Comparative Analysis of Immune Checkpoint Molecules and Their Potential Role in the Transmissible Tasmanian Devil Facial Tumor Disease

Abstract

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.

A novel system to map protein interactions reveals evolutionarily conserved immune evasion pathways on transmissible cancers

“…We chose additional immune checkpoint molecules for FAST protein development (Figure 3A) based on targets of clinical trials and sequence analysis of devil genes 27, 34, 35 . We transfected the FAST protein expression vectors ( Table S2 ) into CHO cells and tested the supernatant against CHO cell lines expressing full-length receptors.…”

“…Interestingly, the CD200 peptides are reported to provide agonistic function through CD200-like activation receptors (CD200R4) rather than by blocking CD200R1 44, 64 . The functional role of CD200-CD200R pathway in devils remains to be elucidated, but the CD200R1 NPLY inhibitory motif and key tyrosine residues are conserved in devil CD200R 27, 65, 66 , demonstrating this motif is conserved over 160 million years of evolutionary history 67 . In addition to agonistic peptides, several other options for countering CD200-CD200R immune inhibition are possible.…”

“…These have proven broadly effective in part because they function across many different MHC types and tumor mutational patterns. However, these pathways have received little attention in transmissible cancers and other naturally occurring cancers in non-model species (Figure 1, Table S1 ) 25–27 . We have previously shown that the inhibitory immune checkpoint molecule programmed death ligand 1 (PDL1) is expressed in the DFT microenvironment and is upregulated by interferon-gamma (IFNγ) in vitro 25 .…”

“…This finding led us to question which other immune checkpoint molecules play a role in immune evasion by the transmissible cancers and the devil’s high spontaneous cancer incidence. Understanding this immune evasion in a natural environment has the potential to help protect this endangered species and identify protein interactions that are conserved across divergent species to improve translational success of animal models 27 . Unfortunately, a persistent limitation for immunology in non-traditional study species is a lack of species-specific reagents.…”

Adoptive CD8+ T cell therapy against cancer:Challenges and opportunities

“…Most of the immune checkpoint molecules are cell surface receptors on activated T cells such as PD-1 [44], CTLA4 [45], and LAG3 [46] etc. and contain tyrosine-based inhibitory motifs (ITIMs) [47] as well as immunoreceptor tyrosine-based inhibitory switch motifs (ITSMs) [48]. The inhibition of PD-1/PD-L1 and CTLA-4 effectively blocked the T cell exhaustion and apoptosis, and facilitated the anti-tumor immune response, which was granted with Nobel Prize in 2018.…”