Journal: Science Advances

Article Title: Immune evasion mediated by PD-L1 on glioblastoma-derived extracellular vesicles

doi: 10.1126/sciadv.aar2766

Figure Lengend Snippet: ( A and B ) PD1 blockade prevents the inhibition of PD-L1 high GSC EVs on PBMCs. Percent change in CD69 expression for CD4 + (A) and CD8 + (B) T cells. PD1 blocking antibody (10 μg/ml) or isotype control (10 μg/ml) was added at day 0 ( n = 7 PBMC donors, means ± SD). ( C and D ) PD1 blockade furthermore prevents the inhibition of PD-L1 high GSC EVs on CD3 + isolated cells. CD3 + CD4 + (C) and CD3 + CD8 + (D) cells ( n = 3) after treatment. ( E ) PD-L1–carrying, palmtdT-labeled PD-L1 high GSC EVs can bind to wells coated with recombinant PD1, whereas PD1 antibody blockade inhibits EV binding. Representative confocal images are shown on the left, whereas quantification is provided on the right. Spots per field of view (FOV) on the y axis represent palmtdT-positive dots. Scale bar, 50 μm; ×500 magnification inserts; quadruplicates as means ± SD. One-way ANOVA, with post hoc Bonferroni’s correction, was used to differentiate multiple groups (**** P < 0.0001, *** P < 0.001, ** P < 0.01, and * P < 0.05).

Article Snippet: Human PD1 Fc fusion protein (2 μg/ml; BPS Bioscience) was coated on high protein-binding 96-well plates (#3590, Costar).

Techniques: Inhibition, Expressing, Blocking Assay, Isolation, Labeling, Recombinant, Binding Assay

Journal: Science Advances

Article Title: Immune evasion mediated by PD-L1 on glioblastoma-derived extracellular vesicles

doi: 10.1126/sciadv.aar2766

Figure Lengend Snippet: ( A ) Glioma GSCs up-regulate PD-L1 in vitro in response to activated PBMC supernatants. PBMCs were stimulated with anti-CD3, and supernatants were collected and co-incubated with GSCs (G44, a PD-L1 low GSC) in the presence or absence of anti–IFN-γ. PD-L1 expression was measured by flow cytometry. DMEM, Dulbecco’s modified Eagle’s medium. ( B ) IFN-γ–mediated increase of PD-L1 expression levels in PD-L1 High and PD-L1 low GSCs as shown by Western blots of four different GSCs. ( C and D ) EVs derived from IFN-γ–treated PD-L1 low GSCs inhibit anti-CD3–stimulated T cell activation, and this can be partially reversed by PD1 blockade. Inhibition potential was measured by the percentage change of CD69 + levels on anti-CD3–stimulated CD3 + CD4 + (C) or CD3 + CD8 + (D) cells, isolated from five human volunteers (means ± SD). Representative dot plots for (C) and (D) can be found in fig. S4C. ( E ) PD-L1 low EVs up-regulated indoleamine 2,3-dioxygenase (IDO) mRNA in PBMCs treated with PD-L1 low EVs. Quantitative polymerase chain reaction (qPCR) expression levels are shown ( n = 3). ( F ) PD-L1 low EVs cause interleukin-10 (IL-10) up-regulation in PBMCs. IL-10 cytokine (left) and qPCR expression levels (right) are shown ( n = 3). ( G and H ) Immunosuppressive molecules IDO and IL-10 primarily derive from the CD3-negative population. IDO (G) and IL-10 (H) mRNA levels are shown after CD3 + magnetic-activated cell sorting ( n = 3). Data sets consist of EVs from four different glioblastoma cell lines with means ± SD. One-way ANOVA, with post hoc Bonferroni’s correction, was used to differentiate multiple groups (**** P < 0.0001, *** P < 0.001, ** P < 0.01, and * P < 0.05). Student’s t test was used to differentiate between two groups, and one-way ANOVA with post hoc Bonferroni’s correction was used for multiple groups (**** P < 0.0001, *** P < 0.001, ** P < 0.01, and * P < 0.05).

Article Snippet: Human PD1 Fc fusion protein (2 μg/ml; BPS Bioscience) was coated on high protein-binding 96-well plates (#3590, Costar).

