NLRC5 regulates expression of MHC-I and provides a target for anti-tumor immunity in transmissible cancers.

PURPOSE: Downregulation of MHC class I (MHC-I) is a common immune evasion strategy of many cancers. Similarly, two allogeneic clonal transmissible cancers have killed thousands of wild Tasmanian devils (Sarcophilus harrisii) and also modulate MHC-I expression to evade anti-cancer and allograft responses. IFNG treatment restores MHC-I expression on devil facial tumor (DFT) cells but is insufficient to control tumor growth. Transcriptional co-activator NLRC5 is a master regulator of MHC-I in humans and mice but its role in transmissible cancers remains unknown. In this study, we explored the regulation and role of MHC-I in these unique genetically mis-matched tumors. METHODS: We used transcriptome and flow cytometric analyses to determine how MHC-I shapes allogeneic and anti-tumor responses. Cell lines that overexpress NLRC5 to drive antigen presentation, and B2M-knockout cell lines incapable of presenting antigen on MHC-I were used to probe the role of MHC-I in rare cases of tumor regressions. RESULTS: Transcriptomic results suggest that NLRC5 plays a major role in MHC-I regulation in devils. NLRC5 was shown to drive the expression of many components of the antigen presentation pathway but did not upregulate PDL1. Serum from devils with tumor regressions showed strong binding to IFNG-treated and NLRC5 cell lines; antibody binding to IFNG-treated and NRLC5 transgenic tumor cells was diminished or absent following B2M knockout. CONCLUSION: MHC-I could be identified as a target for anti-tumor and allogeneic immunity. Consequently, NLRC5 could be a promising target for immunotherapy and vaccines to protect devils from transmissible cancers and inform development of transplant and cancer therapies for humans.

The immunopeptidomes of two transmissible cancers and their host have a common, dominant peptide motif.

Transmissible cancers are malignant cells that can spread between individuals of a population, akin to both a parasite and a mobile graft. The survival of the Tasmanian devil, the largest remaining marsupial carnivore, is threatened by the remarkable emergence of two independent lineages of transmissible cancer, devil facial tumour (DFT) 1 and devil facial tumour 2 (DFT2). To aid the development of a vaccine and to interrogate how histocompatibility barriers can be overcome, we analysed the peptides bound to major histocompatibility complex class I (MHC-I) molecules from Tasmanian devil cells and representative cell lines of each transmissible cancer. Here, we show that DFT1 + IFN-γ and DFT2 cell lines express a restricted repertoire of MHC-I allotypes compared with fibroblast cells, potentially reducing the breadth of peptide presentation. Comparison of the peptidomes from DFT1 + IFNγ, DFT2 and host fibroblast cells demonstrates a dominant motif, despite differences in MHC-I allotypes between the cell lines, with preference for a hydrophobic leucine residue at position 3 and position Ω of peptides. DFT1 and DFT2 both present peptides derived from neural proteins, which reflects a shared cellular origin that could be exploited for vaccine design. These results suggest that polymorphisms in MHC-I molecules between tumours and host can be 'hidden' by a common peptide motif, providing the potential for permissive passage of infectious cells and demonstrating complexity in mammalian histocompatibility barriers.

An oral bait vaccination approach for the Tasmanian devil facial tumor diseases.

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.

Transmissible Cancers and Immune Downregulation in Tasmanian Devil (Sacrophilus harrisii) and Canine Populations.

Known as devil facial tumor disease (DFTD) and canine transmissible venereal tumor (CTVT), transmissible cancer occurs in both Tasmanian devil and canine populations, respectively. Both malignancies show remarkable ability to be transmitted as allografts into subsequent hosts. How DFTD and CTVT avoid detection by immunocompetent hosts is of particular interest, given that these malignancies are rarely seen in other species in nature. Both of these transmissible cancers can downregulate the host immune system, enabling proliferation. DFTD is characterized by epigenetic modifications to the DNA promoter regions of ß2microglobulin, transporters associated with antigen processing 1 and 2, MHC I, and MHC II-crucial proteins required in the detection and surveillance of foreign material. Downregulation during DFTD may be achieved by altering the activity of histone deacetylases. DFTD has caused widespread destruction of devil populations, placing the species on the brink of extinction. CTVT demonstrates a proliferative phase, during which the tumor evades immune detection, allowing it to proliferate, and a regressive phase when hosts mount an effective immune response. Alteration of TGFß signaling in CTVT likely impedes the antigen-processing capabilities of canine hosts in addition to hindering the ability of natural killer cells to detect immune system downregulation. Immunosuppressive cytokines such as CXCL7 may contribute to a favorable microenvironment that supports the proliferation of CTVT. When viewed from an evolutionary paradigm, both DFTD and CTVT may conform to a model of host-parasite coevolution. Furthermore, various genetic features, such as genetically active transposons in CTVT and chromosomal rearrangements in DFTD, play important roles in promoting the survival of these disease agents. Understanding the mode of transmission for these transmissible cancers may shed light on mechanisms for human malignancies and reveal opportunities for treatment in the future.

