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Context Disease is increasingly becoming a driver of wildlife population declines and an extinction risk. Vaccines are one of the most successful health interventions in human history, but few have been tested for mitigating wildlife disease. The transmissible cancer, devil facial tumour disease (DFTD), triggered the Tasmanian devil’s (Sarcophilus harrisii) inclusion on the international endangered species list. In 2016, 33 devils from a DFTD-free insurance population were given an experimental DFTD vaccination before their wild release on the Tasmanian northern coast. Aim To determine the efficacy of the vaccination protocol and the longevity of the induced responses. Method Six trapping trips took place over the 2.5 years following release, and both vaccinated and incumbent devils had blood samples and tumour biopsies collected. Key results In all, 8 of the 33 vaccinated devils were re-trapped, and six of those developed DFTD within the monitoring period. Despite the lack of protection provided by the vaccine, we observed signs of immune activation not usually found in unvaccinated devils. First, sera collected from the eight devils showed that anti-DFTD antibodies persisted for up to 2 years post-vaccination. Second, tumour-infiltrating lymphocytes were found in three of four biopsies collected from vaccinated devils, which contrasts with the ‘immune deserts’ typical of DFTs; only 1 of the 20 incumbent devils with DFTD had a tumour biopsy exhibiting immune-cell infiltrate. Third, immunohistochemical analysis of the vaccinated devils’ tumour biopsies identified the functional immune molecules associated with antigen-presenting cells (MHC-II) and T-cells (CD3), and the immune checkpoint molecule PD-1, all being associated with anti-tumour immunity in other species. Conclusions These results correlate with our previous study on captive devils in which a prophylactic vaccine primed the devil immune system and, following DFTD challenge and tumour growth, immunotherapy induced complete tumour regressions. The field trial results presented here provide further evidence that the devil immune system can be primed to recognise DFTD cells, but additional immune manipulation could be needed for complete protection or induction of tumour regressions. Implications A protective DFTD vaccine would provide a valuable management approach for conservation of the Tasmanian devil.

The wild Tasmanian devil (Sarchophilus harrisii) population has suffered a devastating decline due to two clonal transmissible cancers. Devil facial tumor 1 (DFT1) was first observed in 1996, followed by a second genetically distinct transmissible tumor, devil facial tumor 2 (DFT2), in 2014. DFT1/2 frequently metastasize, with lymph nodes being common metastatic sites. Downregulation of MHC-I by DFT1 cells is a primary means of evading allograft immunity aimed at polymorphic MHC-I proteins. DFT2 cells constitutively express MHC-I, and MHC-I is upregulated on DFT1/2 cells by interferon gamma, suggesting other immune evasion mechanisms may contribute to overcoming allograft and anti-tumor immunity. Human clinical trials have demonstrated PD1/PDL1 blockade effectively treats patients showing increased expression of PD1 in tumor draining lymph nodes, and PDL1 on peritumoral immune cells and tumor cells. The effects of DFT1/2 on systemic immunity remain largely uncharacterized. This study applied the open-access software QuPath to develop a semiautomated pipeline for whole slide analysis of stained tissue sections to quantify PD1/PDL1 expression in devil lymph nodes. The QuPath protocol provided strong correlations to manual counting. PD-1 expression was approximately 10-fold higher than PD-L1 expression in lymph nodes and was primarily expressed in germinal centers, whereas PD-L1 expression was more widely distributed throughout the lymph nodes. The density of PD1 positive cells was increased in lymph nodes containing DFT2 metastases, compared to DFT1. This suggests PD1/PDL1 exploitation may contribute to the poorly immunogenic nature of transmissible tumors in some devils and could be targeted in therapeutic or prophylactic treatments.

The devil facial tumour disease (DFTD) has led to a massive decline in the wild Tasmanian devil (Sarcophilus harrisii) population. The disease is caused by two independent devil facial tumours (DFT1 and DFT2). These transmissible cancers have a mortality rate of nearly 100%. An adenoviral vector-based vaccine has been proposed as a conservation strategy for the Tasmanian devil. This study aimed to determine if a human adenovirus serotype 5 could express functional transgenes in devil cells. As DFT1 cells do not constitutively express major histocompatibility complex class I (MHC-I), we developed a replication-deficient adenoviral vector that encodes devil interferon gamma (IFN-γ) fused to a fluorescent protein reporter. Our results show that adenoviral-expressed IFN-γ was able to stimulate upregulation of beta-2 microglobulin, a component of MHC-I, on DFT1, DFT2, and devil fibroblast cell lines. This work suggests that human adenoviruses can serve as vaccine platform for devils and potentially other marsupials.

