Generation and Testing of Fluorescent Adaptable Simple Theranostic (FAST) Proteins

J Cancer Res Clin Oncol

Patchett

Darby

et al. 2021

Purpose Downregulation of MHC class I (MHC-I) is a common immune evasion strategy of many cancers. Similarly, two allogeneic clonal transmissible cancers have killed thousands of wild Tasmanian devils (Sarcophilus harrisii) and also modulate MHC-I expression to evade anti-cancer and allograft responses. IFNG treatment restores MHC-I expression on devil facial tumor (DFT) cells but is insufficient to control tumor growth. Transcriptional co-activator NLRC5 is a master regulator of MHC-I in humans and mice but its role in transmissible cancers remains unknown. In this study, we explored the regulation and role of MHC-I in these unique genetically mis-matched tumors. Methods We used transcriptome and flow cytometric analyses to determine how MHC-I shapes allogeneic and anti-tumor responses. Cell lines that overexpress NLRC5 to drive antigen presentation, and B2M-knockout cell lines incapable of presenting antigen on MHC-I were used to probe the role of MHC-I in rare cases of tumor regressions. Results Transcriptomic results suggest that NLRC5 plays a major role in MHC-I regulation in devils. NLRC5 was shown to drive the expression of many components of the antigen presentation pathway but did not upregulate PDL1. Serum from devils with tumor regressions showed strong binding to IFNG-treated and NLRC5 cell lines; antibody binding to IFNGtreated and NRLC5 transgenic tumor cells was diminished or absent following B2M knockout. Conclusion MHC-I could be identified as a target for anti-tumor and allogeneic immunity. Consequently, 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.

Section: Plasmid Constructionmentioning

confidence: 99%

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

J Cancer Res Clin Oncol

Patchett

Darby

et al. 2021

“…The sequences were analyzed on 3500xL Genetic Analyzer (ABI) (see Supplementary Table 2 for list of sequencing primers). For detailed step-by-step protocols for plasmid design and construction, reagent recipes, and generation of stable cell lines, see Bio-protocol # e3696 24 .…”

Section: Plasmid Constructionmentioning

confidence: 99%

CIITA induces expression of MHC-I and MHC-II in transmissible cancers

Cheng

Siddle

et al. 2021

Preprint

MHC-I and MHC-II molecules are critical components of antigen presentation and T cell immunity to pathogens and cancer. The transmissible devil facial tumour (DFT) cells that cause Tasmanian devil facial tumour disease (DFTD) exploit MHC-I pathways to overcome immunological anti-tumour and allogeneic barriers. This exploitation underpins the ongoing transmission of DFT cells across the wild Tasmanian devil population. MHC-II expression is crucial for CD4+ T cell activation and is primarily confined to haematopoietic antigen presenting cells. We discovered that the MHC-II transactivator, CIITA, can induce MHC-II expression in non-haematopoietic cells. Transcriptomic analysis of DFT cell lines revealed that CIITA can upregulate several genes of the MHC-I and MHC-II pathways, resulting in protein expression of MHC-I and MHC-II complexes. The induced expression of MHC-II in transmissible cancers signifies that CIITA can function in non-haematopoietic cancer cells and offer a novel strategy to enhance tumour recognition via MHC-II-restricted tumour antigen presentation.

“…For this study, devil NLRC5 was amplified from pAF105 with overlapping ends to the 5' and 3' SfiI sites of the Supplementary Table 3 for list of sequencing primers). For detailed step-by-step protocols for plasmid design and construction, reagent recipes, and generation of stable cell lines, see Bio-protocol # e3986 43 .…”

Section: Plasmid Constructionmentioning

confidence: 99%

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

Patchett

Darby

et al. 2020

Preprint

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 humoral and cellular mechanisms associated with 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 cells 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 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.