Journal: PLoS ONE

Article Title: Interaction of immune checkpoint PD-1 and chemokine receptor 4 (CXCR4) promotes a malignant phenotype in pancreatic cancer cells

doi: 10.1371/journal.pone.0270832

Figure Lengend Snippet: ( A ) PD1 and CXCR4 were detected in PDAC cells and PDOs by western blot. Notably, different cell and PDO lines had unique expression patterns. MOLT-4 was used as a positive control for PD-1 and CXCR4. ( B ) IF staining shows co-expression of PD-1 and CXCR4 in PDAC cells (magnification 40X). ( C ) IF staining shows co-expression of PD-1 and CXCR4 in PDAC PDOs. Consistent with western blot results, hPT1 PDOs had lower expression of CXCR4 compared to hPT4 PDOs (magnification 20X).

Article Snippet: Mouse anti-PD-1-PE antibody (20 μL per test, BD Biosciences, 557946) was used to probe for cell surface PD-1 expression.

Techniques: Western Blot, Expressing, Positive Control, Staining

Journal: PLoS ONE

Article Title: Interaction of immune checkpoint PD-1 and chemokine receptor 4 (CXCR4) promotes a malignant phenotype in pancreatic cancer cells

doi: 10.1371/journal.pone.0270832

Figure Lengend Snippet: Operative human primary PDAC specimens and corresponding PDOs for hPT26 were serially sectioned and stained with multiplex IF. Both primary PDAC tissues and PDOs showed co-expression of PD-1 and CXCR4 ( green + violet→ teal ). These regions corresponded to areas consistent with pancreatic duct cells in primary PDAC tumors and PDOs. α-SMA was used as a marker for CAFs, which are known components of the PDAC TME that express PD-1 and CXCR4 ( red + green→ yellow, red + violet→ pink, red + green + violet→ white ). In primary tumors, CAFs with PD-1 and CXCR4 expression were predictably in regions of desmoplasia characteristic of the TME. α-SMA+ CAFs were similarly noted in areas typical of the TME in PDOs, along the periphery of the 3D organoid structures. Altogether, these results show concomitant expression of PD-1 and CXCR4 in human PDAC.

Article Snippet: Mouse anti-PD-1-PE antibody (20 μL per test, BD Biosciences, 557946) was used to probe for cell surface PD-1 expression.

Techniques: Staining, Multiplex Assay, Expressing, Marker

Journal: PLoS ONE

Article Title: Interaction of immune checkpoint PD-1 and chemokine receptor 4 (CXCR4) promotes a malignant phenotype in pancreatic cancer cells

doi: 10.1371/journal.pone.0270832

Figure Lengend Snippet: Serum-starved PDAC cells were pre-treated with solvent (-) or AMD3100 (1 μM) (+) for 45 min. Cell lysates were immunoprecipitated with IgG control or anti-PD-1 antibodies. (A, B) Immunoprecipitates were immunoblotted (IB) with anti-PD-1 and anti-CXCR4 antibodies, revealing successful pull down of PD-1 and resultant co-IP of CXCR4; 5% of IB lysate (lower panel) was used as input control. Treatment with AMD3100 revealed reduced levels of PD-1-bound CXCR4 in PDAC cells. (C, D) Quantification of CXCR4 immunoprecipitated with PD-1 in MIAPaCa-2 ( B ) and PANC-1 ( D ) cells normalized to β-actin as shown in A and C, respectively. *p < 0.05, ** p < 0.01 compared with IgG. # p < 0.05, ## p < 0.01 compared with solvent control.

Article Snippet: Mouse anti-PD-1-PE antibody (20 μL per test, BD Biosciences, 557946) was used to probe for cell surface PD-1 expression.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: PLoS ONE

Article Title: Interaction of immune checkpoint PD-1 and chemokine receptor 4 (CXCR4) promotes a malignant phenotype in pancreatic cancer cells

doi: 10.1371/journal.pone.0270832

Figure Lengend Snippet: ( A, B ) Immunofluorescence analysis of MIAPaCa-2 and PANC-1 PDAC cells. Treatment with AMD3100 resulted in increased PD-1 cell surface expression compared to controls (magnification 100x). ( C, D ) Flow cytometry analysis of PDAC cells. Cells were treated with AMD3100, isotype antibody, or solvent control and then prepared for flow cytometry analysis of PD-1 membrane expression. Treatment with AMD3100 revealed increased surface expression of PD-1 compared to controls. ( E, F ) Quantification of flow cytometry analysis demonstrated 156% and 53.3% increase in cell surface expression of PD-1 after AMD3100 treatment.

Article Snippet: Mouse anti-PD-1-PE antibody (20 μL per test, BD Biosciences, 557946) was used to probe for cell surface PD-1 expression.

