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