We included nine species in our comparative analysis of checkpoint molecules. Humans and mice were included because checkpoint molecules have been best characterized in these species. We have included the little brown bat (Myotis lucifugus) in our comparative analyses because like devils, the species has recently undergone a dramatic population crash due to disease, and understanding bat immunology has become increasingly important due to the ability of bats to serve as reservoirs for viral pathogens (e.g., rabies, ebola, SARS). We have also included cats (Felis catus) and cattle (Bos taurus) due to their widespread distribution as important domestic animals. See Table 1 for a full list of species names and abbreviations used.

DNA sequences for all genes analyzed here were collected from GenBank and Ensembl and reference numbers for all sequences are available in Table S1 in Supplementary Material (53). All reference sequences current as of 30-Jan-2017, including the most current devil genome (Devil_ref v7.0). The complete coding sequence for the V-domain Ig suppressor of T cell activation (VISTA), CD47, and TIM-3 genes was not available from either GenBank or Ensembl, so sequences were obtained from a de novo transcriptome assembly (Table S2 in Supplementary Material) described below. The full coding sequences were submitted to GenBank with accession numbers: KY856749 (VISTA), KY856750 (CD47), and KY856751 (TIM-3). Where more than one sequence was present we generally chose GenBank transcript X1, unless preliminary alignments suggested that an alternative transcript was a better match to the other species used here. BLAST was used to find additional potential matching sequences for each gene (54). The available reference sequences for several genes that have not been characterized in their respective species contained elongated 5′ regions upstream of the likely start codon, so we manually truncated the amino acid sequences depicted in the alignment figures for the following genes: opossum CD80 and VISTA, bat CD86 and CD47, and hamster B7-H4.

RNA sequencing data were generated as part of a previous study (55). Briefly, RNA was extracted from peripheral blood mononuclear cells (PBMCs) obtained from two disease-free devils. Illumina single-end 100 bp RNA-seq was conducted by the Australian Genome Research Facility. Sequencing generated 82,609,965 and 82,254,904 sequences, respectively. Fastq reads for each sample were assessed with FastQC version 0.11.5 (56), with both samples having acceptable per base sequence quality measurements. Genome-independent de novo assembly of the RNA-seq data was completed using the Trinity platform version 2.2.0 (57, 58) with default settings applied. This generated a reference transcriptome of assembled transcripts collectively from the two samples. The de novo transcriptome assembly was queried with BLASTN version 2.4.0+ (59) with coding sequences of the target genes obtained from Ensembl (53), database version 87.7, and UCSC Genome Browser (60). This identified transcript sequences from the assembled transcriptome of our target genes to use in comparative alignment analysis. Further information on this analysis is detailed in the Data Sheet S1 in Supplementary Material. Access to the raw fastq sequence data, Trinity assembled de novo transcriptome, and BLASTN results are detailed in the Data Sheet S1 in Supplementary Material.

Protein sequences were aligned with CLC sequence viewer using a progressive alignment algorithm with gap open cost = 5, gap extension cost = 1, end gap cost = free (61, 62). Pairwise protein sequence identities between devils and other species were determined using the default parameters on the Clustal Omega web server (63). We used the RasMol coloring scheme to highlight traditional amino acid properties in all sequence alignments, which aids in interpretation of amino differences across species (64).

All sequences were analyzed using the ExPASy server ProtParam tool to predict molecular weight and extinction coefficients (65). The eukaryotic linear motif (ELM) tool was used to predict key signaling motifs (66). The Simple Modular Architecture Research Tool (SMART) and the Phobius web server (67) were used to predict Ig-like domains and membrane topology (68). SignalP was used to confirm predicted signal peptides (69). Disulfide bonds and other unique features of each protein that were annotated on UniProt (70) were used to predict features in devil protein sequences.

