Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: PD-1 , REA1165 , 50 , 130-120-382 , PE , Miltenyi Biotec.

Techniques: Imaging

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: Tropical Medicine and Infectious Disease

Article Title: PD-L2 Blockade Exacerbates Liver Lesion in Mice Infected with Capillaria hepatica through Reducing Alternatively Activated Macrophages

doi: 10.3390/tropicalmed8010046

Figure Lengend Snippet: Late stage of C. hepatica infection or egg-derived antigens induced M2 macrophages through PD-1/PD-L2 pathway. Flow cytometry was performed to measure the expression of PD-L1 and PD-L2 on F4/80 + CD86 + (M1) or F4/80 + CD206 + (M2) macrophages: ( a ) Flow cytometry gating strategy to define F4/80 + macrophage from the infected liver tissues. ( b ) PD-L1 and PD-L2 expression on the macrophages (F4/80 + ) collected from livers of mice infected with C. hepatica . ( c ) Flow cytometry gating strategy to define CD3 + CD4 + infected splenic T-cells. ( d ) PD-1 expression on the CD3 + CD4 + T-cells of spleens from mice infected with C. hepatica . ( e ) Flow cytometry gating strategy to define F4/80 + CD86 + or F4/80 + CD206 + cells from infected liver tissues or RAW246.7 cell line. ( f ) PD-L1 and PD-L2 expression on the macrophages expressing F4/80 + CD86 + (M1) collected from livers of mice infected with C. hepatica . ( g ) PD-L1 and PD-L2 expression on the macrophages expressing F4/80 + CD206 + (M2) collected from livers of mice infected with C. hepatica . ( h ) PD-L1 expression on the M1 (F4/80 + CD86 + ) and M2 (F4/80 + CD206 + ) RAW246.7 cell line co-incubated with AWE in vitro. ( i ) PD-L2 expression on the M1 (F4/80 + CD86 + ) and M2 (F4/80 + CD206 + ) RAW246.7 cell line co-incubated with EE in vitro. Data are expressed as mean ± SEM from three independent experiments ( n = 5 mice per group). * p < 0.05, ** p < 0.01.

Article Snippet: Single-cell suspensions were then blocked with anti-CD16/32 mouse Fc Block (BioLegend, San Diego, CA, USA) and analyzed for the expression of cell surface markers using combinations of the following antibodies: FITC-conjugated anti-F4/80; APC-conjugated anti-CD86 or anti-CD206; PE-conjugated anti-PD-L1; PerCP/Cyanine 5.5-conjugated anti-PD-L2; FITC-conjugated anti-CD3; APC-conjugated anti-CD8 or anti-CD4; or PE-conjugated anti-PD-1 (R&D system, Minneapolis, MN, USA).

Techniques: Infection, Derivative Assay, Flow Cytometry, Expressing, Incubation, In Vitro