ContextDiseases are increasingly contributing to wildlife population declines. Tasmanian devil (Sarcophilus harrisii) populations have locally declined by 82% largely due to the morbidity and mortality associated with two independent transmissible devil facial tumours (DFT1 and DFT2). Toxic baits are often used as a management tool for controlling vertebrate pest populations in Australia, but in other areas of the world oral baits are also used to deliver vaccines or pharmaceuticals to wildlife. Oral vaccine bait products have been distributed for more than 50 years at a landscape scale to protect wildlife from rabies virus and contributed to the elimination of fox rabies from more than ten European nations. An oral bait vaccine to protect against devil facial tumours has been proposed as a management tool to improve the population health, resiliency and fitness of wild Tasmanian devils.AimOur goal was to evaluate the potential use of edible baits as vehicles for vaccine delivery to Tasmanian devils.MethodWe tested placebo versions of baits that are already used in Australia.Key resultsCaptive devils consumed all types of placebo baits but exhibited a preference for ruminant- and fish-based baits. Captive devils also consumed inert capsules inserted into placebo baits. Bait fate trials in the field revealed that baits were generally removed within 24 hours. Tasmanian pademelons (Thylogale billardierii), brushtail possums (Trichosurus vulpecula), and Eastern quolls (Dasyurus viverrinus) were the most common nontarget bait competitors at six private properties in southern Tasmania; wild devils removed approximately 5% of ground baits at these sites. We also evaluated an automated bait dispenser, which reduced nontarget uptake and resulted in over 50% of the baits being removed by devils.ConclusionsThis study demonstrates that captive and wild devils will accept and consume placebo versions of commercial baits. Bait dispensers or modified baits or baiting strategies are needed to increase bait uptake by devils.ImplicationsBait dispensers can be used at a regional scale to deliver baits to Tasmanian devils. These could act as vehicles for preventive or therapeutic vaccines to mitigate the impacts of disease on devil populations.Short summaryThis study aimed to test oral baits as potential vaccine delivery vehicles for Tasmanian devils. Captive and wild devils consumed placebo versions of commercial baits used on mainland Australia. Abundant non-target species, such as brushtail possums, Tasmanian pademelons, and eastern quolls consumed most baits in the wild. Implementation of automated bait dispensers increased bait uptake by devils to over 50% at the same regional field sites.

Immune checkpoint immunotherapies (ICI) are now approved for over 20 types of cancer and there are almost 6000 ongoing clinical trials investigating immuno-modulators as cancer therapies. This review investigated the effect of monoclonal antibody-based immune checkpoint immunotherapies when combined with cytokine therapy. We reviewed published clinical trial results from 2005 to 2020 for studies that used approved monoclonal antibody ICI in combination with the cytokines. Studies that met the search criteria were assessed for treatment efficacy and immunological changes associated with treatment. ICI often fails to result in improved clinical outcomes for patients and lasting protection from cancer recurrence. The use of pro-inflammatory cytokines alongside ICI has been shown to enhance the efficacy of these therapies in vitro and in animal studies. However, the results in human clinical trials are less clear and many clinical trials do not publish results at the end of the trial. A deeper understanding of the molecular interactions between cytokines, tumors, and immune cells is needed to improve overall ICI outcomes and design combination trials. Critical examination of the design and characteristics of previous clinical trials can provide insight into the lack of effective clinical translation for many immunotherapeutic drugs.

Some countries have been crippled by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic while others have emerged with few infections and fatalities; the factors underscoring this macro-epidemiological variation is one of the mysteries of this global catastrophe. Variation in immune responses influence SARS-CoV-2 transmission and mortality, and factors shaping this variation at the country level, in addition to other socio-ecological drivers, may be important. Here, we construct spatially explicit Bayesian models that combine data on prevalence of endemic diseases and other socio-ecological characteristics to quantify patterns of confirmed deaths and cases across the globe before mass vaccination. We find that the prevalence of parasitic worms, human immunodeficiency virus and malaria play a surprisingly important role in predicting country-level SARS-CoV-2 patterns. When combined with factors such as population density, our models predict 63% (56-67) and 76% (69-81) of confirmed cases and deaths among countries, respectively. While our findings at this macro-scale are necessarily associative, they highlight a need for studies to consider factors, such as infection by other pathogens, on global SARS-CoV-2 dynamics. These relationships are vital for developing countries that already have the highest burden of endemic disease and are becoming the most affected by the SARS-CoV-2 pandemic.

MHC-I and MHC-II molecules are critical components of antigen presentation and T cell immunity to pathogens and cancer. The two monoclonal transmissible devil facial tumours (DFT1, DFT2) exploit MHC-I pathways to overcome immunological anti-tumour and allogeneic barriers. This exploitation underpins the ongoing transmission of DFT cells across the wild Tasmanian devil population. We have previously shown that constitutive expression of NLRC5 can induce stable upregulation of MHC-I on DFT1 and DFT2 cells, but unlike IFNG-treated cells, NLRC5 does not upregulate PDL1. MHC-II expression is crucial for CD4+T cell activation and is primarily confined to haematopoietic antigen presenting cells. Transcriptomic analysis of DFT1 and DFT2 cell lines showed that several genes of the MHC-I and MHC-II pathways were upregulated in response to constitutive overexpression of the class II transactivator (CIITA) gene. This was further supported by upregulation of MHC-I protein on DFT1 and DFT2 cells, but interestingly MHC-II protein was upregulated only on DFT1 cells. The functional significance of the MHC upregulation on DFT cells was shown using serum from devils with natural or immunotherapy-induced DFT1 regressions; binding of serum IgG was stronger in CIITA-transfected cells than wild type cells, but was less than binding to NLRC5 transfected cells. This new insight into regulation of MHC-I and MHC-II in cells that naturally overcome allogeneic barriers can inform vaccine, immunotherapy, and tissue transplant strategies for human and veterinary medicine.