Devil Facial Tumours: Towards a Vaccine.

The Tasmanian devil is the only mammalian species to harbour two independent lineages of contagious cancer. Devil facial tumour 1 (DFT1) emerged in the 1990s and has caused significant population declines. Devil facial tumour 2 (DFT2) was identified in 2014, and evidence indicates that this new tumour has emerged independently of DFT1. While DFT1 is widespread across Tasmania, DFT2 is currently found only on the Channel Peninsula in south east Tasmania. Allograft transmission of cancer cells should be prevented by major histocompatibility complex (MHC) molecules. DFT1 avoids immune detection by downregulating MHC class I expression, which can be reversed by treatment with interferon-gamma (IFNγ), while DFT2 currently circulates in hosts with a similar MHC class I genotype to the tumour. Wild Tasmanian devil numbers have not recovered from the emergence of DFT1, and it is feared that widespread transmission of DFT2 will be devastating to the remaining wild population. A preventative solution for the management of the disease is needed. Here, we review the current research on immune responses to devil facial tumours and vaccine strategies against DFT1 and outline our plans moving forward to develop a specific, effective vaccine to support the wild Tasmanian devil population against the threat of these two transmissible tumours.

TNF May Negatively Regulate Phagocytosis of Devil Facial Tumour Disease Cells by Activated Macrophages.

Introduction: Macrophage phagocytosis of pathogens and tumour cells is an important early event in protection against infectious disease and cancer. As tumour necrosis factor α (TNF) is an important cytokine in macrophage activation, we investigated the involvement of TNF in macrophage phagocytosis of tumour cells. Methods: We used Devil Facial Tumour Disease (DFTD) cancer cells as the target tumour cells. The Tasmanian devil (Sarcophilus harrisii) population is threatened by the transmissible DFTD. Using DFTD cells provided the opportunity to determine if these cells can be phagocytosed and investigate requirement for TNF. As effector cells, bone marrow derived macrophages (BMDMs), generated from C57BL/6 wild type (B6.WT) and C57BL/6 TNF-/- (B6.TNF-/-) mice were used. Phagocytosis of DFTD cells was investigated by confocal microscopy and flow cytometry. Results: DFTD cells were consistently phagocytosed by B6.WT and B6.TNF-/- BMDMs with similar efficiency in vitro. Consequently the DFTD cells are not resistant to phagocytosis. Following activation by exposure to IFNγ and LPS or LPS alone, B6.TNF-/- BMDMs had higher phagocytic efficiency and lower nitric oxide (NO) production compared to wild-type controls. In addition, NO seems to be unlikely to be the involved in phagocytosis efficiency in IFNγ and LPS activated B6.TNF-/- macrophages and consequences thereof. Conclusion: Our results indicate that TNF is not required for IFNγ and LPS or LPS alone activation of macrophage phagocytosis. TNF may negatively regulate macrophage phagocytosis of tumour cells.

Lessons learnt from the Tasmanian devil facial tumour regarding immune function in cancer.