Devil CTLA-4 and CD28 are predicted to be 25 and 28 kDa, respectively, type-I transmembrane proteins with a single Ig-like V-type extracellular domain (ECD) (Figure 1). Protein sequence identity for CTLA-4 between devils and opossums was 86% and ranged from 63 to 69% between devils and placental mammals (Figure 2; Table 2). Percent identity for CD28 among species was lower than for CTLA-4, with 72% between devils and opossums and ranging from 54 to 57% between devils and placental mammals (Figure 3). Devil CTLA-4 had approximately 29% amino acid sequence identity with CD28. The CTLA-4 and CD28 coreceptors CD80 and CD86 are predicted to both be 35 kDa type-I transmembrane proteins that contain Ig-like V-type and an Ig-like C2-type domains. Devil CD80 and CD86 share only 21% sequence identity with each other, and the identity across the species analyzed here ranges from 34 to 55% and 32–49% for CD80 (Figure 4; Table 2) and CD86 (Figure 5; Table 2), respectively; the percent identity for CD80 and CD86 across species is the lowest of any of the checkpoint molecules compared here. All intra- and intermolecular disulfide bonds that have been identified in humans for CTLA-4, CD28, CD80, and CD86 are likely present in all the orthologs analyzed here, as we observed 100% conservation of cysteine residues at these sites in all species tested (Figures 2–5).

Devil VISTA (PD-1H, C10orf54, Gi24, DD1α, Dies1) is a type-I transmembrane protein in the Ig superfamily predicted to be 33 kDa. The full coding sequence for this gene was previously not available in the devil genome (Devil_ref v7.0), but our de novo RNA transcriptome assembly uncovered a full coding devil VISTA transcript. Devil VISTA shares 60–74% sequence identity at the protein level with the other species analyzed here (Figure 6; Table 2). The ELM tool predicted a TRAF1/2-binding site in the ECD of devil VISTA_SIQE.

4-1BB (TNFRSF9, CD137) is a predicted 31 kDa member of the tumor necrosis factor (TNF) receptor family expressed predominantly as a type-I transmembrane homodimer (74, 75). 4-1BB ligand (4-1BBL, TNFSF9, CD137L) is a TNF family member of 20 kDa, expressed as a type II transmembrane homotrimer (Figure 1). SMART analysis of devil 4-1BB identified two TNFR-Cys repeat domains, whereas human 4-1BB contains four TNFR-Cys repeats (Figure 7). All cysteine repeats in the four human TNFR-Cys domains were conserved in devils, suggesting that devil 4-1BB might also contain four TNFR-Cys domains despite not being predicted by the SMART program. Indeed, all 20 cysteine residues involved in disulfide bonds in human 4-1BB were also present in devil 4-1BB. Overall sequence identity among species compared here ranged from 43 to 75%, with mouse and hamster 4-1BB having the lowest percent identities with devil 4-1BB (Table 2).

We identified a putative inhibitory domain in devil 4-1BB_LPYFLK based on alignment with a potential inhibitory domain in human 4-1BB_LLYIFK (76, 77). The putative inhibitory domain resembles an ITIM domain but lacks the I, L, or V in the terminal amino acid position. To date, this potential inhibitory domain has only been reported in humans, but several species analyzed here have similar motifs. Additionally, mice have a 4-1BB_CXCP motif in the cytoplasmic domain that facilitates binding of CD4 and CD8 molecules to p56^lck, a protein tyrosine kinase that is important for optimal T cell responses (78, 79); of the nine species compared here, mice are the only species that have the 4-1BB_CXCP sequence. Our analysis using the ELM tool predicted two TRAF1/2 ligand-binding sites (devil 4-1BB_TAQEE, 4-1BB_PEEE), which align with the known TRAF1/2-binding sites in human and mouse 4-1BB (80).