Devil facial tumour disease (DFTD) comprises two genetically distinct transmissible cancers (DFT1 and DFT2) endangering the survival of the Tasmanian devil (Sarcophilus harrisii) in the wild. DFT1 first arose from a cell of the Schwann cell lineage; however, the tissue-of-origin of the recently discovered DFT2 cancer is unknown. In this study, we compared the transcriptome and proteome of DFT2 tumours to DFT1 and normal Tasmanian devil tissues to determine the tissue-of-origin of the DFT2 cancer. Our findings demonstrate that DFT2 expresses a range of Schwann cell markers and exhibits expression patterns consistent with a similar origin to the DFT1 cancer. Furthermore, DFT2 cells express genes associated with the repair response to peripheral nerve damage. These findings suggest that devils may be predisposed to transmissible cancers of Schwann cell origin. The combined effect of factors such as frequent nerve damage from biting, Schwann cell plasticity and low genetic diversity may allow these cancers to develop on rare occasions. The emergence of two independent transmissible cancers from the same tissue in the Tasmanian devil presents an unprecedented opportunity to gain insight into cancer development, evolution and immune evasion in mammalian species.

Individual hosts differ extensively in their competence for parasites, but traditional research has discounted this variation, partly because modeling such heterogeneity is difficult. This discounting has diminished as tools have improved and recognition has grown that some hosts, the extremely competent, can have exceptional impacts on disease dynamics. Most prominent among these hosts are the superspreaders, but other forms of extreme competence (EC) exist and others await discovery; each with potentially strong but distinct implications for disease emergence and spread. Here, we propose a framework for the study and discovery of EC, suitable for different host–parasite systems, which we hope enhances our understanding of how parasites circulate and evolve in host communities., Highlights A few members of host populations, so-called superspreaders, have disproportionate impacts on the risk of infectious disease emergence and spread. Several other forms of EC exist; some of which might be exceptionally protective. To discover and understand forms of EC, it is imperative to describe the distribution of, and covariation among, traits of individual hosts that mediate the many stages of host–parasite interactions. Here, we provide a framework to do so, emphasizing how interplay among host traits related to parasite exposure behavior, susceptibility, replicability of parasites on/in hosts, and transmissibility, comprise host competence. We hope this framework helps reveal new forms of EC and informs and improves management of disease risk.

Immune checkpoint immunotherapies (ICI) are now approved for over 20 types of cancer and there are almost 6000 ongoing clinical trials investigating immuno-modulators as cancer therapies. This review investigated the effect of monoclonal antibody-based immune checkpoint immunotherapies when combined with cytokine therapy. We reviewed published clinical trial results from 2005 to 2020 for studies that used approved monoclonal antibody ICI in combination with the cytokines. Studies that met the search criteria were assessed for treatment efficacy and immunological changes associated with treatment.ICI often fails to result in improved clinical outcomes for patients and lasting protection from cancer recurrence. The use of pro-inflammatory cytokines alongside ICI has been shown to enhance the efficacy of these therapies in vitro and in animal studies. However, the results in human clinical trials are less clear and many clinical trials do not publish results at the end of the trial. A deeper understanding of the molecular interactions between cytokines, tumors, and immune cells is needed to improve overall ICI outcomes and design combination trials. Critical examination of the design and characteristics of previous clinical trials can provide insight into the lack of effective clinical translation for many immunotherapeutic drugs.

Immunological tools have enormous potential to help us understand and manage wildlife diseases in a rapidly changing world. Here, we present a series of case studies in which immunological tools have been used to inform conservation actions. Additionally, we identify opportunities to improve the utility of ecoimmunological tools to conservation practitioners., Ecoimmunology is a rapidly developing field that explores how the environment shapes immune function, which in turn influences host–parasite relationships and disease outcomes. Host immune defence is a key fitness determinant because it underlies the capacity of animals to resist or tolerate potential infections. Importantly, immune function can be suppressed, depressed, reconfigured or stimulated by exposure to rapidly changing environmental drivers like temperature, pollutants and food availability. Thus, hosts may experience trade-offs resulting from altered investment in immune function under environmental stressors. As such, approaches in ecoimmunology can provide powerful tools to assist in the conservation of wildlife. Here, we provide case studies that explore the diverse ways that ecoimmunology can inform and advance conservation efforts, from understanding how Galapagos finches will fare with introduced parasites, to using methods from human oncology to design vaccines against a transmissible cancer in Tasmanian devils. In addition, we discuss the future of ecoimmunology and present 10 questions that can help guide this emerging field to better inform conservation decisions and biodiversity protection. From better linking changes in immune function to disease outcomes under different environmental conditions, to understanding how individual variation contributes to disease dynamics in wild populations, there is immense potential for ecoimmunology to inform the conservation of imperilled hosts in the face of new and re-emerging pathogens, in addition to improving the detection and management of emerging potential zoonoses.

