Journal: Oncology Letters

Article Title: Role of programmed death ligands in effective T-cell interactions in extranodal natural killer/T-cell lymphoma

doi: 10.3892/ol.2014.2356

Figure Lengend Snippet: Representative immunohistochemical streptavidin-peroxidase staining in extranodal natural killer/T-cell lymphoma (upper row) and rhinitis tissues (lower row). The positive cases of (A and D) programmed death 1, (B and E) PD-L1 and (C and F) PD-L2 (magnification, ×200). PD-L, programmed death ligand.

Article Snippet: The antigen retrieval was conducted in 0.01 mol/l citrate (pH 6.0) and the slides were incubated overnight with rabbit anti-human PD-L1 polyclonal antibody (1:120; Proteintech, Chicago, IL, USA), rabbit anti-human PD-L2 polyclonal antibody (1:150) and mouse anti-human PD-1 monoclonal antibody (mAb; 1:100) (both ZSGB-BIO, Beijing, China) and phosphate-buffered saline was used as a blank control.

Techniques: Immunohistochemical staining, Staining

Journal: Oncology Letters

Article Title: Role of programmed death ligands in effective T-cell interactions in extranodal natural killer/T-cell lymphoma

doi: 10.3892/ol.2014.2356

Figure Lengend Snippet: PD-1 expression in (A) CD4 + and (B) CD8 + T-cell subsets in 20 ENKL patients was significantly increased compared with that in 10 HVs (P<0.05). Representative PD-1 expression in (C) CD4 + and (D) CD8 + T-cell subsets in six ENKL patients was (E) downregulated with chemotherapy. (F) T-helper cell type 1 cytokine (IL-2 and IFN-γ) mean production levels in the serum of 20 ENKL patients were significantly lower than those in 10 HVs (P<0.05). PD1, programmed death 1; ENKL, extranodal natural killer/T-cell lymphoma; HVs, healthy volunteers; IL-2 interleukin 2; IFN-γ, interferon γ.

Article Snippet: The antigen retrieval was conducted in 0.01 mol/l citrate (pH 6.0) and the slides were incubated overnight with rabbit anti-human PD-L1 polyclonal antibody (1:120; Proteintech, Chicago, IL, USA), rabbit anti-human PD-L2 polyclonal antibody (1:150) and mouse anti-human PD-1 monoclonal antibody (mAb; 1:100) (both ZSGB-BIO, Beijing, China) and phosphate-buffered saline was used as a blank control.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Role of programmed death ligands in effective T-cell interactions in extranodal natural killer/T-cell lymphoma

doi: 10.3892/ol.2014.2356

Figure Lengend Snippet: (A) Purity of CD8 + T cells separated by magnetic-activated cell sorting was 99%. (B) Purity of CD8 + PD-1 + T cells was 96.2% following the stimulation of allogeneic CD8 + T cells with phytohemagglutinin for 48 h. (C) SNK-6 cells were used as the control group and, following the coculture of SNK-6 cells and CD8 + T cells for 72 h, a significant inhibitory effect of PD-L1 on allogeneic CD8 + T-helper type 1 cytokine (IL-2 and IFN-γ) secretion was observed; (A and B) P<0.05. (D) CD8 + T-cell apoptosis in groups A and B was not altered significantly compared with activated CD8 + T cells at 72 h (P>0.05). (E) SNK-6 cells were used as the control group and cells harvested at 0, 24, 48 and 72 h were analyzed by flow cytometry gating CFSE + events. The proliferation index was not significantly different among the groups (P>0.05). PD-1, programme death 1; PD-L. programmed death ligand; IL-2 interleukin 2; IFN-γ, interferon γ; CFSE, carboxy-fluorescein succinimidyl ester.

Article Snippet: The antigen retrieval was conducted in 0.01 mol/l citrate (pH 6.0) and the slides were incubated overnight with rabbit anti-human PD-L1 polyclonal antibody (1:120; Proteintech, Chicago, IL, USA), rabbit anti-human PD-L2 polyclonal antibody (1:150) and mouse anti-human PD-1 monoclonal antibody (mAb; 1:100) (both ZSGB-BIO, Beijing, China) and phosphate-buffered saline was used as a blank control.

