Journal: Immune Network

Article Title: Adenosine Blockage in Tumor Microenvironment and Improvement of Cancer Immunotherapy

doi: 10.4110/in.2019.19.e23

Figure Lengend Snippet: Clinical trials with adenosine pathway inhibitors combined with an immune check inhibitor in cancer

Article Snippet: A2AR , PFB509 , Novartis/Palobiofarma , I, Ib , NSCLC , PDR001 (anti-PD-1) , NCT02403193.

Techniques: Clinical Proteomics

Journal: bioRxiv

Article Title: MR1-restricted MAIT cells from the human lung mucosal surface have distinct phenotypic, functional, and transcriptomic features that are preserved in HIV infection

doi: 10.1101/2020.11.19.389858

Figure Lengend Snippet: Peripheral blood MAIT cells display a pro-inflammatory profile in healthy participants. (A) Gating strategy used to define cytokine producing CD3+CD4-MR1 5-OP-RU tetramer-positive cells observed in the unstimulated (top row) and stimulated (bottom row) conditions. Ex vivo staining of peripheral blood and bronchoalveolar MAIT cells for the production of (B) inducible IFN-γ ( P = 0.0156), (C) inducible IL-17 and (D) constitutive granzyme B following 6 hour stimulation with PMA/ionomycin (n = 8). Frequency of (E) PD-1 (P < 0.0001) and (F) TIM-3 ( P = 0.0417) expressing peripheral blood (red) and bronchoalveolar (blue) MAIT cells (n = 18). Bars represent medians and error bars represent interquartile ranges. Statistical difference was determined using the Mann-Whitney U test.

Article Snippet: The first included 1:100 of anti-CD3 PE-CF594 (UCHT1, BD Biosciences), 1:50 of anti-CD4 BV711 (OKT4, BioLegend), 1:200 of anti-CD8 APC-H7 (SK1, BD Biosciences), 1:50 of anti-CD26 FITC (BA5b, BioLegend), 1:50 of anti-CD161 PE-Cy7 (HP-3G10, BioLegend), and 1:50 of anti-PD-1 BV421 (EH12.1, BD Biosciences).

Techniques: Ex Vivo, Staining, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: MR1-restricted MAIT cells from the human lung mucosal surface have distinct phenotypic, functional, and transcriptomic features that are preserved in HIV infection

doi: 10.1101/2020.11.19.389858

Figure Lengend Snippet: Preservation of bronchoalveolar MAIT cell phenotype and function in HIV infection. (A) MR1 tetramer staining of CD3+CD4-cells showing depletion peripheral blood MAIT cells ( P = 0.0349) and (B) CD161 and CD26 staining of peripheral blood MAIT cells ( P = 0.0202 and 0.0104). (C) Depletion of bronchoalveolar MAIT cells ( P = 0.0471) and (D) CD161 and CD26 staining of bronchoalveolar MAIT cells ( P = 0.0176) [HIV-negative (n = 18) and HIV-positive (n = 12) participants are shown]. Intracellular cytokine staining for (E) inducible IFN-γ, (F) inducible IL-17 ( P = 0.0247) and (G) constitutive granzyme B production in HIV-negative (n = 8) and HIV-positive (n = 9) peripheral blood and bronchoalveolar MAIT cells following 6 hour stimulation with PMA/ionomycin. Frequency of (H) PD-1 and (I) TIM-3 ( P = 0.0171) expressing MR1 tetramer+ cells in the peripheral blood and bronchoalveolar lavage fluid of HIV-negative and HIV-positive individuals. Bars represent medians and error bars represent interquartile ranges. Statistical difference was determined using the Mann-Whitney U test.

Article Snippet: The first included 1:100 of anti-CD3 PE-CF594 (UCHT1, BD Biosciences), 1:50 of anti-CD4 BV711 (OKT4, BioLegend), 1:200 of anti-CD8 APC-H7 (SK1, BD Biosciences), 1:50 of anti-CD26 FITC (BA5b, BioLegend), 1:50 of anti-CD161 PE-Cy7 (HP-3G10, BioLegend), and 1:50 of anti-PD-1 BV421 (EH12.1, BD Biosciences).

Techniques: Preserving, Infection, Staining, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Rapid development of SARS-CoV-2 receptor binding domain-conjugated nanoparticle vaccine candidate

doi: 10.1101/2020.11.03.366138

Figure Lengend Snippet: (A) Germinal center B cells are marked out from the drained lymph node using B220* (CD45R), IgD-low, GL7* and CD95* as cell marker. Ratio of the positive cells are presented. (B) T follicular helper (Tfh) cells are marked out using CD4*, CD44*, PD-1* and CXCR5* as cell marker. Ratio of the positive cells are presented. (C) (D) Cytokine-secreting CD4* and CD8* T cells are marked out using CD4* and IFN-γ*/IL-2*/TNF-α* as cell markers.

Article Snippet: Immunized mice were sacrificed by CO 2 inhalation 12 days after the 2 nd boost immunization (40 days).To simultaneously identify germinal center B cells and T follicular helper cells of mice drained lymph nodes, cell suspensions of draining lymph nodes from mice sacrificed were stained with fixable viability stain 780 (BD Biosciences) and blocked with anti-CD16/32 antibody (BD Biosciences), followed by labeling with anti-B220-BV421 (BD Biosciences), anti-IgD-PE (BD Biosciences), anti-GL7-Alexa Fluor 647 (BD Biosciences), anti-CD95-FITC (BD Biosciences), anti-CD4-BV510 (BD Biosciences), anti-CD44-BV786 (BD Biosciences), anti-ICOS-PE-Cyanine7 (BD Biosciences), anti-CXCR5-PE-CF594 (BD Biosciences) and anti-PD-1-APC-R700 (BD Biosciences) in PBS in the presence of 2 % BSA.

Techniques: 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: ( 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