Genetic and genomic technologies have facilitated a greater understanding of the Tasmanian devil immune system and the origins, evolution and spread of devil facial tumour disease (DFTD). DFTD is a contagious cancer that has caused significant declines in devil populations across Tasmania. Immune responses to DFTD are rarely detected, allowing the cancer to pass between individuals and proliferate unimpeded. Early immunosenscence in devils appears to decrease anti-tumour immunity in older animals compared to younger animals, which may increase susceptibility to DFTD and explain high DFTD prevalence in this age group. Devils also have extremely low major histocompatibility complex (MHC) diversity, and multiple alleles are shared with the tumour, lowering histocompatibility barriers which may have contributed to DFTD evolution. DFTD actively evades immune attack by down-regulating cell-surface MHC I molecules, making it effectively invisible to the immune system. Altered MHC I profiles should activate natural killer (NK) cell anti-tumour responses, but these are absent in DFTD infection. Recent immunisation and immunotherapy using modified DFTD cells has induced an anti-DFTD immune response and regression of DFTD in some devils. Knowledge gained from immune responses to a transmissible cancer in devils will ultimately reveal useful insights into immunity to cancer in humans and other species.

Two Decades of the Impact of Tasmanian Devil Facial Tumor Disease.

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.

The Origins and Vulnerabilities of Two Transmissible Cancers in Tasmanian Devils.

Transmissible cancers are clonal lineages that spread through populations via contagious cancer cells. Although rare in nature, two facial tumor clones affect Tasmanian devils. Here we perform comparative genetic and functional characterization of these lineages. The two cancers have similar patterns of mutation and show no evidence of exposure to exogenous mutagens or viruses. Genes encoding PDGF receptors have copy number gains and are present on extrachromosomal double minutes. Drug screening indicates causative roles for receptor tyrosine kinases and sensitivity to inhibitors of DNA repair. Y chromosome loss from a male clone infecting a female host suggests immunoediting. These results imply that Tasmanian devils may have inherent susceptibility to transmissible cancers and present a suite of therapeutic compounds for use in conservation.

Heat shock proteins expressed in the marsupial Tasmanian devil are potential antigenic candidates in a vaccine against devil facial tumour disease.

The Tasmanian devil (Sarcophilus harrisii), the largest extant carnivorous marsupial and endemic to Tasmania, is at the verge of extinction due to the emergence of a transmissible cancer known as devil facial tumour disease (DFTD). DFTD has spread over the distribution range of the species and has been responsible for a severe decline in the global devil population. To protect the Tasmanian devil from extinction in the wild, our group has focused on the development of a prophylactic vaccine. Although this work has shown that vaccine preparations using whole DFTD tumour cells supplemented with adjuvants can induce anti-DFTD immune responses, alternative strategies that induce stronger and more specific immune responses are required. In humans, heat shock proteins (HSPs) derived from tumour cells have been used instead of whole-tumour cell preparations as a source of antigens for cancer immunotherapy. As HSPs have not been studied in the Tasmanian devil, this study presents the first characterisation of HSPs in this marsupial and evaluates the suitability of these proteins as antigenic components for the enhancement of a DFTD vaccine. We show that tissues and cancer cells from the Tasmanian devil express constitutive and inducible HSP. Additionally, this study suggests that HSP derived from DFTD cancer cells are immunogenic supporting the future development of a HSP-based vaccine against DFTD.

Immunization Strategies Producing a Humoral IgG Immune Response against Devil Facial Tumor Disease in the Majority of Tasmanian Devils Destined for Wild Release.

Devil facial tumor disease (DFTD) is renowned for its successful evasion of the host immune system. Down regulation of the major histocompatabilty complex class I molecule (MHC-I) on the DFTD cells is a primary mechanism of immune escape. Immunization trials on captive Tasmanian devils have previously demonstrated that an immune response against DFTD can be induced, and that immune-mediated tumor regression can occur. However, these trials were limited by their small sample sizes. Here, we describe the results of two DFTD immunization trials on cohorts of devils prior to their wild release as part of the Tasmanian Government's Wild Devil Recovery project. 95% of the devils developed anti-DFTD antibody responses. Given the relatively large sample sizes of the trials (N = 19 and N = 33), these responses are likely to reflect those of the general devil population. DFTD cells manipulated to express MHC-I were used as the antigenic basis of the immunizations in both trials. Although the adjuvant composition and number of immunizations differed between trials, similar anti-DFTD antibody levels were obtained. The first trial comprised DFTD cells and the adjuvant combination of ISCOMATRIX™, polyIC, and CpG with up to four immunizations given at monthly intervals. This compared to the second trial whereby two immunizations comprising DFTD cells and the adjuvant combination ISCOMATRIX™, polyICLC (Hiltonol®) and imiquimod were given a month apart, providing a shorter and, therefore, more practical protocol. Both trials incorporated a booster immunization given up to 5 months after the primary course. A key finding was that devils in the second trial responded more quickly and maintained their antibody levels for longer compared to devils in the first trial. The different adjuvant combination incorporating the RNAase resistant polyICLC and imiquimod used in the second trial is likely to be responsible. The seroconversion in the majority of devils in these anti-DFTD immunization trials was remarkable, especially as DFTD is hallmarked by its immune evasion mechanisms. Microsatellite analyzes of MHC revealed that some MHC-I microsatellites correlated to stronger immune responses. These trials signify the first step in the long-term objective of releasing devils with immunity to DFTD into the wild.