Techniques: Immunofluorescence, Expressing, Flow Cytometry

Journal: PLoS ONE

Article Title: Interaction of immune checkpoint PD-1 and chemokine receptor 4 (CXCR4) promotes a malignant phenotype in pancreatic cancer cells

doi: 10.1371/journal.pone.0270832

Figure Lengend Snippet: ( A ) Treatment with AMD3100 or pembrolizumab alone did not alter cell migration. However, exposure to CXCL12 promoted cell migration in both lines as expected. The addition of AMD3100 or pembrolizumab to CXCL12-treated cells resulted in inhibition of cell migration in both cell lines, demonstrating that inhibition of PD-1 or CXCR4 can block CXCL12-induced migration. All images at 10x magnification. ( B ) Quantification of transwell migration assays revealed that migration was significantly inhibited in CXCL12-treated cells when exposed to AMD3100 and pembrolizumab, demonstrating that combined CXCR4 and PD-1 inhibition abrogated CXCL12-induced migration. ***p<0.001 vs. control; ##p<0.01, ###p<0.001 vs. CXCL12.

Article Snippet: Mouse anti-PD-1-PE antibody (20 μL per test, BD Biosciences, 557946) was used to probe for cell surface PD-1 expression.

Techniques: Migration, Inhibition, Blocking Assay

Journal: PLoS ONE

Article Title: Interaction of immune checkpoint PD-1 and chemokine receptor 4 (CXCR4) promotes a malignant phenotype in pancreatic cancer cells

doi: 10.1371/journal.pone.0270832

Figure Lengend Snippet: ( A ) MIAPaCa-2 PD-1 KD was most successful in decreasing PD-1 in construct #2. ( B ) PD-1 KD cells demonstrated decreased migration even when cells were exposed to CXCL12 (100 ng/mL). ( C ) Quantification of transwell migration assays demonstrated a 107% decrease in migration in PD-1 KD cells compared to KD control. When treated with CXCL12, PD-1 KD cells had a 99% less migration than KD controls. (*** p <0.001 vs. KD control; ### p <0.001 vs. PD-1 KD; &&& p <0.001 vs. KD control + CXCL12).

Article Snippet: Mouse anti-PD-1-PE antibody (20 μL per test, BD Biosciences, 557946) was used to probe for cell surface PD-1 expression.

Techniques: Construct, Migration

Journal: Journal of Inflammation Research

Article Title: Tumor-Infiltrating PD-L1+ Neutrophils Induced by GM-CSF Suppress T Cell Function in Laryngeal Squamous Cell Carcinoma and Predict Unfavorable Prognosis

doi: 10.2147/JIR.S347777

Figure Lengend Snippet: Immune cell infiltration profiles in LSCC. ( A ) Representative polychromatic dot plots displayed CD45+ leukocytes, CD3+ T cells, CD4+ T cells, CD8+ T cells γδT cells (γδTCR+) and neutrophils (CD66b+) in peripheral blood, tumor tissues and normal tissues from LSCC patients or healthy controls. ( B ) Calculative distribution of lymphocytes, neutrophils, NLR, CD3+T cells, CD4+T cells, CD8+T cells, γδT cells, and CD4+T cells /CD8 + T cells ratio in peripheral blood, tumor tissues and normal tissues. ( C ) Representative PD-1 expression on CD4+T/CD8+T/γδT cells and PDL-1 expression on CD66b+ neutrophils from the peripheral blood, tumor tissues and normal tissues. ( D ) Statistics analysis of PD-1+ CD4+T, PD-1+ CD8+T, PD-1+ γδT cells and PDL-1+ CD66b+ neutrophils. ( E ) Association analysis of TANs, NLR, CD3+ T cells or CD8+ T cells and tumor staging. ( F ) Linear correlation between TANs and CD3+ T cells, CD4+T cells, CD8+ T cells, or γδT cells. ( G ) Linear correlation between PD-L1+ TANs and PD-1+ CD4+ T cells, PD-1+ CD8+ T cells, or PD-1+ γδT cells. Each dot represents an independent data point as determined by flow cytometry. ns = p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For flow cytometry, cell suspensions from human peripheral blood and tissue samples were stained with following antibodies cocktail at 4°C for 40 mins: Percp-Cy TM 5.5 mouse anti-human CD45 (564,105, Clone HI30, BD Pharmingen TM , San Diego, USA), BV510 mouse anti-human CD3 (564,713, Clone HIT3α, BD Pharmingen TM , San Diego, USA), APC-R700 mouse anti-human CD4 (564,975, Clone RPA-T4, BD Pharmingen TM , San Diego, USA), APC-Cy TM 7 mouse anti-human CD8 (557,834, Clone SK1, BD Pharmingen TM , San Diego, USA), PE mouse anti-human TCR γδ (555,717, Clone B1, BD Pharmingen TM , San Diego, USA), Alexa Fluor ® 647 mouse anti-human CD66b (561,645, Clone G10F5, BD Pharmingen TM , San Diego, USA), BV421 mouse anti-human PD-1 (564,323, Clone MIH4, BD Horizon TM , San Diego, USA), PE-Cy TM 7 mouse anti-human CD274 (558,017, Clone MIH1, BD Pharmingen TM , San Diego, USA).

Techniques: Expressing, Flow Cytometry

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