Human CD47, also known as integrin-associated protein for its role in cell adhesion, is a cell surface protein that contains five transmembrane segments and a single Ig-like V-type domain (81, 82). The most up-to-date gene sequences for devil CD47 in Ensembl (ENSSHAT00000014546.1) did not contain a full coding sequence (i.e., no start codon) and the GenBank (XM_012544496) gene had an extended 5′ region that did not align well with other species. Our de novo RNA transcriptome assembly uncovered a full coding transcript for CD47 from devil PBMCs that aligned well with other species analyzed here. Our analysis suggests that the 34 kDa devil CD47 also contains five transmembrane segments and a single Ig-like domain (Figure 8). The four cysteines needed for the disulfide bonds reported in human CD47 are conserved in all species examined here, and overall sequence identity across all species compared here ranges from 54 to 87% (Table 2). Curiously, the ELM tool predicted an ITIM in the devil CD47_LKYHVV ECD. All other species except bats and hamsters also contain the putative ITIM domains, but the ELM tool classified the ITIM as an excluded motif due to the putative ITIM being located in ECD of the protein. We also identified a putative TRAF2-binding site in the third devil intracellular domain of CD47_KAVEE that was conserved across all species examined here except bats.

Devil SIRP-α is a predicted 56 kDa protein. Human SIRP-α binds to CD47 as either a monomer or a dimer (83, 84) and forms three intramolecular disulfide bonds. All six of the cysteines that form intramolecular disulfide bonds in humans are conserved in devils (Figure S1 in Supplementary Material). The percent sequence identity for SIRP-α ranged from 53 to 69%. Analysis of devil SIRP-α revealed two putative ITIM and one ITSM domain. The ITIM_ITYADL shares 100% identity with all species except cattle, and the ITIM_LTYADL is 100% conserved across all species. The devil ITSM_SQTEYASI matches 5/7 amino acids with all other species; to our knowledge no ITSM has previously been reported in SIRP-α. We identified two potential SIRP-β1 orthologs in devils (ENSSHAT00000016950, XM_012553634.1) that contained ITIM motifs (SIRP-β1_VSYNLI) in the ECD. The putative SIRP-β1 (ENSSHAT00000016950) ITIM aligned with an SIRP-β1_VSYNIT sequence in SIRP-α that contains the key tyrosine residue for ITIMs but did not contain the canonical 3′ I/V/L.

Devil CD200 is predicted to be a 31 kDa type-I transmembrane protein that shares 52–66% sequence identity across the species compared here (Figure 9; Table 2). The cysteine residues that form intramolecular and intermolecular disulfide bonds necessary for binding as either a monomer or homodimer to CD200 receptor (CD200R) are 100% conserved across all species examined here (Figure 9) (85). Additionally, the Ig-like V-type and Ig-like C2-type domains of human and mouse CD200 were identified by the SMART tool. Many key motifs, including endocytic sorting signals, and TRAF2, SH2, and SH3 binding motifs, are present in all species compared here. Our analysis using ELM predicts a potential ITSM only in devil CD200. However, the devil CD200_TLQE TRAF1/2-binding site and CD200_NKTEYVVI ITSM are both located in the predicted ECD of devil CD200, so empirical testing is needed to determine if these are functional sites. Although not analyzed in depth here, CD200R and CD200R-like proteins were identified in the nine species compared here. The primary reference sequence for each species contained the key CD200R_NPLY motif (86), which has been shown to be a key motif for CD200R inhibitory function (87).

TIM-3, also known as T-cell immunoglobulin and mucin domain-containing protein 3, hepatitis A virus cellular receptor 2, and CD366, is a surface protein expressed on T cells, Tregs, monocytes, macrophages, and DCs in mice and humans [reviewed in Ref. (88)]. The most up-to-date gene sequences for devil TIM-3 in Ensembl (ENSSHAT00000018107.1) and GenBank (XM_012552117.1) had truncated 3′ region of the coding sequence, and the Ensembl gene had an extended 5′ region that did not align with other species. Our de novo RNA transcriptome assembly uncovered a full coding devil TIM-3 transcript from devil PBMCs that aligned well with other species analyzed here. Analysis of the devil TIM-3 predicted sequence suggests that it is a 24 kDa protein with a Ig-like V-type domain and mucin domain in the extracellular region of the protein (Figure 10). Percent amino acid sequence identity between devils and the other eight species analyzed here ranges from 42 to 53%, which is the lowest identity for any molecule analyzed here except for CD80 and CD86 (Table 2). Despite this relatively low conservation of protein sequence, many of the key TIM-3 features identified in mice and humans appear to be present in devil TIM-3.

