequenced devil MHC haplotypes revealed three copy number variations that are likely to significantly affect immune response and suggest that future work should focus on the role of copy number variations in disease susceptibility in this species.

Class I genes have split from the MHC in the tammar wallaby.

Genes within the Major Histocompatibility Complex (MHC) are critical to the immune response and immunoregulation. Comparative studies have revealed that the MHC has undergone many changes throughout evolution yet in tetrapods the three different classes of MHC genes have maintained linkage, suggesting that there may be some functional advantage obtained by maintaining this clustering of MHC genes. Here we present data showing that class II and III genes, the antigen processing gene TAP2, and MHC framework genes are found together in the tammar wallaby on chromosome 2. Surprisingly class I loci were not found on chromosome 2 but were mapped to ten different locations spread across six chromosomes. This distribution of class I loci in the wallaby on nearly all autosomes is not a characteristic of all marsupials and may be a relatively recent phenomenon. It highlights the need for the inclusion of more than one marsupial species in comparative studies and raises questions regarding the functional significance of the clustering of MHC genes.