human pd 1  (R&D Systems)

Journal: Cells

Article Title: Differential Response of MDA-MB-231 and MCF-7 Breast Cancer Cells to In Vitro Inhibition with CTLA-4 and PD-1 through Cancer-Immune Cells Modified Interactions

doi: 10.3390/cells10082044

Figure Lengend Snippet: Analysis of MDA-MB-231 and MCF-7 breast cancer-associated parameters. Proliferation status of CFSE-labeled MDA-MB-231/MCF-7 breast cancer cells following 24-h incubation with PBMC alone, and with the addition of CTLA-4 or PD-1 inhibitors ( a ). Cell cycle status of MDA-MB-231/MCF-7 breast cancer cells following 24-h incubation with PBMC alone, and with the addition of CTLA-4 or PD-1 inhibitors, based on the frequency of cancer cells in G1- (upper graph) and S-phase (lower graph) ( b ). Cytotoxic effects of PBMC alone, and with the addition of CTLA-4 or PD-1 inhibitors, on MDA-MB-231/MCF-7 breast cancer cells following 24-h incubation, based on the activity of LDH in supernatants ( c ) ( n = 10). Evaluation of viable cancer cells based on dead cells exclusion with 7AAD ( d ). Asterisks above individual cancer cell line columns show the level of significance with colors indicating whether the difference is reported when the specific column is compared to cancer cells alone (black) or co-cultured with PBMC (light grey) (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Moreover, brackets with significance demonstrated are applied when there was a difference in response between MDA-MB-231 and MCF-7 cells.

Article Snippet: Additionally, the experimental layout included MDA-MB-231 of MCF-7 co-cultured with PBMC in the presence of 3 µg/mL anti-CTLA-4 (clone AS32; monoclonal IgG1; company provided reactivity to the extracellular domain of the human CTLA-4) (anti-CTLA-4/-CD152, Invitrogen, Waltham, MA, USA) or 3 µg/mL anti-PD-1 (polyclonal IgG; company provided reactivity to the extracellular domain of the human PD-1) (anti-PD-1/-CD279, R&D Systems) antibodies, and wells with cancer cells alone, cancer cells with 25 ng/mL of colcemid (negative control for proliferation) (Colcemid, Biowest, Nuaille, France) and PBMC alone.

Techniques: Labeling, Incubation, Activity Assay, Cell Culture

Journal: Cells

Article Title: Differential Response of MDA-MB-231 and MCF-7 Breast Cancer Cells to In Vitro Inhibition with CTLA-4 and PD-1 through Cancer-Immune Cells Modified Interactions

doi: 10.3390/cells10082044

Figure Lengend Snippet: Evaluation of CTLA-4, PD-1, PD-L1, CD80, and CD86 immune checkpoint proteins on the studied cells. Acquired data included frequency of cells expressing selected markers and mean expression (MFI) on single cells, analyzed within MDA-MB-231/MCF-7 cancer cells ( n = 8) ( a ) and lymphocytes including CD4+, CD8+, and the total pool of lymphocytes ( n = 7) ( b ). The first two rows demonstrate significant differences between MDA-MB-231/MCF-7 or different lymphocyte subsets within each of the immune checkpoint proteins tested. Left column, rows 3–4, demonstrate the profile of immune checkpoint proteins within MCF-7 and MDA-MB-231, respectively. In the right column, rows 3–5, differences between studied checkpoint proteins are indicated for all studied lymphocyte subsets separately (statistically significant differences are indicated with asterisks) (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Additionally, the experimental layout included MDA-MB-231 of MCF-7 co-cultured with PBMC in the presence of 3 µg/mL anti-CTLA-4 (clone AS32; monoclonal IgG1; company provided reactivity to the extracellular domain of the human CTLA-4) (anti-CTLA-4/-CD152, Invitrogen, Waltham, MA, USA) or 3 µg/mL anti-PD-1 (polyclonal IgG; company provided reactivity to the extracellular domain of the human PD-1) (anti-PD-1/-CD279, R&D Systems) antibodies, and wells with cancer cells alone, cancer cells with 25 ng/mL of colcemid (negative control for proliferation) (Colcemid, Biowest, Nuaille, France) and PBMC alone.