Inducible IFN-γ Expression for MHC-I Upregulation in Devil Facial Tumor Cells.

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.

The toll-like receptor ligands Hiltonol® (polyICLC) and imiquimod effectively activate antigen-specific immune responses in Tasmanian devils (Sarcophilus harrisii).

Devil facial tumour disease (DFTD) describes two genetically distinct transmissible tumours that pose a significant threat to the survival of the Tasmanian devil. A prophylactic vaccine could protect devils from DFTD transmission. For this vaccine to be effective, potent immune adjuvants will be required. Toll-like receptors (TLRs) promote robust immune responses in human cancer studies and are highly conserved across mammalian species. In this study, we investigated the proficiency of TLR ligands for immune activation in the Tasmanian devil using in vitro mononuclear cell stimulations and in vivo immunisation trials with a model antigen. We identified two such TLR ligands, polyICLC (Hiltonol®) (TLR3) and imiquimod (TLR7), that in combination induced significant IFNγ production from Tasmanian devil lymphocytes in vitro. Immunisation with these ligands and the model antigen keyhole limpet haemocyanin activated robust antigen-specific primary, secondary and long-term memory IgG responses. Our results support the conserved nature of TLR signaling across mammalian species. PolyICLC and imiquimod will be trialed as immune adjuvants in future DFTD vaccine formulations.

Regression of devil facial tumour disease following immunotherapy in immunised Tasmanian devils.

Devil facial tumour disease (DFTD) is a transmissible cancer devastating the Tasmanian devil (Sarcophilus harrisii) population. The cancer cell is the 'infectious' agent transmitted as an allograft by biting. Animals usually die within a few months with no evidence of antibody or immune cell responses against the DFTD allograft. This lack of anti-tumour immunity is attributed to an absence of cell surface major histocompatibility complex (MHC)-I molecule expression. While the endangerment of the devil population precludes experimentation on large experimental groups, those examined in our study indicated that immunisation and immunotherapy with DFTD cells expressing surface MHC-I corresponded with effective anti-tumour responses. Tumour engraftment did not occur in one of the five immunised Tasmanian devils, and regression followed therapy of experimentally induced DFTD tumours in three Tasmanian devils. Regression correlated with immune cell infiltration and antibody responses against DFTD cells. These data support the concept that immunisation of devils with DFTD cancer cells can successfully induce humoral responses against DFTD and trigger immune-mediated regression of established tumours. Our findings support the feasibility of a protective DFTD vaccine and ultimately the preservation of the species.

Rapid evolutionary response to a transmissible cancer in Tasmanian devils.

Although cancer rarely acts as an infectious disease, a recently emerged transmissible cancer in Tasmanian devils (Sarcophilus harrisii) is virtually 100% fatal. Devil facial tumour disease (DFTD) has swept across nearly the entire species' range, resulting in localized declines exceeding 90% and an overall species decline of more than 80% in less than 20 years. Despite epidemiological models that predict extinction, populations in long-diseased sites persist. Here we report rare genomic evidence of a rapid, parallel evolutionary response to strong selection imposed by a wildlife disease. We identify two genomic regions that contain genes related to immune function or cancer risk in humans that exhibit concordant signatures of selection across three populations. DFTD spreads between hosts by suppressing and evading the immune system, and our results suggest that hosts are evolving immune-modulated resistance that could aid in species persistence in the face of this devastating disease.

Mitogen-activated Tasmanian devil blood mononuclear cells kill devil facial tumour disease cells.

