Dataset Information

A_2AR eGFP reporter mouse enables elucidation of A_2AR expression dynamics during anti-tumor immune responses.

ABSTRACT: There is significant clinical interest in targeting adenosine-mediated immunosuppression, with several small molecule inhibitors having been developed for targeting the A_2AR receptor. Understanding of the mechanism by which A_2AR is regulated has been hindered by difficulty in identifying the cell types that express A_2AR due to a lack of robust antibodies for these receptors. To overcome this limitation, here an A_2AR eGFP reporter mouse is developed, enabling the expression of A_2AR during ongoing anti-tumor immune responses to be assessed. This reveals that A_2AR is highly expressed on all tumor-infiltrating lymphocyte subsets including Natural Killer (NK) cells, NKT cells, γδ T cells, conventional CD4⁺ and CD8⁺ T lymphocytes and on a MHCII^hiCD86^hi subset of type 2 conventional dendritic cells. In response to PD-L1 blockade, the emergence of PD-1⁺A_2AR^- cells correlates with successful therapeutic responses, whilst IL-18 is identified as a cytokine that potently upregulates A_2AR and synergizes with A_2AR deficiency to improve anti-tumor immunity. These studies provide insight into the biology of A_2AR in the context of anti-tumor immunity and reveals potential combination immunotherapy approaches.

SUBMITTER: Todd KL

PROVIDER: S-EPMC10620403 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

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A2AR eGFP reporter mouse enables elucidation of A2AR expression dynamics during anti-tumor immune responses.

Todd Kirsten L KL Lai Junyun J Sek Kevin K Huang Yu-Kuan YK Newman Dane M DM Derrick Emily B EB Koay Hui-Fern HF Nguyen Dat D Hoang Thang X TX Petley Emma V EV Chan Cheok Weng CW Munoz Isabelle I House Imran G IG Lee Joel N JN Kim Joelle S JS Li Jasmine J Tong Junming J N de Menezes Maria M Scheffler Christina M CM Yap Kah Min KM Chen Amanda X Y AXY Dunbar Phoebe A PA Haugen Brandon B Parish Ian A IA Johnstone Ricky W RW Darcy Phillip K PK Beavis Paul A PA

Nature communications 20231101 1

There is significant clinical interest in targeting adenosine-mediated immunosuppression, with several small molecule inhibitors having been developed for targeting the A2AR receptor. Understanding of the mechanism by which A2AR is regulated has been hindered by difficulty in identifying the cell types that express A2AR due to a lack of robust antibodies for these receptors. To overcome this limitation, here an A2AR eGFP reporter mouse is developed, en ...[more]

PMID: 37914685

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Project description:BackgroundDystrophin is a rod-shaped cytoplasmic protein that provides sarcolemmal stability as a structural link between the cytoskeleton and the extracellular matrix via the dystrophin-associated protein complex (DAPC). Mutations in the dystrophin-encoding DMD gene cause X-linked dystrophinopathies with variable phenotypes, the most severe being Duchenne muscular dystrophy (DMD) characterized by progressive muscle wasting and fibrosis. However, dystrophin deficiency does not only impair the function of skeletal and heart muscle but may also affect other organ systems such as the brain, eye, and gastrointestinal tract. The generation of a dystrophin reporter mouse would facilitate research into dystrophin muscular and extramuscular pathophysiology without the need for immunostaining.ResultsWe generated a Dmd (EGFP) reporter mouse through the in-frame insertion of the EGFP coding sequence behind the last Dmd exon 79, which is known to be expressed in all major dystrophin isoforms. We analyzed EGFP and dystrophin expression in various tissues and at the single muscle fiber level. Immunostaining of various members of the DAPC was done to confirm the correct subsarcolemmal location of dystrophin-binding partners. We found strong natural EGFP fluorescence at all expected sites of dystrophin expression in the skeletal and smooth muscle, heart, brain, and retina. EGFP fluorescence exactly colocalized with dystrophin immunostaining. In the skeletal muscle, dystrophin and other proteins of the DAPC were expressed at their correct sarcolemmal/subsarcolemmal localization. Skeletal muscle maintained normal tissue architecture, suggesting the correct function of the dystrophin-EGFP fusion protein. EGFP expression could be easily verified in isolated myofibers as well as in satellite cell-derived myotubes.ConclusionsThe novel dystrophin reporter mouse provides a valuable tool for direct visualization of dystrophin expression and will allow the study of dystrophin expression in vivo and in vitro in various tissues by live cell imaging.

Project description:We describe the utility of a tandem-tagged autophagy reporter mouse model (CAG-RFP-EGFP-MAP1LC3B) in investigating basal macroautophagic/autophagic flux in the neural retina. Western blot, in situ hybridization, immunohistochemistry, and confocal microscopy showed that CAG promoter-driven expression of RFP-EGFP-MAP1LC3B increased "cytosolic" RFP-EGFP-LC3B-I levels, whereas RFP-EGFP-LC3B-II decorates true phagosomes. We verified that the electroretinographic (ERG) responses of tandem-tagged LC3B mice were comparable to those of age-matched controls. Optimized microscope settings detected lipofuscin autofluorescence in retinas of abca4-/- mice. The majority of retinal phagosomes in the reporter mice exhibited only RFP (not EGFP) fluorescence, suggesting rapid maturation of phagosomes. Only ~1.5% of the total phagosome population was EGFP-labeled; RFP-labeled (mature) phagosomes colocalized with lysosomal markers LAMP2 and CTSD. In the outer retina, phagosome sizes were as follows (in µm2, ave ± SEM): RPE, 0.309 ± 0.015; photoreceptor inner segment-myoid, 0.544 ± 0.031; and outer nuclear layer, 0.429 ± 0.011. Detection of RPE phagosomes by fluorescence microscopy is challenging, due to the presence of melanin. Increased lipofuscin autofluorescence, such as observed in the abca4-/- mouse model of Stargardt disease, is a strong confounding factor when attempting to study autophagy in the RPE. In addition to RPE and photoreceptor cells, phagosomes were detected in inner retinal cell types, microglia, astrocytes, and endothelial cells. We conclude that the tandem-tagged LC3B mouse model serves as a useful system for studying autophagy in the retina. This utility, however, is dependent upon several technical and biological factors, including microscope settings, transgene expression, choice of fluorophores, and lipofuscin autofluorescence.Abbreviations: ACTB: actin, beta; AIF1: allograft inflammatory factor 1; ATG: autophagy related; CTSD: cathepsin D; DAPI: (4',6-diamido-2-phenylindole); DIC: differential interference contrast; EGFP: enhanced green fluorescent protein; ELM: external limiting membrane; ERG: electroretinography; GCL: ganglion cell layer; GLUL: glutamine-ammonia ligase (glutamine synthetase); INL: inner nuclear layer; IS-E/M: inner segment - ellipsoid/myoid; ISH: in situ hybridization; LAMP2: lysosomal-associated membrane protein 2; L.I.: laser Intensity; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; O.C.T.: optimal cutting temperature; OS: outer segment; ONL: outer nuclear layer; PE: phosphatidylethanolamine; RFP: red fluorescent protein; R.O.I.: region of interest; RPE: retinal pigment epithelium.

Dataset Information

A_2AR eGFP reporter mouse enables elucidation of A_2AR expression dynamics during anti-tumor immune responses.

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