Disease is increasingly becoming a driver of wildlife population declines and extinction risk. Vaccines have been one of the most successful health interventions in human history, but few have been tested for mitigating wildlife disease. The transmissible cancer, devil facial tumour disease (DFTD), triggered the Tasmanian devil’s (Sarcophilus harrisii) inclusion on the international endangered species list. Development of a protective DFTD vaccine would provide a valuable management approach for conservation of the species. In 2016, 33 devils from a DFTD-free insurance population were given an experimental DFTD vaccination prior to their release on the north coast of Tasmania. The release site was already home to an incumbent population of devils, including some individuals with DFTD. To determine the efficacy of the vaccination protocol and the longevity of the response it induced, six trapping trips took place at the site over the 2.5 years following release. Eight of the 33 vaccinated devils were re-trapped, and six of those developed DFTD within the monitoring period. Despite the apparent lack of protection provided by the vaccine for the re-trapped devils, we observed several signs of immune activation not usually found in unvaccinated devils. Firstly, sera collected from the eight devils showed that anti-DFTD antibodies persisted for up to two years post vaccination. Secondly, tumour infiltrating lymphocytes were found in three out of four biopsies collected from vaccinated devils which contrasts with the “immune deserts” typical of DFT’s; only one out of twenty incumbent devils with DFTD trapped during the same period had a tumour biopsy exhibiting immune cell infiltrate. Thirdly, immunohistochemical analysis of tumour biopsies from the vaccinated devils identified the functional immune molecules associated with antigen presenting cells (MHC-II) and T cells (CD3), and the immune checkpoint molecule PD-1, all associated with anti-tumour immunity in other species. These results correlate with our previous study on captive devils in which a prophylactic vaccine primed the devil immune system and, following DFTD challenge and tumour growth, immunotherapy induced complete tumour regressions. The field trial results presented here provide further evidence that the devil immune system can be primed to recognise DFTD cells, but additional immune manipulation could be needed for complete protection or induction of tumour regressions.

A simple cut-and-paste reagent development method applicable to any species reveals checkpoint molecules on transmissible cancers., Around 40% of humans and Tasmanian devils (Sarcophilus harrisii) develop cancer in their lifetime, compared to less than 10% for most species. In addition, devils are affected by two of the three known transmissible cancers in mammals. Immune checkpoint immunotherapy has transformed human medicine, but a lack of species-specific reagents has limited checkpoint immunology in most species. We developed a cut-and-paste reagent development system and used the fluorescent fusion protein system to show that immune checkpoint interactions are conserved across 160,000,000 years of evolution, CD200 is highly expressed on transmissible tumor cells, and coexpression of CD200R1 can block CD200 surface expression. The system’s versatility across species was demonstrated by fusing a fluorescent reporter to a camelid-derived nanobody that binds human programmed death ligand 1. The evolutionarily conserved pathways suggest that naturally occurring cancers in devils and other species can be used to advance our understanding of cancer and immunological tolerance.

Immune checkpoint immunotherapy is a pillar of human oncology treatment with potential for non-human species. The first checkpoint immunotherapy approved for human cancers targeted the CTLA4 protein. CTLA4 can inhibit T cell activation by capturing and internalizing CD80 and CD86 from antigen presenting cells, a process called trans-endocytosis. Similarly, CD28 can capture CD80 and CD86 via trogocytosis and retain the captured ligands on the surface of the CD28-expressing cells. The wild Tasmanian devil (Sarcophilus harrisii) population has declined by 77% due to transmissible cancers that evade immune defenses despite genetic mismatches between the host and tumours. We used a live cell-based assay to demonstrate that devil CTLA4 and CD28 can capture CD80 and CD86. Mutation of evolutionarily conserved motifs in CTLA4 altered functional interactions with CD80 and CD86 in accordance with patterns observed in other species. These results suggest that checkpoint immunotherapies can be translated to evolutionarily divergent species.HighlightsKey immune checkpoint receptor-ligand interactions are conserved in marsupials.Live cell-based assays show Tasmanian devil CD28 and CTLA4 can capture CD80 and CD86in transfrom adjacent cells.Mutation of the conserved CTLA4MYPPPYligand binding motif to CTLA4MYPPPAreduces binding to CD80 and intercellular protein transfer.Removal of conserved CTLA4YVKMprotein recycling binding motif in CTLA4 results in bidirectional intercellular protein transfer between CTLA4 and CD80.Highly successful human immune checkpoint immunotherapies have the potential to be translated for veterinary and conservation medicine.

This protocol provides a step-by-step method to create recombinant fluorescent fusion proteins that can be secreted from mammalian cell lines. This builds on many other recombinant protein and fluorescent protein techniques, but is among the first to harness fluorescent fusion proteins secreted directly into cell culture supernatant. This opens new possibilities that are not achievable with proteins produced in bacteria or yeast, such as direct use of the fluorescent protein-secreting cells in live co-culture assays. The Fluorescent Adaptable Simple Theranostic (FAST) protein system includes a histidine purification tag and a tobacco etch virus (TEV) cleavage site, allowing the purification tag and fluorescent protein to be removed for therapeutic use. This protocol is split into five parts: (A) In silico characterization of the gene-of-interest (GOI) and protein-of-interest (POI); (B) design of the expression vector; (C) assembly of the expression vector; (D) transfection of a eukaryotic cell line with the expression vector; (E) testing of the recombinant protein. This extensive protocol can be completed with only polymerase chain reaction (PCR) and cell culture training. Additionally, each part of the protocol can be used independently.

