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. 2017 Sep 21;8(1):647.
doi: 10.1038/s41467-017-00799-8.

PD-L1 Is an Activation-Independent Marker of Brown Adipocytes

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Free PMC article

PD-L1 Is an Activation-Independent Marker of Brown Adipocytes

Jessica R Ingram et al. Nat Commun. .
Free PMC article

Abstract

Programmed death ligand 1 (PD-L1) is expressed on a number of immune and cancer cells, where it can downregulate antitumor immune responses. Its expression has been linked to metabolic changes in these cells. Here we develop a radiolabeled camelid single-domain antibody (anti-PD-L1 VHH) to track PD-L1 expression by immuno-positron emission tomography (PET). PET-CT imaging shows a robust and specific PD-L1 signal in brown adipose tissue (BAT). We confirm expression of PD-L1 on brown adipocytes and demonstrate that signal intensity does not change in response to cold exposure or β-adrenergic activation. This is the first robust method of visualizing murine brown fat independent of its activation state.Current approaches to visualise brown adipose tissue (BAT) rely primarily on markers that reflect its metabolic activity. Here, the authors show that PD-L1 is expressed on brown adipocytes, does not change upon BAT activation, and that BAT volume in mice can be measured by PET-CT with a radiolabeled anti-PD-L1 antibody.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
PD-L1 immuno-PET reveals expression in brown adipose tissue. a, b PET-CT 3D projection images of 6-week-old C57BL/6 mice (left panel), PET-CT axial slice through the brown adipose tissue (BAT) (top right panel) with companion CT axial image (bottom right panel). a 18F-B3 image from a WT mouse. b 18F-B3 image from a PD-L1 knockout (KO). c PD-L1 and PD-1 transcript levels measured by RNAseq in WT BAT. d Magnification of 3D projection of thorax from a. Arrows show BAT deposits. fh H&E stained tissue sections 10× magnification. e, f Principal interscapular BAT deposit from WT e and PD-L1 KO f mice. g, h small parathoracic BAT deposits from WT g and PD-L1 KO h mice. i, j 18F-B3 PET-CT 3D projection image of a 6-week-old BALB/c WT mouse i and 6-month-old C57BL/6 WT mouse j. k 18F- B3 image from a WT mouse injected with subcutaneous B16 melanoma (arrow). l 18F-FDG image of 5-week-old C57BL/6 mouse. Images are all window-leveled to the same intensity. Three biological replicates were performed for each experiment. Error bars indicate standard error of mean (SEM)
Fig. 2
Fig. 2
Non-hematopoietic cells in BAT express PD-L1. a PET-CT axial slice through BAT imaged with 64Cu-B3. (Top panels) WT (left) and PD-L1 KO (right) mice. (Bottom panels) Bone marrow chimeras; WT bone marrow into PD-L1 KO mice (left), and PD-L1 KO bone marrow into WT mice (right). N = 3 per chimera. b Confocal microscopy on brown fat resected from WT mice injected with Alexa647-labeled B3 or a non-specific control VHH. c Flow cytometry using the indicated antibody on cultured pre-adipocytes isolated from WT, PD-L1 KO, and PD-1 KO BAT. d Mice were imaged by PET-CT two hours after injection with 64Cu-B3. A 3D projection image comparing the torso and head of imaged WT, PD-L1 KO, and PD-1 KO mice is shown. e Quantification of background subtracted BAT signal from C57BL/6 mice analyzed as in d. Two mice were analyzed per group. f PD-L1 transcript levels measured by RNAseq in WT and PD-1 KO BAT g QPCR for PD-L1, PD-1 and Ucp1 transcripts on in vitro differentiated brown adipocytes. X axis indicates the day of culture after the induction differentiation. Each result is representative of at least two independent experiments. Error bars indicate SEM
Fig. 3
Fig. 3
PD-L1 is expressed on brown adipocytes. a QPCR for the immune genes CD11b and CD4 in the floating fraction (FF) (left panel) and stromovascular fraction (SVF) (middle panel), and the brown fat gene UCP-1 in the FF from isolated brown fat and white fat. Y-axis shows relative expression normalized to 18S. b PD-L1 flow cytometry using αPD-L1 or B3 as indicated on CD45 + populations extracted from BAT SVF or spleens of WT mice. PD-L1 is only clearly detectable in splenic macrophages and myeloid DCs. Y axis is normalized cell counts. X axis shows MFI for each antibody or VHH as indicated in the figure legend. c Quantification of the data in b including B cell and T cell populations. d PD-1 flow cytometry on CD45 + cells from WT spleen and BAT SVF. e The lipid fraction was analyzed by flow cytometry and brown adipocytes were identified as CD45− large granular cells using the gating strategy shown on a representative sample from a WT mouse stained with isotype control antibody. The great majority of the small granular counts are from free lipid derived from ruptured adipocytes. f Flow cytometry using anti-PD-L1 or isotype control antibody on FF CD45− brown adipocytes isolated as in e compared to CD45 + cells from the SVF. g Flow cytometry on brown adipocytes as in f isolated from FF WT and PD-L1 KO mice. Each result is representative of at least two independent experiments with six pooled BAT samples per experiment
Fig. 4
Fig. 4
PD-L1 is an activation-independent marker of BAT. a 64Cu-B3 PET-CT 3D projection image of age and sex matched C57BL/6 mice held at room temperature (left panel) or held at 4C (right panel) for 90 min prior to injection of radiolabeled B3. Imaging was performed 60 min after 64Cu-B3 injection. b PET signal of BAT normalized to spleen from matched animals treated and imaged as in a. c Ucp-1 qPCR on RNA isolated from BAT excised from mice included in b. d 64Cu-B3 PET-CT 3D projection image acquired as in a of age and sex matched C57BL/6 mice treated with 10 mg/kg of the β-adrenergic agonist CL316243 given i.p. 45 min before injection of radiolabeled B3 (right panel) or left untreated (left panel, naive). e PET signal of BAT normalized to spleen and muscle from matched animals treated and imaged as in d. f Immunoblot for UCP-1 (top panel) on lystates from BAT excised from the mice included in d with quantification of normalized UCP-1 signal intensity (bottom panel) Numbers indicate biological replicates. g, h 18F-FDG g or 64Cu-B3 h PET-CT 3D projection image acquired on naive or CL316243-treated mice as in c. The mice in g, h are identical but treated and imaged 24 h apart (18F-FDG followed by 64Cu-B3) to allow for 18F radiodecay. i-k Matched animals were treated and imaged as g, h and PET signal was normalized and quanitifed. i 18F-FDG signal of BAT normalized to muscle and liver. j 64Cu-B3 signal of BAT normalized to spleen. k signal ratio of 18F-FDG to 64Cu-B3 for each mouse represented in i, j. Error bars throughout show SEM. Statistical significance was determined by a Student’s t-test. Three biological replicates were performed for each condition in each experiment

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