A unique feature of human TIM-3 is the double cleft which facilitates binding to multiple ligands (89, 90). The six cysteines that form three intramolecular disulfide bonds in the Ig-like region of human TIM-3 (89) are all present in devil TIM-3 (Figure 10), suggesting that devil TIM-3 folding and structure might be similar to human TIM-3. Analysis of human TIM-3 sequences identified five key amino acids for the two ligand-binding clefts; four of the five key amino acids were conserved in devil TIM-3. All species analyzed here also have two critical tyrosine residues in the TIM-3 cytoplasmic domain that align with mouse TIM-3_Y256 and TIM-3_Y263 (Figure 10), which are phosphorylated following ligation by GAL-9 and are associated with increased NFAT/AP-1 and NF-κB activity (91). A TIM-3_TIEE TRAF1/2-binding site is located between these tyrosine residues in all species except for cats, dogs, and cattle (Figure 10). Also, mouse TIM-1, TIM-3, and TIM-4 all bind to phosphatidylserine on the surface of apoptotic cells via a conserved metal ion-dependent ligand-binding site (MILIBS). The mouse TIM-3_RIQFPGLMND MILIBS motif is 100% conserved in cats, dogs, and cattle TIM-3, whereas devils have a phenylalanine instead of a methionine in this motif. Whether this TIM-3_M114F affects phosphatidylserine binding and clearance of apoptotic cells in devils is unknown.

In humans and mice, LAG-3 (CD223) is an inhibitory type-I transmembrane protein that is structurally homologous to CD4 (92). Human and mice LAG-3 each contain one Ig-like V-type and three Ig-like C-type domains, whereas devil LAG-3 is predicted to be a 54 kDa protein with three Ig-like domains. However, a 100 amino acid (LAG3_330–429) region downstream of the three predicted Ig-like domains could contain a fourth Ig-like domain that was not predicted by the SMART prediction tool (68, 93). Devil LAG-3 percent sequence identity with the other eight species analyzed here ranges from 43 to 73% (Table 2). In humans and mice, the LAG-3_KIEELE (Figure S2 in Supplementary Material) motif in the cytoplasmic domain is critical for downstream inhibitory signaling interactions, but this exact sequence is not present in the other species analyzed here. The critical lysine (LAG-3_K468) in this motif is present in devil (LAG-3_KAEEME) and all other mammals tested here, with the exception of opossums (LAG-3_IAEQME).

Devil V-set domain-containing T-cell activation inhibitor 1, or more commonly referred to as B7 homolog 4 (B7-H4), is a 30 kDa type-I membrane protein that contains two Ig-like V-type domains in the extracellular region (Figure 11). Devil B7-H4 protein shares 87% sequence identity with human B7-H4 and ranges from 80 to 94% for the other seven species analyzed here (Table 2). Based on homology to human and mouse B7-H4 and analysis of devil B7-H4 using cNLS Mapper (score of 3.4) (94, 95), it is likely that devil B7-H4_KKRGH contains a bipartite nuclear localization signal (NLS). Interestingly, B7-H4 has only two amino acids in the cytoplasmic domain when expressed as a surface protein.

Despite an estimated 162 million years of evolution since the last common ancestor of placental and non-placental mammals, most of the checkpoint molecules we analyzed have orthologs in Tasmanian devils. Importantly, many of the key signaling motifs and ligand-binding sites are highly conserved in the genes analyzed, particularly cysteine residues for intra- and intermolecular disulfide bonds and transmembrane and intracellular tyrosine residues for receptor tyrosine kinases. The strong conservation of the key protein regions suggests that inhibitory and stimulatory function of these molecules, which is relatively well-documented in humans and mice, is conserved in devils. Importantly, by following established procedures and focusing on the checkpoint molecules that have proven most clinically effective in humans, we can rapidly characterize key checkpoint molecules in devils and incorporate potent immunomodulatory agents into our DFT disease vaccine-development efforts.