Techniques: FACS, Flow Cytometry

Journal: Materials Today Bio

Article Title: Developing immune-regulatory materials using immobilized monosaccharides with immune-instructive properties

doi: 10.1016/j.mtbio.2020.100080

Figure Lengend Snippet: Flow cytometry data are shown as a heat map summarizing the expression profile of three surface markers (CD86, CD40, and CD274) on DCs after incubation on immobilized monosaccharide libraries in the presence of a TLR4 ligand (LPS). Unstimulated (immature) DCs were used as a negative control. All changes are expressed as a percentage of expression levels in mature (LPS-stimulated) DCs. A reduction in CD40 expression was observed in most conditions with the highest reduction in fucose-contained libraries compared to LPS alone. CD86 expression was higher in a number of conditions (particularly mixers of Gal1 and Gal2); however, these did not reach statistical significance. CD274 (PD-L1) expression showed a significant increase in Man1 and Gal2 combinations. Data are shown as mean ± SD of three independent donors where ∗ p <0.05, ∗∗ p <0.01, ∗∗∗ p <0.001, and ∗∗∗∗ p <0.0001.

Article Snippet: DCs were then incubated with labeled antibodies CD274 (APC clone REA1197), CD40 PE (clone HB14), and CD86 FITC (clone FM95) and isotype-matched mouse antibody controls (all from Miltenyi Biotec) for 20 min in the dark at 4 °C.

Techniques: Flow Cytometry, Expressing, Incubation, Negative Control

Journal: Scientific Reports

Article Title: Selective HDAC6 inhibitors improve anti-PD-1 immune checkpoint blockade therapy by decreasing the anti-inflammatory phenotype of macrophages and down-regulation of immunosuppressive proteins in tumor cells

doi: 10.1038/s41598-019-42237-3

Figure Lengend Snippet: The up-regulation of PD-L1 in anti-PD-1 treated mice is mediated by IFNγ. ( A ) C57BL/6 mice were subcutaneously injected with 1 × 10 6 SM1 murine melanoma tumor cells. Mice were treated with 15 mg/kg anti-PD-1 or a vehicle control for 21 days. Tumor nodules were isolated to evaluate the expression of IFNγ by qRT-PCR ( B ), and PD-L1, PD-L2, B7-H3, B7-H4, OX40L, and GAPDH by immunoblot ( C ). SM1 melanoma cells were treated in vitro with NextA or vehicle and then co-cultured with CD3/CD28 activated splenocytes in the presence or absence of IFNγ blocking antibody at 1:1000 and 1:100 dilutions. Then, the expression of PD-L1 was analyzed by qRT-PCR ( D ), and the expression of IFNγ by ELISA ( E ).

Article Snippet: The antibodies used for immunoblotting included: PD-L1 (ProSci, 4059), PD-L2 (ProSci, 4063), CD70 (Abcam, ab175389), B7-H3 (ThermoFisher Scientific, PA551098), B7-H4 (Abbiotec, 250473), Galectin-9 (Abcam, ab9630), ICOS-L (Abcam, ab138354), alpha-Tubulin (Cell Signaling, 3873) and acetyl-alpha Tubulin (Cell Signaling, 3971).

Techniques: Injection, Control, Isolation, Expressing, Quantitative RT-PCR, Western Blot, In Vitro, Cell Culture, Blocking Assay, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Selective HDAC6 inhibitors improve anti-PD-1 immune checkpoint blockade therapy by decreasing the anti-inflammatory phenotype of macrophages and down-regulation of immunosuppressive proteins in tumor cells