Introduction: The Tasmanian devil (Sarcophilus harrisii) is the largest extant carnivorous marsupial. Since 1996, its population has declined by 77% primarily due to a clonal transmissible tumor, known as devil facial tumor (DFT1) disease. In 2014, a second transmissible devil facial tumor (DFT2) was discovered. DFT1 and DFT2 are nearly 100% fatal.Areas covered: We review DFT control approaches and propose a rabies-style oral bait vaccine (OBV) platform for DFTs. This approach has an extensive safety record and was a primary tool in large-scale rabies virus elimination from wild carnivores across diverse landscapes. Like rabies virus, DFTs are transmitted by oral contact, so immunizing the oral cavity and stimulating resident memory cells could be advantageous. Additionally, exposing infected devils that already have tumors to OBVs could serve as an oncolytic virus immunotherapy. The primary challenges may be identifying appropriate DFT-specific antigens and optimization of field delivery methods.Expert opinion: DFT2 is currently found on a peninsula in southern Tasmania, so an OBV that could eliminate DFT2 should be the priority for this vaccine approach. Translation of an OBV approach to control DFTs will be challenging, but the approach is feasible for combatting ongoing and future disease threats.

The Tasmanian devil, a marsupial carnivore, has been restricted to the island state of Tasmania since its extinction on the Australian mainland about 3000 years ago. In the past two decades, this species has experienced severe population decline due to the emergence of devil facial tumor disease (DFTD), a transmissible cancer. During these 20 years, scientists have puzzled over the immunological and evolutionary responses by the Tasmanian devil to this transmissible cancer. Targeted strategies in population management and disease control have been developed as well as comparative processes to identify variation in tumor and host genetics. A multi-disciplinary approach with multi-institutional teams has produced considerable advances over the last decade. This has led to a greater understanding of the molecular pathogenesis and genomic classification of this cancer. New and promising developments in the Tasmanian devil’s story include evidence that most immunized, and some wild devils, can produce an immune response to DFTD. Furthermore, epidemiology combined with genomic studies suggest a rapid evolution to the disease and that DFTD will become an endemic disease. Since 1998 there have been more than 350 publications, distributed over 37 Web of Science categories. A unique endemic island species has become an international curiosity that is in the spotlight of integrative and comparative biology research.

Around 40% of humans and captive Tasmanian devils (Sarcophilus harrisii) develop cancer, compared to less than 10% for most species. Tasmanian devils also suffer from two of the three known naturally-occurring transmissible cancers detected in vertebrate species. Transmissible cancers are a unique form of cancer in which tumor cells act as an infectious pathogen and an allograft. The two different transmissible devil facial tumours (DFT1 and DFT2) overcome immunological barriers (e.g. major histocompatibility complex) and have killed thousands of devils. Immune checkpoint immunotherapy has revolutionized human oncology in recent years. However, in non-model species the role of immune checkpoints in transmissible and non-transmissible cancers remains largely unexplored due to a lack of species-specific reagents. To overcome this, we developed a "cut-and-paste" Fluorescent Adaptable Simple Theranostic (FAST) protein system, adaptable for any vertebrate species. This method facilitated rapid confirmation of seven receptor-ligand interactions between twelve immune checkpoint proteins in Tasmanian devils, thus filling a 162 million year gap in our understanding of the evolution of the mammalian immune system. We used this system to investigate the checkpoint molecule CD200, which can inhibit natural killer cell responses to cancer and facilitate graft tolerance in humans and mice. CD200 was highly expressed on DFT cells and can be used to identify DFT cells in devil blood. Understanding how clonal tumor cells graft onto new hosts, evade immune defenses and metastasize within hosts will help to identify evolutionarily conserved immunological principles relevant to transplant immunology, cancer, and infectious disease for human and veterinary medicine.

The Tasmanian devil (Sarcophilus harrisii) is the only mammalian species known to be affected by multiple transmissible cancers. Devil facial tumours 1 and 2 (DFT1 and DFT2) are independent neoplastic cell lineages that produce large, disfiguring cancers known as devil facial tumour disease (DFTD). The long-term persistence of wild Tasmanian devils is threatened due to the ability of DFTD cells to propagate as contagious allografts and the high mortality rate of DFTD. Recent studies have demonstrated that both DFT1 and DFT2 cancers originated from founder cells of the Schwann cell lineage, an uncommon origin of malignant cancer in humans. This unprecedented finding has revealed a potential predisposition of Tasmanian devils to transmissible cancers of the Schwann cell lineage. In this review, we compare the molecular nature of human Schwann cells and nerve sheath tumours with DFT1 and DFT2 to gain insights into the emergence of transmissible cancers in the Tasmanian devil. We discuss a potential mechanism, whereby Schwann cell plasticity and frequent wounding in Tasmanian devils combine with an inherent cancer predisposition and low genetic diversity to give rise to transmissible Schwann cell cancers in devils on rare occasions.