Our analysis shows for the first time that the key CTLA-4_MYPPPY and CD28_MYPPPY are conserved in devils. Given that the MYPPPY motif present in chickens facilitates binding to mammalian CD80 and that a single leucine for methionine substitution in pig CTLA-4_LYPPPY is sufficient to disrupt binding of porcine CTLA-4 to human CD80 (35, 72, 96), the evolutionary conservation of CTLA-4_MYPPPY and CD28_MYPPPY in devils suggests that the competitive binding of CTLA-4 and CD28 for CD80/86 should be present in devils.

In addition to the conserved binding motifs, the key CD28_SDYMNN stimulatory domain (97) and CTLA_GVYVKM inhibitory domains are also conserved in devils, suggesting that the complementary stimulatory and inhibitory functions of CD28 and CTLA-4 are present. The CTLA-4_GVYVKM motif, in association with T-cell receptor-interacting molecule (TRIM), regulates surface expression of CTLA-4 (98). The TRIM-mediated recycling of CTLA-4 from the surface provides the means for cell-extrinsic inhibitory function by trans-endocytosis of CD80/86 (99). By trans-endocytosing, or in other words stripping CD80/86 from the surface of an opposing cell (i.e., APCs) and degrading the CD80/86 molecules in the CTLA-4-expressing cell, the potential of costimulation via CD80/86 binding to CD28 is eliminated. Whether trans-endocytosis or trogocytosis (100) occurs in devils is unknown. It could be readily tested by developing CD80 transgenic cell lines that have a fluorescent protein, such as green fluorescent protein (GFP), fused to the 3′ tail of CD80. Flow cytometric or immunofluorescence observations of GFP transferred from the transgenic cell line to the CTLA-4 expressing cells would demonstrate that this inhibitory pathway is conserved in devils.

The complex interactions involving CTLA-4 are further complicated by some anti-CTLA-4 monoclonal antibody effects that are mediated through Fc receptor signaling (11, 101, 102). Tregs expressing high levels of CTLA-4 can be killed via antibody-dependent cell-mediated cytotoxicity, thus reducing the inhibition of antitumor immunity. This could be a useful tactic for increasing T cell responses against neoantigens in DFT cells, but little is known about IgG isotypes and Fc receptors in devils (103). To our knowledge only a single IgG subclass has been identified in marsupials (103), and the number and type of Fc receptors are unknown. Investigations into devil isotypes and Fc receptors are needed to aid development of potential monoclonal antibody immunotherapies for devils. Additionally, we identified a potential TRAF1/2-binding site in devil CD80 and a TRAF6-binding site in devil CD86. TRAF6 is associated with NF-κB activation and production of IL-6 and IL-8, whereas TRAF2 is associated with IκB-kinases. The binding site for TRAF2 but not TRAF6 is required CD40-mediated antibody class switching (104–106). Specific targeting of CD80 or CD86 by monoclonal antibodies or soluble ligands could help to determine if additional IgG subclasses exist in devils and to understand the potential role of IL-6 and IL-8 in immune evasion by DFT cells.

In addition to binding to both CTLA-4 and CD28 in humans and mice, CD80 binds to PD-L1 with a greater affinity than to CD28, but less than affinity of CD80 for CTLA and PD-L1 for PD-1 (107, 108). In humans, overexpression of CD80 blocks PD-L1 expression by sequestering PD-L1 in the cytoplasm while simultaneously providing costimulation (109). Additional pleiotropic effects of CD80 include its ability to stimulate NK cells through both CD28-dependent and -independent pathways (110–112). As DFT1 cells are poorly immunogenic and do not express MHC-I, the ability to stimulate NK cells using CD80 could provide immunotherapeutic potential.