doi: 10.1038/s41598-019-42237-3

Figure Lengend Snippet: NextA improves the anti-tumor activity of anti-PD-1 immune checkpoint blockade. ( A ) C57BL/6 mice were subcutaneously injected with 1 × 10 6 SM1 murine melanoma tumor cells. Mice were treated with a vehicle control, 3 mg/kg anti-PD-1, 15 mg/kg NextA, or a combination of both agents for 25 days. ( B ) Kaplan-Meier survival plot of the previous study. ( C ) Individual group plots representation for the previous study. ( D – H ) Tumors were collected at the end point from mice treated with a vehicle control, anti-PD-1, NextA, or combination of both agents and the presence of the costimulatory markers PD-L1, PD-L2, OX40L, MHC class I, and MHC class II was evaluated by flow cytometry. ( I ) The expression of PD-L1, PD-L2 and GAPDH was evaluated by immunoblot. ( J ) Tumors were collected at the end point from mice treated with vehicle control, anti-PD-1, NextA, or a combination of both agents and the presence of M1 and M2 surface markers were evaluated by flow cytometry. M1/M2 ratios were calculated from three independent populations from M1 and M2. ( K – O ) Total RNA was isolated from the same aforementioned sample conditions and qRT-PCR was done to evaluate the expression of IFNγ, IL-2, IL-12, IL-10 and TGFβ.

Article Snippet: The antibodies used for immunoblotting included: PD-L1 (ProSci, 4059), PD-L2 (ProSci, 4063), CD70 (Abcam, ab175389), B7-H3 (ThermoFisher Scientific, PA551098), B7-H4 (Abbiotec, 250473), Galectin-9 (Abcam, ab9630), ICOS-L (Abcam, ab138354), alpha-Tubulin (Cell Signaling, 3873) and acetyl-alpha Tubulin (Cell Signaling, 3971).

Techniques: Activity Assay, Injection, Control, Flow Cytometry, Expressing, Western Blot, Isolation, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Selective HDAC6 inhibitors improve anti-PD-1 immune checkpoint blockade therapy by decreasing the anti-inflammatory phenotype of macrophages and down-regulation of immunosuppressive proteins in tumor cells

doi: 10.1038/s41598-019-42237-3

Figure Lengend Snippet: Schematic representation of the role of HDAC6 in the TME. Combination treatment of anti-PD-1 blocking antibody and selective HDAC6i. ( A ) Treatment with anti-PD-1 antibody enhances T cell function and the release of pro-inflammatory cytokines. These mediators induce the production of several immunosuppressive proteins in tumor cells, including PD-L1 and PD-L2. Additionally, anti-PD-1 treatment reduces the pro-inflammatory phenotype of macrophages. ( B ) The addition of selective HDAC6i neutralizes the negative feedback induced by IFNγ and de-activates immune inhibitory check-point signals.

Article Snippet: The antibodies used for immunoblotting included: PD-L1 (ProSci, 4059), PD-L2 (ProSci, 4063), CD70 (Abcam, ab175389), B7-H3 (ThermoFisher Scientific, PA551098), B7-H4 (Abbiotec, 250473), Galectin-9 (Abcam, ab9630), ICOS-L (Abcam, ab138354), alpha-Tubulin (Cell Signaling, 3873) and acetyl-alpha Tubulin (Cell Signaling, 3971).

Techniques: Blocking Assay, Cell Function Assay

Journal: Scientific Reports

Article Title: Selective HDAC6 inhibitors improve anti-PD-1 immune checkpoint blockade therapy by decreasing the anti-inflammatory phenotype of macrophages and down-regulation of immunosuppressive proteins in tumor cells

doi: 10.1038/s41598-019-42237-3

Figure Lengend Snippet: Primers used for qRT-PCR.

Article Snippet: The antibodies used for immunoblotting included: PD-L1 (ProSci, 4059), PD-L2 (ProSci, 4063), CD70 (Abcam, ab175389), B7-H3 (ThermoFisher Scientific, PA551098), B7-H4 (Abbiotec, 250473), Galectin-9 (Abcam, ab9630), ICOS-L (Abcam, ab138354), alpha-Tubulin (Cell Signaling, 3873) and acetyl-alpha Tubulin (Cell Signaling, 3971).

Techniques: Sequencing