Journal: Nature neuroscience

Article Title: Control of tumor-associated macrophages and T cells in glioblastoma via AHR and CD39

doi: 10.1038/s41593-019-0370-y

Figure Lengend Snippet: a, Left: representative immunofluorescence images of tissue array from human gliomas stained for TMEM119 (red), CD68 (green), CD39 (cyan) and nuclei (blue). Scale bars, 20 μm. Right: quantification of the total number of CD39+ CD68+ TMEM119Neg cells in 33 glioma samples classified as grade 1 (n = 2), grade 2 (n = 14), grade 3 (n = 8) or grade 4 (n = 9). Kruskal–Wallis test. b, Flow cytometry analysis of CD39 expression in peripheral infiltrated macrophages in TAMs (LinNeg CD11b+ CD45Hi) gated from WT and AHRLysM mice 17 days after GL261 cell implantation (n = 3 independent mice). Representative histogram shown on the left and quantification analysis on the right. c, AHR recruitment to the Entpd1 promoter as assessed by ChIP in KYN- or TCM-stimulated BMDMs in the presence or absence of an AHR inhibitor (n = 3 technical replicates). d, Relative expression of luciferase activity in HEK293 cells transfected with a luciferase reporter driven by the ENTPD1 promoter alone, or with a construct coding for AHR, and stimulated TCM for 24 h. RPMI condition was the reference sample (n = 3 technical replicates). e, Representative images from intracranial tumors in WT and CD39LysM mice 21 days after GL261-luciferase cell implantation (n = 4 independent mice). f, Survival curve analysis of WT and CD39LysM mice implanted intracranially with GL261-luciferase cells (n = 8 independent mice). g,h, Total number of TILs (CD3+ CD8+) (g) and percentage of IFN-γ+ cells in TILs (h) gated from WT and CD39LysM mice 15 days after GL261-luciferase cell implantation (n = 3 independent mice). i–k, Nanostring analysis of sorted CD8+ TILs from WT and CD39LysM mice 15 days after GL261-luciferase cell implantation (pool of 5 mice per group). Heatmap of gene expression for all genes (i) and of T cell dysfunction-related genes (j). k, Ratio of the expression of genes related to CD8+ T cell dysfunction in WT and CD39LysM mice. l–n, Naive CD8+ T cells purified using beads activated with anti-CD3 and anti-CD28 monoclonal antibodies in the presence or absence of adenosine for 48 h (n = 3 technical replicates for all analyses). Expression levels of T cell effector and dysfunction genes were analyzed by qPCR (l), frequency of IFN-γ+ cells in CD8+ T cells by FACS (m) and cytokines production by ELISA (n). Data in b–h and l–n are representative of two independent experiments; similar results were obtained. Data in a–e, g, h, l–n are shown as mean ± s.e.m. P values were determined by two-sided Student’s t-tests (b, e, g, h, l–n) or one-way ANOVA (a, c and d), and survival analysis was performed using a Kaplan–Meier plot using a log-rank (Mantel–Cox) test (f).

Article Snippet: The following primary antibodies were used in this study: mouse anti-L-KYN (1:100; no. IS003 clone 3D4-F2, ImmuSmol); goat anti-CD14 (1:100; no. LS-B3012–50, LifeSpan Biosciences); rabbit anti-TMEM119 (1:500; no. ab185333, Abcam); mouse anti-CD39 (1:100; no. 328202, Biolegend); goat anti-AHR (1:100; no. ab186070, Abcam); mouse anti-AHR (1:100; no. ab2769, Abcam); mouse anti-CD68 (1:100; no. ab955, Abcam); rabbit anti-CD8 (0.175 μg ml −1 ; no. 790–4460, Ventana Labs); rabbit anti-CD4 (0.5 μg ml −1 ; no. 790–4423, Ventana Labs); and rat anti-CD3 (1.2 μg ml −1 ; no. MCA1477, Bio-Rad).

Techniques: Immunofluorescence, Staining, Flow Cytometry, Expressing, Luciferase, Activity Assay, Transfection, Construct, Purification, Enzyme-linked Immunosorbent Assay