Soluble CD80 can also effectively block PD-1:PD-L1 interactions and provide costimulation (113, 114). Interestingly, in addition to the full-length devil CD80, we identified a devil CD80 splice variant that lacks a transmembrane domain (TMD) and likely codes for soluble version of this key checkpoint molecule. Humans are also known to express soluble CD80 and soluble PD-L1, and high plasma levels of PD-L1 are associated with poor prognosis in B cell lymphomas (115–117). The anti-PD-L1 monoclonal antibodies we have previously developed could be used to screen devil plasma for soluble PD-L1 and possibly serve as a prognostic test for DFT disease.

Despite the potential for enhanced immunotherapies targeting the 4-1BB/L pathway, actual progress has been slow, in part due to differences between mice and humans. 4-1BB costimulation in both mice and humans leads to enhanced T cell-mediated immunity, but the effects are contrasting in human and mouse NK cells. In mice, 4-1BB stimulation leads to enhanced NK cell effector mechanisms, but in humans 4-1BB signaling inhibits NK effector activity and reverse signaling through 4-1BBL on tumor cells can induce IL-10 and TNF-α (77, 118). This is potentially mediated by a putative inhibitory motif 4-1BB_LLYIFK in the human cytoplasmic domain that resembles an ITIM domain (76, 77). Interestingly, devils have a similar putative 4-1BB_LPYFLK inhibitory domain that includes the key tyrosine residue, but opossum 4-1BB does not contain the putative inhibitory motif (4-1BB_LSFLLQ). Comparative testing of opossum and devil 4-1BB should be able to determine the inhibitory capacity of these motifs. Devil 4-1BB also contains conserved TRAF1/2-binding sites that in humans are necessary for recruiting TRAF1 and TRAF2 and inducing expression of pro-survival genes, including Bcl-2, Bcl-XL, and Bfl-1 and decreasing expression of Bim, a pro-apoptotic molecule (119–121). Altogether, devil 4-1BB has key features that more closely resemble human 4-1BB, and thus devil 4-1BB might stimulate T cell function but inhibit NK cell function.

As expression of 4-1BB on T cells is largely dependent on activation state, combining agonistic anti-4-1BB immunotherapies with stimulatory cytokines presents an opportunity for promoting immune memory. For instance, CD28- or IL-15-mediated upregulation of 4-1BB combined with agonistic anti-4-1BB monoclonal antibodies or recombinant 4-1BBL could promote antigen-independent survival and expansion of memory CD8 T cells (122, 123); this could be critical for long-term memory against repeated exposure to DFT cells in the wild. Also of particular relevance to the DFT disease is that blockade of 4-1BB:4-1BBL pathways leads to prolonged allograft survival for heart (124), skin (124), eye (125), and intestinal (126) tissues in other species.

CD47 binds to several coreceptors, including SIRP-γ, THSB1, and THSB2, which induce either activation or inhibition signals (174, 175). An additional SIRP-family protein, SIRP-β1, does not bind to CD47 and no ligand for SIRP-β1 has been identified to date in humans (176). The SIRP-family proteins are hypothesized to be paired receptors that have evolved to reduce pathogen exploitation of conserved motifs found in inhibitory receptors, essentially a decoy target system that facilitates pathogen control. Pathogens that trigger inhibitory signals via conserved motifs in inhibitory receptors would thus also trigger activation signals when exploiting the similar motifs in the activating receptor (177, 178). Analysis of devil SIRP-α revealed two predicted cytoplasmic ITIM and one ITSM inhibitory domains, suggesting the inhibitory capacity of the CD47:SIRP-α interaction is conserved in devils and could be an immunotherapy target. The devil SIRP-α ITSM was not detected in any other species, so empirical testing will be needed to clarify the potential for targeting the CD47:SIRP-α pathway in devils.

Like the SIRP-family proteins, members of the CD200R family are hypothesized to have evolved as paired activating and inhibitory receptors. Specifically, herpesviruses, cytomegaloviruses, pox viruses, and adenoviruses inhibit macrophage activation via CD200 mimics that bind to CD200R-family members (179–181). In recent years, CD200 has received greater interest as an inhibitory checkpoint molecule associated with antitumor immunity. CD200 is expressed on a wide range of tumors, including lymphomas (182), myelomas (183), leukemias (184), and gliomas (185), and CD200 expression is correlated with expression of epithelial cancer stem cell markers on prostate, brain, breast, and colon cancers (186, 187).

