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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.

Immune checkpoint molecules function as a system of checks and balances that enhance or inhibit immune responses to infectious agents, foreign tissues, and cancerous cells. Immunotherapies that target immune checkpoint molecules, particularly the inhibitory molecules programmed cell death 1 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), have revolutionized human oncology in recent years, yet little is known about these key immune signaling molecules in species other than primates and rodents. The Tasmanian devil facial tumor disease is caused by transmissible cancers that have resulted in a massive decline in the wild Tasmanian devil population. We have recently demonstrated that the inhibitory checkpoint molecule PD-L1 is upregulated on Tasmanian devil (Sarcophilus harrisii) facial tumor cells in response to the interferon-gamma cytokine. As this could play a role in immune evasion by tumor cells, we performed a thorough comparative analysis of checkpoint molecule protein sequences among Tasmanian devils and eight other species. We report that many of the key signaling motifs and ligand-binding sites in the checkpoint molecules are highly conserved across the estimated 162 million years of evolution since the last common ancestor of placental and non-placental mammals. Specifically, we discovered that the CTLA-4 (MYPPPY) ligand-binding motif and the CTLA-4 (GVYVKM) inhibitory domain are completely conserved across all nine species used in our comparative analysis, suggesting that the function of CTLA-4 is likely conserved in these species. We also found that cysteine residues for intra- and intermolecular disulfide bonds were also highly conserved. For instance, all 20 cysteine residues involved in disulfide bonds in the human 4-1BB molecule were also present in devil 4-1BB. Although many key sequences were conserved, we have also identified immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and immunoreceptor tyrosine-based switch motifs (ITSMs) in genes and protein domains that have not been previously reported in any species. This checkpoint molecule analysis and review of salient features for each of the molecules presented here can serve as road map for the development of a Tasmanian devil facial tumor disease immunotherapy. Finally, the strategies can be used as a guide for veterinarians, ecologists, and other researchers willing to venture into the nascent field of wild immunology.