Journal: PLoS ONE

Article Title: Therapeutic Effects of Autologous Tumor-Derived Nanovesicles on Melanoma Growth and Metastasis

doi: 10.1371/journal.pone.0033330

Figure Lengend Snippet: A. C57BL/6 mouse-derived bone marrow DCs efficiently take up DiI-labeled NVs (10 µg/ml), as assessed by FACS analysis. The numbers indicate mean fluorescence intensities. B. FACS analysis reveals that DCs upregulate the expression of maturation markers CD40, CD80, and CD86 after 24 h of incubation with NVs (2 µg/ml) and polyI:C (10 µg/ml). The numbers in the panels indicate mean fluorescence intensities. C. IL-12p70 levels in the supernatants of the cultured DCs, as assessed by ELISA. D. Splenocytes were collected from the mice immunized with NVs (10 µg) and/or polyI:C (50 µg), and the levels of secreted IL-12p70 were determined after in vitro re-stimulation with NVs. E. Serum samples were collected and analyzed by ELISA for the presence of anti-tumor IgG 2a . * P <0.05; ** P <0.01; *** P <0.001.

Article Snippet: Briefly, flat-bottom 96-well plates were coated overnight with 1 μg of NV, blocked with 1% BSA, and incubated with a 1∶10 dilution of mouse serum for the detection of anti-NV IgG 2a antibodies (Bethyl Labs, Montgomery, TX, USA).

Techniques: Derivative Assay, Labeling, Fluorescence, Expressing, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, In Vitro

Journal: Oncoimmunology

Article Title: A novel regulation of PD-1 ligands on mesenchymal stromal cells through MMP-mediated proteolytic cleavage

doi: 10.1080/2162402X.2015.1091146

Figure Lengend Snippet: Binding of PD-1-Fc fusion protein on 30 Gray-irradiated infant foreskin fibroblasts is abrogated by anti-PD-1 ligand mAbs. The binding of PD-1-Fc fusion protein (PD-1-Fc) to γ-FFs was analyzed before (i and ii) and after (from iii to viii) exposure to blocking anti-PD-L1 (clone 29E2A3) and/or anti-PD-L2 mAbs (clone 24F10C12). Representative dot plots showing isotype control (i, iii, v, and vii) and PD-1-Fc binding (ii, iv, vi, and viii) on γ-FFs non pre-incubated (A) or pre-incubated with PD-L1 (B), PD-L2 (C), and both PD-L1 and PD-L2 (D) blocking mAbs. (A–D) Representative flow histogram overlay of PE-GAH-Fc control (filled histograms) and of PD-1-Fc + PE-GAH-Fc (open histograms). Mean Fluorescence Intensity (MFI) values are indicated upper left for negative control and upper right for PD-1-Fc. (E) Frequency and (F) MFI of positive cells showing binding to PD-1-Fc fusion protein. PD-1-Fc binding was neutralized by anti-PD-L1 (open circle), anti-PD-L2 alone (open square) or combined to 50 µg/mL anti-PD-L1 (closed triangle) blocking mAbs at the indicated concentrations. Closed circle on dotted lines represent isotype-matched controls (mixed IgG 2a (50µg/mL)/IgG 2b ) and large dotted lines represent PE-conjugated goat anti-human immunoglobulin Fc portion (PE-GAH-Fc) alone. Data are representative of n = 3 independent experiments (n = 2 γ-FF donors). Bars represent the SD. ** p < 0 .01 and *** p < 0 .001.

Article Snippet: The following color- and isotype-matched control antibodies were used to confirm the staining specificities: PE-conjugated mouse IgG 1 (BD BioScience, clone X40), PE-conjugated mouse IgG 2a (BD BioScience, clone X39), APC-conjugated mouse IgG 1κ (BD PharMingen, clone MOPC-21), PerCP-Cy5.5-conjugated mouse IgG 1 (BD BioScience, clone X40).

Techniques: Binding Assay, Irradiation, Blocking Assay, Incubation, Fluorescence, Negative Control

Journal: Oncoimmunology

Article Title: A novel regulation of PD-1 ligands on mesenchymal stromal cells through MMP-mediated proteolytic cleavage

doi: 10.1080/2162402X.2015.1091146

Figure Lengend Snippet: PD-L1 and PD-L2 blockade by mAbs reverts the inhibition of T cell proliferation and the induction of T cell apoptosis triggered by 30 Gray-irradiated infant foreskin fibroblasts. (A1, A2) PHA was added to a co-culture of γ-FFs pre-treated or not with TNFα and CD3 + T cells at different ratios of 2.5 γ-FFs:100 T cells and 5 γ-FFs:100 T cells. Flow cytometry analysis of the number of the total bulk CD3 + T cells, co-labeled with anti-CD4 and anti-CD8 mAbs was performed after 7 d of co-culture in the presence or not of anti-PD-L1 (clone 29E23A3)/anti-PD-L2 (clone 24F10C12 ) mAbs or isotype control antibodies (mouse IgG 2a /IgG 2b ). Results are mean ± SD of the number of CD4 + cells and CD8 + T cells within the CD3 + T cells and are representative of three independent experiments. Bars represent the SD, ** p < 0 .01. (B1, B2) The blocking capacity of mixed anti-PD-L1 (clone 29E23A3) and anti-PD-L2 mAbs (clone 24F10C12), compared to mixed isotype controls (mouse IgG 2a /IgG 2b ) was determined by the difference of percentage of early apoptotic CD3 + T cells (DAPI − /Annexin V + T cells) in presence or in absence of γ-FFs at two ratios of 2.5 γ-FFs:100 T cells and 5 γ-FFs:100 T cells. 17 h after PHA stimulation in the conditions as indicated, T cells were co-labeled with anti-CD4-FITC mAb and anti-CD8-PerCP-Cy5.5 mAb, stained with PE-Annexin V, and then analyzed by flow cytometry. Results are representative of n = 3 independent experiments. Bars represent the SD. * p < 0 .05, ** p < 0 .01.

Article Snippet: The following color- and isotype-matched control antibodies were used to confirm the staining specificities: PE-conjugated mouse IgG 1 (BD BioScience, clone X40), PE-conjugated mouse IgG 2a (BD BioScience, clone X39), APC-conjugated mouse IgG 1κ (BD PharMingen, clone MOPC-21), PerCP-Cy5.5-conjugated mouse IgG 1 (BD BioScience, clone X40).

Techniques: Inhibition, Irradiation, Co-Culture Assay, Flow Cytometry, Labeling, Blocking Assay, Staining

Journal: Communications Biology

Article Title: The superantigens SpeC and TSST-1 specifically activate TRBV12-3/12-4 + memory T cells

doi: 10.1038/s42003-023-04420-1

Figure Lengend Snippet: a , b Frequency of CD69 + (gray filled circles), TNF-α + (red filled circles), IL-2 + (black filled circles), or IFN-γ + cells (green filled circles) among TRBV12-3/12-4 + CD4 + ( a ) or CD8 + T cells ( b ) from human PBMCs pretreated with anti-human HLA-DR or an isotype control antibody (IgG 2a ) and then stimulated for 24 h with SpeC or TSST-1. Each dot represents one donor. Data are shown as mean ± SEM. * p < 0.05, ** p < 0.01. One-way ANOVA with Tukey’s post-hoc test.

Article Snippet: In mechanistic experiments, PBMCs were preincubated for 30 min with 10 μg/mL anti-human HLA-DR (clone L243; BioLegend) or 10 μg/mL mouse IgG 2a (clone MOPC-173; BioLegend).

Techniques: Control