Expression of CD200 on tumor cells has been associated with impaired NK cell function, increased frequencies of Tregs, suppression of memory T cells, decreased CTL activity, and induction of tolerance to allografts (184, 188, 189). Interestingly, CD200 expression has also been associated with Th2-skewed cytokine profiles and prolonged allograft survival (190–193). The transplantable EMT6 mouse breast cancer line expresses low levels of CD200 in vitro, but upregulates CD200 following inoculation into immunocompetent mice (194); this is similar to PD-L1, which is often expressed at low levels in vitro, but rapidly upregulated in immune active environments. Given the ongoing transmission of DFT for at least 20 years, immunohistochemical analysis of archived DFT tissue samples should determine expression levels of CD200 in the tumor microenvironment, and understanding this potential inhibitory pathway could help direct vaccine-development efforts.

Like devil PD-1, devil CD200 and SIRP-α are predicted to contain ITSM domains capable of regulating TCR signal transduction. ITSMs have been reported for both PD-1 and SIRP-α, but to our knowledge this is the first report of an ITSM in CD200. Interestingly, the CD200 ITSM was predicted only for devils and not any of the other eight species analyzed here, although it is possible that other isoforms of CD200 exist. There have only been a limited number molecules that have been predicted to contain ITSMs: BTLA; 2B4 (CD244); Siglec7; Siglec9; SLAMF1 (CD150); Ly9 (SLAMF3); CD84 (SLAMF5); SLAMF6 (CD352); and SLAMF7 (195, 196). Paired inhibitory and stimulatory receptors are hypothesized to have evolved in response to exploitation of inhibitory receptors by pathogens. Thus, pathogen motifs that utilize the inhibitory receptor will also trigger the stimulatory signal, so it is interesting that the three of the families that contain ITSMs are also predicted to be paired-receptor families (SIRP, CD200, Siglecs). The original ITSM was identified in CD150 and was named “switch-motif” due to its ability to switch from recruitment of SHP-2 to SHIP in the presence of SH2D1A (17, 197). This flexible switching aspect of the paired receptors could be an additional evolved response to exploitation of inhibitory receptors by common pathogens.

The short cytoplasmic tail of CD200 and known soluble forms in humans suggests that the primary inhibition through CD200 is mediated via ligation of CD200Rs. However, as mentioned above we identified a potential inhibitory ITSM in the extracellular region of CD200_NKTEYVVI. Interestingly, our analysis predicted putative inhibitory motifs in the extracellular region of several checkpoint molecules: devil CD47_LKYHVV ITIM near the border of the first extracellular and TMDs; devil SIRP-β1_VSYNLI extracellular ITIM; dog CD80_VKYGDL extracellular ITIM; and human CD86_IEYDGI extracellular ITIM.

To our knowledge, the function of ITIMs and ITSMs has only been explored in intracellular regions of checkpoint molecules. However, despite most checkpoint molecules being considered transmembrane proteins, the majority of checkpoint molecules covered in this analysis are only surface expressed in particular contexts. For instance, CTLA-4 protein can be detected in the cytoplasm prior to T cell activation, but then is transiently moved to the cell surface following activation. Trans-endocytosis of ITIM-containing CD80 (dog) or CD86 (human) could allow the ECD ITIMs to affect intracellular signaling. Also, CD80, CD86, CD200, CD47, all of which were identified to contain extracellular inhibitory domains, are part of multi-receptor or paired-receptor interactions and the potential role of ECD ITIMs and ITSMs may have been overlooked. This is potentially an understudied aspect of molecular and cellular immunology that warrants further investigation.

