Biology subjects

Darby, J. M.

Publications and source records attributed to Darby, J. M..

6 recordsLinked to original sources

bioRxiv2024

Isolation of an infectious mammalian chu-like virus from tumor cells of the endangered Tasmanian devil (Sarcophilus harrisii)

Jingchuvirales (negative-sense RNA viruses) were initially discovered in invertebrates, with both exogenous and endogenous jingchuviruses subsequently identified in fish, reptiles and mammals. To date, jingchuviruses have only been described metagenomically. By screening primary tumor tissues and tumor cell lines from the endangered Tasmanian devil (Sarcophilus harrisii), we isolated Tasmanian devil chu-like virus (TDCV) from cultures of Tasmanian devil facial tumor disease (DFTD) cells. Cell infection experiments demonstrated active virus replication in Tasmanian devil tumor cells, but not mosquito cells. The absence of viral replication in fibroblasts in cell culture and the lack of RNA detection in several organs suggested that replication was associated with tumor cells. Phylogenetic analysis revealed that TDCV likely represents a novel virus family. This is the first isolation of a jingchuvirus, demonstrating their capacity to infect mammalian cells, and providing in vitro avenues to understand the biology of TDCV and its association with tumor cell infection.

microbiology↗

bioRxiv2023

Conversion of mouse-derived hybridomas to Tasmanian devil recombinant IgG antibodies

The hybridoma method for production of monoclonal antibodies has been a cornerstone of biomedical research for several decades. Here we convert the monoclonal antibody sequence from mouse-derived hybridomas into a "devilised" recombinant antibody with devil IgG heavy chain and IgK light chain. The chimeric recombinant antibody can be used in functional assays, immunotherapy, and to improve understanding of antibodies and Fc receptors in Tasmanian devils. The process can be readily modified for other species.

immunology↗

bioRxiv2022

A human adenovirus encoding IFN-γ can transduce Tasmanian devil facial tumour cells and upregulate MHC-I

The devil facial tumour disease (DFTD) has led to a massive decline in the wild Tasmanian devil (Sarcophilus harrisii) population. The disease is caused by two independent devil facial tumours (DFT1 and DFT2). These transmissible cancers have a mortality rate of nearly 100%. An adenoviral vector-based vaccine has been proposed as a conservation strategy for the Tasmanian devil. This study aimed to determine if a human adenovirus serotype 5 could express functional transgenes in devil cells. As DFT1 cells do not constitutively express major histocompatibility complex class I (MHC-I), we developed a replication-deficient adenoviral vector that encodes devil interferon gamma (IFN-{gamma}) fused to a fluorescent protein reporter. Our results show that adenoviral-expressed IFN-{gamma} was able to stimulate upregulation of beta-2 microglobulin, a component of MHC-I, on DFT1, DFT2, and devil fibroblast cell lines. This work suggests that human adenoviruses can serve as vaccine platform for devils and potentially other marsupials.

immunology↗

bioRxiv2020

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

Downregulation of major histocompatibility complex I (MHC-I) on tumor cells is a primary means of immune evasion by many types of cancer. Additionally, MHC-I proteins are a primary target of immune-mediated transplant rejection. Transmissible tumors that overcome allograft rejection mechanisms and evade anti-tumor immunity have killed thousands of wild Tasmanian devils (Sarcophilus harrisii). Interferon gamma (IFNG) upregulates surface MHC-I expression on devil facial tumor (DFT) cells but is not sufficient to induce tumor regressions. Transcriptome analysis of IFNG-treated DFT cells revealed strong upregulation of NLRC5, a master regulator of MHC-I in humans and mice. To explore the role of NLRC5 in transmissible cancers, we developed DFT cell lines that constitutively overexpress NLRC5. Transcriptomic results suggest that the role of NLRC5 as a master regulator of MHC-I is conserved in devils. Furthermore, NLRC5 was shown to drive the expression of many components of the antigen presentation pathway. To determine if MHC-I is a target of allogeneic immune responses, we tested serum from devils with anti-DFT responses including natural DFT regressions against DFT cells. Antibody binding occurred with cells treated with IFNG and overexpressed NLRC5. However, CRISPR/Cas9-mediated knockout of MHC-I subunit beta-2-microglobulin (B2M) eliminated antibody binding to DFT cells. Consequently, MHC-I could be identified as a target for anti-tumor and allogeneic immunity and provides mechanistic insight into MHC-I expression and antigen presentation in marsupials. 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.

immunology↗

bioRxiv2020

Tasmanian devil CD28 and CTLA4 capture CD80 and CD86 from adjacent cells

Immune checkpoint immunotherapy is a pillar of human oncology with potential for non-human species. The first checkpoint immunotherapy approved for human cancers targeted the CTLA4 protein. CTLA4 can inhibit T cell activation by capturing and internalizing CD80 and CD86 from antigen presenting cells, a process called trans-endocytosis. Similarly, CD28 can capture CD80 and CD86 via trogocytosis and retain the captured ligands on the surface of the CD28-expressing cells. The Tasmanian devil (Sarcophilus harrisii) population has declined by 77% due to transmissible cancers that evade immune defenses despite genetic mismatches between the host and tumours. We used a live cell-based assay to demonstrate that devil CTLA4 and CD28 can capture CD80 and CD86. Mutation of evolutionarily conserved motifs in CTLA4 altered functional interactions with CD80 and CD86 in accordance with patterns observed in other species. These results suggest the potential for checkpoint immunotherapies can be translated to evolutionarily divergent species.

immunology↗

bioRxiv2019

Immune checkpoint proteins are conserved across 160 million years of evolution and are expressed on transmissible cancers

Immune checkpoint immunotherapy has revolutionized medicine, but translational success for new treatments remains low. Around 40% of humans and Tasmanian devils (Sarcophilus harrisii) develop cancer in their lifetime, compared to less than 10% for most species. Additionally, devils are affected by two of the three known transmissible cancers in mammals. Unfortunately, little is known about of immune checkpoints in devils and other non-model species, largely due to a lack of species-specific reagents. We developed a simple cut-and-paste reagent development method applicable to any vertebrate species and show that immune checkpoint interactions are conserved across 160 million years of evolution. The inhibitory checkpoint molecule CD200 is highly expressed on devil facial tumor cells. We are the first to demonstrate that co-expression of CD200R1 can block CD200 expression. The evolutionarily conserved pathways suggest that naturally occurring cancers in devils and other species can serve as models for understanding cancer and immunological tolerance. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/831404v3_ufig1.gif" ALT="Figure 1"> View larger version (108K): org.highwire.dtl.DTLVardef@14ff10eorg.highwire.dtl.DTLVardef@15acfd4org.highwire.dtl.DTLVardef@102a37dorg.highwire.dtl.DTLVardef@3b726c_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