The Tasmanian devil facial tumor (DFT) disease has led to an 80% reduction in the wild Tasmanian devil (Sarcophilus harrisii) population since 1996. The limited genetic diversity of wild devils and the lack of MHC-I expression on DFT cells have been implicated in the lack of immunity against the original DFT clonal cell line (DFT1). Recently, a second transmissible tumor of independent origin (DFT2) was discovered. Surprisingly, DFT2 cells do express MHC-I, but DFT2 cells appear to be on a trajectory for reduced MHC-I expression in vivo. Thus, much of the ongoing vaccine-development efforts and conservation plans have focused on MHC-I. A major limitation in conservation efforts is the lack of species-specific tools to understand Tasmanian devil gene function and immunology. To help fill this gap, we developed an all-in-one Tet-Off vector system to regulate expression of IFN-γ in DFT cells (DFT1.Tet/IFN-γ). IFN-γ can have negative effects on cell proliferation and viability; thus, doxycycline was used to suppress IFN-γ production whilst DFT1.Tet/IFN-γ cells were expanded in cell culture. Induction of IFN-γ following removal of doxycycline led to upregulation of MHC-I but also the inhibitory checkpoint molecule PD-L1. Additionally, DFT1.Tet/IFN-γ cells were capable of stimulating MHC-I upregulation on bystander wild type DFT cells in co-culture assays in vitro. This system represents a major step forward in DFT disease immunotherapy and vaccine development efforts, and ability to understand gene function in devils. Importantly, the techniques are readily transferable for testing gene function in DFT2 cells and other non-traditional species.

Understanding the factors influencing mosquito distribution is important for effective surveillance and control of nuisance and disease vector mosquitoes. The goal of this study was to determine how trap height and distance to the city center influenced the abundance and species of mosquitoes collected in Adelaide, South Australia. Mosquito communities were sampled at two heights (

Beginning in 1997 Michigan State University Mara Hyena Project investigators observed waxing and waning progression of oral and genital masses during long-term behavioral observations of a population of wild spotted hyenas (Crocuta crocuta) from the Masai Mara Game Reserve, Kenya. From 1999-2000, we darted adult spotted hyenas to obtain routine physiologic and hematologic data and collected small, raised, lobulated, pigmented masses from the oral or genital areas of eight animals. Microscopically, masses consisted of variably thickened epidermis with thick elongate rete pegs, prominent stratum spinosum, and few koilocytes, consistent with papillomavirus-induced lesions. Immunohistochemistry on formalin-fixed, paraffin-embedded papilloma tissue revealed positive intranuclear labeling for papillomavirus antigen in the superficial stratum granulosum and in sloughing keratin layers of multiple samples. Polymerase chain reaction on DNA extracts from tumor tissue amplified a papillomavirus-specific 418 base pair amplicon in the E1 ORF. Basic Local Alignment Search Tool analysis of the sequenced amplicon suggests a novel hyaenid papillomavirus. Confirmatory complete genomic sequencing was performed later by the Rega Institute in Belgium. To our knowledge, this is the first report of a papillomavirus in a Hyaenidae species. Spotted hyena social behavior might facilitate oral-genital transmission of papillomavirus in this population. Refereed/Peer-reviewed

The devil facial tumor disease (DFTD) is caused by clonal transmissible cancers that have led to a catastrophic decline in the wild Tasmanian devil (Sarcophilus harrisii) population. The first transmissible tumor, now termed devil facial tumor 1 (DFT1), was first discovered in 1996 and has been continually transmitted to new hosts for at least 20 years. In 2015, a second transmissible cancer [devil facial tumor 2 (DFT2)] was discovered in wild devils, and the DFT2 is genetically distinct and independent from the DFT1. Despite the estimated 136,559 base pair substitutions and 14,647 insertions/deletions in the DFT1 genome as compared to two normal devil reference genomes, the allograft tumors are not rejected by the host immune system. Additionally, genome sequencing of two sub-strains of DFT1 detected greater than 15,000 single-base substitutions that were found in only one of the DFT1 sub-strains, demonstrating the transmissible tumors are evolving and that generation of neoantigens is likely ongoing. Recent evidence in human clinical trials suggests that blocking PD-1:PD-L1 interactions promotes antitumor immune responses and is most effective in cancers with a high number of mutations. We hypothesized that DFTD cells could exploit the PD-1:PD-L1 inhibitory pathway to evade antitumor immune responses. We developed recombinant proteins and monoclonal antibodies (mAbs) to provide the first demonstration that PD-1 binds to both PD-L1 and PD-L2 in a non-placental mammal and show that PD-L1 is upregulated in DFTD cells in response to IFN-gamma. Immunohistochemistry showed that PD-L1 is rarely expressed in primary tumor masses, but low numbers of PD-L1+ non-tumor cells were detected in the microenvironment of several metastatic tumors. Importantly, in vitro testing suggests that PD-1 binding to PD-L1 and PD-L2 can be blocked by mAbs, which could be critical to understanding how the DFT allografts evade the immune system. Refereed/Peer-reviewed

1. Social rank can profoundly affect many aspects of mammalian reproduction and stress physiology, but little is known about how immune function is affected by rank and other socioecological factors in free-living animals. 2. In this study, we examine the effects of sex, social rank and reproductive status on immune function in long-lived carnivores that are routinely exposed to a plethora of pathogens, yet rarely show signs of disease. 3. Here, we show that two types of immune defences, complement-mediated bacterial killing capacity (BKC) and total IgM, are positively correlated with social rank in wild hyenas, but that a third type, total IgG, does not vary with rank. 4. Female spotted hyenas, which are socially dominant to males in this species, have higher BKC, and higher IgG and IgM concentrations, than do males. 5. Immune defences are lower in lactating than pregnant females, suggesting the immune defences may be energetically costly. 6. Serum cortisol and testosterone concentrations are not reliable predictors of basic immune defences in wild female spotted hyenas. 7. These results suggest that immune defences are costly and multiple socioecological variables are important determinants of basic immune defences among wild hyenas. The effects of these variables should be accounted for when attempting to understand disease ecology and immune function. Refereed/Peer-reviewed