Although human LAG-3 has a higher affinity for MHC-II than CD4, the inhibitory effects of LAG-3 in humans and mice are thought to be mediated primarily via negative regulation of TCR:CD3 signal transduction rather than competition for MHC-II binding (198, 199). In addition to binding to MHC-II, LAG-3 also binds to C-type lectin domain family 4 member G (CLEC4G, LSECTin) and galectin 3 (Gal-3, LGALS3). In mice, Gal-3 expression can suppress CD8 antitumor responses and expansion of plasmacytoid DCs (200). Tumor cell expression of Gal-3 has also been associated with poor prognosis in non-small-cell lung cancer (201), Hodgkin lymphoma (202), and acute myeloid leukemia (203), but the opposite effect has also been reported for breast (204) and gastric (205) cancer.

Of particular interest for devils is the potential for LAG-3 to facilitate anti-DFT immunity. Immunization of mice with LAG-3+ tumor cells, or irradiated tumor cells along with soluble LAG-3-Ig resulted in tumor regression or reduced tumor growth (206). In addition, soluble LAG-3-Ig binding to MHC-II reliably induced DC maturation, which is indicated by upregulation of MHC-II, costimulatory molecules CD40, CD80, CD83, CD86, and IL-12 and TNF-α cytokines (207, 208). In allogenic bone marrow transplant models, LAG-3 blockade abrogates CD8 T cell tolerance to tissue transplants (209), which suggests that LAG-3 blockade could be a target for inducing CD8 T cell responses to allogenic DFT cells. Additionally, because LAG-3 signaling is bidirectional, DFT expressed Gal-3 could inhibit T and NK cell responses via direct Gal-3:LAG-3 interactions. Finally, LAG-3 and PD-1 are coexpressed in exhausted T cells in viral infection and on TILs, and blockade of both LAG-3 and PD-1 has been demonstrated to have synergistic effects in mouse models (33, 210–212). Altogether, there is a strong potential for LAG-3 to stimulate anti-DFT immunity due to the potential for overexpression of LAG-3 on tumor cells and/or administration of soluble LAG-3-Ig to serve as an adjuvant, and to use monoclonal antibodies that block LAG-3 on T cells to reduce T cell inhibition.

Despite more than a decade of searching, the identity of the B7-H4 coreceptor remains unknown. Further complicating the functional role of B7-H4 protein is that it is commonly expressed in the cytoplasm and nucleus of cancer cells, and nuclear localization of B7-H4 is associated with cell cycle progression and proliferation of cancer cells (95). B7-H4 is upregulated to the cell surface on T and B cells by several mitogens (213, 214) and on monocytes, TAMs, and DCs in response to IL-6 and IL-10 (215, 216).

Despite the receptor for B7-H4 being unknown, the inhibitory function of B7-H4 in regard to T cell responses and apoptosis has been well-documented (213, 214, 217). Binding of B7-H4-Ig to the putative coreceptor inhibits T cell activation, which can be abrogated by monoclonal blockade of B7-H4 (213, 214). B7-H4 protein has been detected on breast, lung, ovarian, prostate, renal cell, and uterine endometrioid cancer cells and is associated with decreased survival [reviewed in Ref. (20, 218)]. The high sequence identity of B7-H4 between humans and devils (87%) suggests that B7-H4 function might be similar in devils, and thus could be used as a prognostic indicator for DFT in addition to the potential for anti-B7-H4 monoclonal antibody immunotherapy.

The molecules described above demonstrated strong homology to other mammals, but another key inhibitory molecule in humans and mice, BTLA4, has not yet been identified in devils. Multiple BTLAs have been identified in bony fish (Teleosts), but have not been identified in amphibians or aves (24). Our search of GenBank and Ensembl and also our de novo assembly of a transcriptome from devil PBMCs failed to identify a BTLA gene and full coding sequence for the TIGIT gene in devils. Several TIGIT coreceptors are present in the devil genome and three TIGIT transcript variants reported in the opossum genome. Given that TIGIT expression appears to be tightly regulated in humans and mice (219), we believe it likely that a functional TIGIT gene will eventually be detected in devils through targeted laboratory methods (i.e., RACE PCR).