Devil facial tumour disease (DFTD) is a transmissible cancer that has brought the host species, the Tasmanian devil, to the brink of extinction. The cancer cells avoid allogeneic immune recognition by downregulating cell surface major histocompatibility complex (MHC) I expression. This should prevent CD8(+) T cell, but not natural killer (NK) cell, cytotoxicity. The reason why NK cells, normally reactive to MHC-negative cells, are not activated to kill DFTD cells has not been determined. The immune response of wild devils to DFTD, if it occurs, is uncharacterised. To investigate this, we tested 12 wild devils with DFTD, and found suggestive evidence of low levels of antibodies against DFTD cells in one devil. Eight of these devils were also analysed for cytotoxicity, however, none showed evidence for cytotoxicity against cultured DFTD cells. To establish whether mimicking activation of antitumour responses could induce cytotoxic activity against DFTD, Tasmanian devil peripheral blood mononuclear cells (PBMCs) were treated with either the mitogen Concanavalin A, the Toll-like receptor agonist polyinosinic:polycytidylic acid or recombinant Tasmanian devil IL-2. All induced the PBMC cells to kill cultured DFTD cells, suggesting that activation does not occur after encounter with DFTD cells in vivo, but can be induced. The identification of agents that activate cytotoxicity against DFTD target cells is critical for developing strategies to protect against DFTD. Such agents could function as adjuvants to induce functional immune responses capable of targeting DFTD cells and tumours in vivo.

Immunoglubolin dynamics and cancer prevalence in Tasmanian devils (Sarcophilus harrisii).

Immunoglobulins such as IgG and IgM have been shown to induce anti-tumour cytotoxic activity. In the present study we therefore explore total serum IgG and IgM expression dynamics in 23 known-aged Tasmanian devils (Sarcophilus harrisii) of which 9 where affected by Devil Facial Tumour Disease (DFTD). DFTD is clonally transmissible cancer that has caused massive declines in devil numbers. Our analyses revealed that IgM and IgG expression levels as well as IgM/IgG ratios decreased with increasing devil age. Neither age, sex, IgM nor IgG expression levels affected devil DFTD status in our analyses. However, devils with increased IgM relative to IgG expression levels had significantly lower DFTD prevalence. Our results therefore suggest that IgM/IgG ratios may play an important role in determining devil susceptibility to DFTD. We consequently propose that our findings warrant further studies to elucidate the underpinning(s) of devil IgM/IgG ratios and DFTD status.

Demonstration of immune responses against devil facial tumour disease in wild Tasmanian devils.

Devil facial tumour disease (DFTD) is a recently emerged fatal transmissible cancer decimating the wild population of Tasmanian devils (Sarcophilus harrisii). Biting transmits the cancer cells and the tumour develops in the new host as an allograft. The literature reports that immune escape mechanisms employed by DFTD inevitably result in host death. Here we present the first evidence that DFTD regression can occur and that wild devils can mount an immune response against the disease. Of the 52 devils tested, six had serum antibodies against DFTD cells and, in one case, prominent T lymphocyte infiltration in its tumour. Notably, four of the six devils with serum antibody had histories of DFTD regression. The novel demonstration of an immune response against DFTD in wild Tasmanian devils suggests that a proportion of wild devils can produce a protective immune response against naturally acquired DFTD. This has implications for tumour-host coevolution and vaccine development.

Toll-like receptor signaling is functional in immune cells of the endangered Tasmanian devil.

Devil facial tumour disease (DFTD) is a fatally transmissible cancer that threatens the Tasmanian devil population. As Tasmanian devils do not produce an immune response against DFTD cells, an effective vaccine will require a strong adjuvant. Activation of innate immune system cells through toll-like receptors (TLRs) could provide this stimulation. It is unknown whether marsupials, including Tasmanian devils, express functional TLRs. We isolated RNA from peripheral blood mononuclear cells and, with PCR, detected transcripts for TLRs 2, 3, 4, 5, 6, 7, 8, 9, 10 and 13. Stimulation of the mononuclear cells with agonists to these TLRs increased the expression of downstream TLR signaling products (IL1α, IL6, IL12A and IFNß). Our data provide the first evidence that TLR signaling is functional in the mononuclear cells of the Tasmanian devil. Future DFTD vaccination trials will incorporate TLR agonists to enhance the immune response against DFTD.