PD-L1 Is Not Constitutively Expressed on Tasmanian Devil Facial Tumor Cells but Is Strongly Upregulated in Response to IFN-γ and Can Be Expressed in the Tumor Microenvironment

Abstract

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-γ. 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.

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

“…In line with this hypothesis, a recent study reported that overexpressing several checkpoint molecules, including CD200, PDL1, and CD47, in mouse embryonic stem cells could be used to generate teratomas that could establish long-term allograft tolerance in fully immunocompetent hosts 60 . We have previously reported that PDL1 mRNA and protein are upregulated on DFT2 cells in response to IFNγ 25 , and our transcriptome results show that CD47 is expressed at moderate to high levels in DFT cells. Here we show that overexpression of CD200R1 on DFT1 eliminates binding of our polyclonal anti-CD200 antibodies, suggesting that DFT cells overexpressing CD200R1 could be used to test the role of CD200 in allograft tolerance.…”

“…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 .…”

“…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.…”

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

“…We have not included the analysis of PD-1, PD-L1, and PD-L2 here, as we have previously published this information (52). Understanding the broad role of checkpoint molecules in these complex diseases has the potential to shed light on fundamental aspects of immune evasion, transplant tolerance and rejection, and infectious disease; this is logically relevant to both human and veterinary medicine.…”

Possible mechanisms of transmissible cancers in Tasmanian devils

“…It is known that cancer can weaken the immune system, however, cancer rarely acts as an infectious disease [28]. DFTD is extremely unusual that the allograft tumors are not rejected by the host immune system [29].…”