Techniques: In Vitro, Incubation, Expressing, Flow Cytometry, Modification, Western Blot, Derivative Assay, Activation Assay, Inhibition, Isolation, Real-time Polymerase Chain Reaction, FACS

Journal: Scientific Reports

Article Title: PGE2 displays immunosuppressive effects during human active tuberculosis

doi: 10.1038/s41598-021-92667-1

Figure Lengend Snippet: PGE2 regulates the expression of surface receptors on PBMC and neutrophils from HD and TB patients. ( A – E ) PBMC from HD (n = 7) and TB patients (n = 11) were stimulated with Mtb -Ag (10 µg/ml) in the presence or absence of PGE2 (2 µM) for 5 days. Afterwards, the surface expression of ( A ) SLAMF1 and ( B ) CD31 on CD3 + T cells and ( C ) CD80, ( D ) MHC-I and ( E ) MHC-II on CD14 + mononuclear phagocytes were determined by flow cytometry. Each bar represents the mean percentage of ( A – C ) CD3 + or ( D,E ) CD14 + Receptor + cells ± SEM or relative Mean Fluorescence Intensity (rMFI) ± SEM. ( F – H ) Human purified neutrophils from HD (n = 7) and TB patients (n = 6) were stimulated with Mtb -Ag (10 µg/ml) in the presence or absence of PGE2 (2 µM) for 2 h. Then, ( F ) SLAMF1, ( G ) PD-L1 and ( H ) PD-L2 expressions were evaluated by flow cytometry. Bars represent the mean values of the percentage of Receptor + neutrophils ± SEM. Statistical differences were calculated using one-way ANOVA and post hoc Dunnett’s multiple comparison test. * p < 0.05. # p < 0.05; Mann–Whitney nonparametric test for unpaired samples.

Article Snippet: To determine the expression of immune receptors on lymphocytes, mononuclear phagocytes and neutrophils, cells stimulated with Mtb -Ag (10 μg/mL) treated or not with PGE2 (2 µM) were blocked in PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na 2 HPO 4 , and 2 mM KH 2 PO 4 )-FBS 5% for 15 min and then stained for surface expression with fluorophore-marked antibodies against SLAMF1 (BD, 559572), CD31 (BD, 560984), CD80 (Biolegend, 305207), Major Histocompatibility Complex (MHC) -I (Biolegend, HLA-A2, 343306), MHC-II (Biolegend, HLA-DQ, 318106), PD-L1 (eBioscience, MIH1), PD-L2 (BD, MIH18), CD3 (Biolegend, 300306) and CD14 (Biolegend, 367116).

Techniques: Expressing, Flow Cytometry, Fluorescence, Purification, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mannose-Capped Lipoarabinomannan (LAM) from Mycobacterium tuberculosis Induces CD4 + T cell Anergy via GRAIL

doi: 10.4049/jimmunol.1500710

Figure Lengend Snippet: LAM does not affect receptor expression on LAM-anergized P25 TCR-Tg CD4+ T cells. (A) 1×106 CD4+ T cells were pre-treated with either LAM or left untreated (none) and co-cultured with Ag85B-pulsed APCs. After 48 h, cells were collected, washed and labeled with anti-PD1, anti-Lag3 or anti-Tim 3, anti-CD28, anti-CD40L mAbs. Intracellular staining was performed for CTLA4. Gating was performed on live CD4+ T cells. Alternatively, after 48 h cells were collected, washed and cultured in IL-7 for 5 d. Cells were then stained for surface expression of TCR and CD3. Gating was performed on live CD4+ T cells. Representative histograms of at least three independent experiments are shown. (B) The mean fluorescence intensity (MFI) of PD-1 staining in LAM-treated (LAM) and untreated (none) T cells was quantified after 48 h of primary stimulation. Data are means +/− SEM of three independent experiments.

Article Snippet: The following mAbs and isotype controls were purchased for analysis of receptor expression: anti-CD3-PE, anti-CD4-APC, anti-CD28-APC, anti-Tim3-PE, anti-CTLA4-PE, anti-CD25-alexa Fluor-488, anti-FoxP3-PE, anti-Annexin V-Alexa Fluor 450, anti-cholera toxin subunit B-Alexa Fluor 647, anti-CD3-Alexa Fluor 647, anti-mouse IgG-Alexa Fluor 488, and LIVE/DEAD violet and yellow cell stain (all from ebioscience); anti-PD1-PE (BD Pharmingen); anti-Lag3-APC (Biolegend); anti-TCR-Vβ-PE and anti-CD40L-PE (Miltenyi Biotech).

Techniques: Expressing, Cell Culture, Labeling, Staining, Fluorescence