Ross River virus (RRV) is responsible for the most notifications of human arboviral infection in Australia. Seroprevalence and experimental infection studies have implicated macropods (e.g., kangaroos) as the major reservoir hosts. However, transmission ecology varies spatially, and infections in urban areas have prompted the question of what animals serve as reservoirs in regions where macropods are scarce. In South Australia (SA), human infection rates for RRV vary greatly by region as do vector and reservoir abundance. We hypothesized that mosquito abundance and feeding patterns would vary among ecoregions of SA and could help explain divergent human case rates. To test our hypothesis, we amplified and sequenced a 457 base pair region of the cytochrome B segment of mitochondrial DNA from blood fed mosquitoes collected in three main ecoregions of SA and identified sequences using a BLAST search in NCBI. Domestic livestock made up the vast majority of bloodmeals from the region with the highest human infection rate. Livestock are generally not considered to be important reservoir hosts for RRV, but our results suggest they may have a role in transmission ecology in some places. Surprisingly, none of the 199 bloodmeal samples were identified as macropod in origin. In the context of these findings, we consider the possible RRV vectors and reservoir hosts in these regions and propose that diverse spatial and temporal transmission ecologies occur in SA, depending on vector and reservoir availability. Refereed/Peer-reviewed

CD137 has long been recognized as a costimulatory receptor for growth and functional maturation of recently activated T cells in the presence of T-cell receptor signal. In this report, we present the fact that, in the absence of MHC and antigen, triggering of CD137 by an agonist monoclonal antibody induces vigorous growth of both CD8+ and CD4+ T cells with memory phenotype, whereas it does not affect naive T cells. Moreover, T cells with memory phenotype accumulate progressively in transgenic mice overexpressing CD137 ligand. CD137-mediated proliferation of memory T cells is directly through CD137 on T cells and does not require IL-15 and IFN-γ. Our results define a new role of CD137 signal in the growth of memory T cells.

Decoy lymphotoxin β receptor (LTβR) has potent immune inhibitory activities and thus represents a promising biologic for the treatment of inflammation, autoimmune diseases, and graft-versus-host disease (GVHD). As this reagent interrupts multiple molecular interactions, including LTβ-LTβR and LIGHT-HVEM/LTβR, underlying molecular mechanisms have yet to be fully understood. In this study, we demonstrate that blockade of the LIGHT-HVEM pathway is sufficient to induce amelioration of GVHD in mouse models. Anti–host cytotoxic T lymphocyte (CTL) activity following in vivo transfer of allogeneic lymphocytes was completely abrogated when LIGHT- or HVEM-deficient (KO) T cells were used as donor cells. Accordingly, survival of the recipient mice following the transfer of allogeneic bone marrow cells plus LIGHT-KO or HVEM-KO T cells was significantly prolonged. In the absence of LIGHT-HVEM costimulation, alloreactive donor T cells undergo vigorous apoptosis while their proliferative potential remains intact. Furthermore, we prepared a neutralizing monoclonal antibody (mAb) specific to HVEM and showed that administration of anti–HVEM mAb profoundly ameliorated GVHD and led to complete hematopoietic chimerism with donor cells. Collectively, our results demonstrate an indispensable role of LIGHT-HVEM costimulation in the pathogenesis of GVHD and illustrate a novel target for selective immunotherapy in allogeneic bone marrow transplantation.

LIGHT is an important costimulatory molecule for T cell immunity. Recent studies have further implicated its role in innate immunity and inflammatory diseases, but its cellular and molecular mechanisms remain elusive. We report here that LIGHT is upregulated and functions as a proinflammatory cytokine in 2 independent experimental hepatitis models, induced by concanavalin A and Listeria monocytogenes. Molecular mutagenesis studies suggest that soluble LIGHT protein produced by cleavage from the cell membrane plays an important role in this effect through the interaction with the lymphotoxin-beta receptor (LTbetaR) but not herpes virus entry mediator. NK1.1+ T cells contribute to the production, but not the cleavage or effector functions, of soluble LIGHT. Importantly, treatment with a mAb that specifically interferes with the LIGHT-LTbetaR interaction protects mice from lethal hepatitis. Our studies thus identify a what we believe to be a novel function of soluble LIGHT in vivo and offer a potential target for therapeutic interventions in hepatic inflammatory diseases.

B7-H3 is a B7 family molecule with T cell costimulatory function in vitro. The in vivo role of B7-H3 in the stimulation of tumor immunity is unclear. We report here that expression of B7-H3 by transfection of the mouse P815 tumor line enhances its immunogenicity, leading to the regression of tumors and amplification of a tumor-specific CD8+ CTL response in syngeneic mice. Tumor cells engineered to express B7-H3 elicit a rapid clonal expansion of P1A tumor Ag-specific CD8+ CTL in lymphoid organs in vivo and acquire the ability to directly stimulate T cell growth, division, and development of cytolytic activity in vitro. Our results thus establish a role for B7-H3 in the costimulation of T cell immune responses in vivo.

