Journal: Scientific Reports

Article Title: Immunoregulation induced by autologous serum collected after acute exercise in obese men: a randomized cross-over trial

doi: 10.1038/s41598-020-78750-z

Figure Lengend Snippet: CD4 + and CD8+ T cells expressing PD-1 or CTLA-4 following in vitro incubation with autologous serum from before (white bars) and after (black bars) two types of exercise. The frequencies of CD4 + CTLA-4 + ( 4A ), CD4 + PD-1 + ( 4B ) and CD8 + PD-1 + ( 4C ) T cells were analyzed by flow cytometry after in vitro PBMC stimulation with serum collected before and immediately after high-intensity interval exercise (HIIE) or exhaustive exercise. Statistical analysis conducted by two-way repeated measurements ANOVA with Bonferroni’s post hoc ( p < 0.05).

Article Snippet: Briefly, 2 × 10 5 PBMCs were stained with monoclonal antibodies (all anti-human) conjugated with specific fluorochromes: CD4 FITC, CD8 PerCP, CD25 Pe, CD39 PerCP-7, CD73 APC (all from Ebioscience, USA); CD125 (CTLA-4) Pe, CD279 (PD-1) Pe (all from Biogems, USA).

Techniques: Expressing, In Vitro, Incubation, Flow Cytometry

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