Previous research has shown that spotted hyenas (Crocuta crocuta) regularly survive exposure to deadly pathogens such as rabies, canine distemper virus, and anthrax, suggesting that they have robust immune defenses. Toll-like receptors (TLRs) recognize conserved molecular patterns and initiate a wide range of innate and adaptive immune responses. TLR genes are evolutionarily conserved, and assessing TLR expression in various tissues can provide insight into overall immunological organization and function. Studies of the hyena immune system have been minimal thus far due to the logistical and ethical challenges of sampling and preserving the immunological tissues of this and other long-lived, wild species. Tissue samples were opportunistically collected from captive hyenas humanely euthanized for a separate study. We developed primers to amplify partial sequences for TLRs 1-10, sequenced the amplicons, compared sequence identity to those in other mammals, and quantified TLR expression in lymph nodes, spleens, lungs, and pancreases. Results show that hyena TLR DNA and protein sequences are similar to TLRs in other mammals, and that TLRs 1-10 were expressed in all tissues tested. This information will be useful in the development of new assays to understand the interactions among the hyena immune system, pathogens, and the microbial communities that inhabit hyenas. Refereed/Peer-reviewed

B7-H4 is an immunoglobulin superfamily molecule and shown to be inhibitory for T-cell responses. To explore physiologic roles of B7-H4, we created B7-H4–deficient (KO) mice by genetic targeting. B7-H4KO mice are healthy and their T- and B-cell responses to polyclonal antigens are in normal range. However, B7-H4KO mice are more resistant to infection by Listeria monocytogenes than their littermates. Within 3 days after infection, bacterial colonies in livers and spleens are significantly lower than the controls, suggesting a role of B7-H4 in enhancing innate immunity. Further studies demonstrate that neutrophils increase in peripheral organs of B7-H4KO mice more so than their littermates but their bactericidal functions remain unchanged. Augmented innate resistance is completely dependent on neutrophils, even in the absence of adaptive immunity. In vitro B7-H4 inhibits the growth of bone marrow–derived neutrophil progenitors, suggesting an inhibitory function of B7-H4 in neutrophil expansion. Our results identify B7-H4 as a negative regulator of the neutrophil response to infection and provide a new target for manipulation of innate immunity.

B7-H1 is an immunoglobulin-like immune suppressive molecule broadly detectable on the majority of human and rodent cancers, and its functions have been attributed to delivering an inhibitory signal to its counter-receptor programmed death-1 (PD-1) on T cells. Here we report that B7-H1 on cancer cells receives a signal from PD-1 to rapidly induce resistance against T cell–mediated killing because crippling signaling capacity of B7-H1 but not PD-1 ablates this resistance. Importantly, loss of B7-H1 signaling is accompanied by increased susceptibility to immune-mediated tumoricidal activity. In addition to resistance against T-cell destruction, B7-H1+ cancer cells also become refractory to apoptosis induced by Fas ligation or the protein kinase inhibitor Staurosporine. Our study reveals a new mechanism by which cancer cells use a receptor on immune cells as a ligand to induce resistance to therapy.

Although self-reactive T-cell precursors can be eliminated upon recognition of self-antigen presented in the thymus, this central tolerance process is often incomplete, and additional mechanisms are required to prevent autoimmunity. Recent studies indicates that the interaction between B7-H1 and its receptor PD-1 on activated T cells plays an important role in the inhibition of T-cell responses in peripheral organs. Here, we show that, before their exit to the periphery, T cells in lymphoid organs rapidly up-regulate PD-1 upon tolerogen recognition. Ablation of the B7-H1 and PD-1 interaction when T cells are still in lymphoid organs prevents anergy. Furthermore, blockade of B7-H1 and PD-1 interaction could render anergic T cells responsive to antigen. Our results thus reveal previously unappreciated roles of B7-H1 and PD-1 interaction in the control of initiation and reversion of T-cell anergy.

Evolutionary processes have shaped the vertebrate immune system over time, but proximal mechanisms control the onset, duration, and intensity of immune responses. Based on testing of the hygiene hypothesis, it is now well known that microbial exposure is important for proper development and regulation of the immune system. However, few studies have examined the differences between wild animals in their natural environments, in which they are typically exposed to a wide array of potential pathogens, and their conspecifics living in captivity. Wild spotted hyenas (Crocuta crocuta) are regularly exposed to myriad pathogens, but there is little evidence of disease-induced mortality in wild hyena populations, suggesting that immune defenses are robust in this species. Here we assessed differences in immune defenses between wild spotted hyenas that inhabit their natural savanna environment and captive hyenas that inhabit a captive environment where pathogen control programs are implemented. Importantly, the captive population of spotted hyenas was derived directly from the wild population and has been in captivity for less than four generations. Our results show that wild hyenas have significantly higher serum antibody concentrations, including total IgG and IgM, natural antibodies, and autoantibodies than do captive hyenas; there was no difference in the bacterial killing capacity of sera collected from captive and wild hyenas. The striking differences in serum antibody concentrations observed here suggest that complementing traditional immunology studies, with comparative studies of wild animals in their natural environment may help to uncover links between environment and immune function, and facilitate progress towards answering immunological questions associated with the hygiene hypothesis. Refereed/Peer-reviewed