Techniques: Expressing

Journal: Cells

Article Title: Differential Response of MDA-MB-231 and MCF-7 Breast Cancer Cells to In Vitro Inhibition with CTLA-4 and PD-1 through Cancer-Immune Cells Modified Interactions

doi: 10.3390/cells10082044

Figure Lengend Snippet: Evaluation of CTLA-4 and PD-1 levels in lymphocytes in response to co-culture with MDA-MB-231/MCF-7. Changes in frequency of CTLA-4+ and PD-1+ cells within CD4+ and CD8+ lymphocytes ( a ), expression of these markers presented as mean fluorescence intensity (MFI) ( b ), and percentage of CTLA-4+ or PD-1+ CD4+ and CD8+ cells within total pool of lymphocytes ( c ) ( n = 8). Asterisks above individual cancer cell line columns show the level of significance with colors indicating whether a difference is reported when the specific column is compared to cancer cells alone (black) or co-cultured with PBMC (light grey) (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Moreover, brackets with significance demonstrated are applied when there was a difference in response between MDA-MB-231 and MCF-7 cells.

Article Snippet: Additionally, the experimental layout included MDA-MB-231 of MCF-7 co-cultured with PBMC in the presence of 3 µg/mL anti-CTLA-4 (clone AS32; monoclonal IgG1; company provided reactivity to the extracellular domain of the human CTLA-4) (anti-CTLA-4/-CD152, Invitrogen, Waltham, MA, USA) or 3 µg/mL anti-PD-1 (polyclonal IgG; company provided reactivity to the extracellular domain of the human PD-1) (anti-PD-1/-CD279, R&D Systems) antibodies, and wells with cancer cells alone, cancer cells with 25 ng/mL of colcemid (negative control for proliferation) (Colcemid, Biowest, Nuaille, France) and PBMC alone.

Techniques: Co-Culture Assay, Expressing, Fluorescence, Cell Culture

Journal: Cells

Article Title: Differential Response of MDA-MB-231 and MCF-7 Breast Cancer Cells to In Vitro Inhibition with CTLA-4 and PD-1 through Cancer-Immune Cells Modified Interactions

doi: 10.3390/cells10082044

Figure Lengend Snippet: Granzyme B and perforin production in PBMC following co-culture with MDA-MB-231/MCF-7 and CTLA-4/PD-1 blockage. Changes in frequency of granzyme B+ and perforin+ cells were presented as the frequency within total lymphocytes ( a ), CD4+ and CD8+ lymphocytes ( b ), and percentage of granzyme B+ or perforin+ CD4+/CD8+ cells within the total pool of lymphocytes ( c ) ( n = 6). Asterisks above individual cancer cell line columns show the level of significance with colors indicating whether the difference is reported when the specific column is compared to cancer cells alone (black) or co-cultured with PBMC (light grey) (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Moreover, brackets with significance demonstrated are applied when there was a difference in response between MDA-MB-231 and MCF-7 cells.

Article Snippet: Additionally, the experimental layout included MDA-MB-231 of MCF-7 co-cultured with PBMC in the presence of 3 µg/mL anti-CTLA-4 (clone AS32; monoclonal IgG1; company provided reactivity to the extracellular domain of the human CTLA-4) (anti-CTLA-4/-CD152, Invitrogen, Waltham, MA, USA) or 3 µg/mL anti-PD-1 (polyclonal IgG; company provided reactivity to the extracellular domain of the human PD-1) (anti-PD-1/-CD279, R&D Systems) antibodies, and wells with cancer cells alone, cancer cells with 25 ng/mL of colcemid (negative control for proliferation) (Colcemid, Biowest, Nuaille, France) and PBMC alone.

Techniques: Co-Culture Assay, Cell Culture

Journal: Stem Cell Research & Therapy

Article Title: Mechanisms utilized by feline adipose-derived mesenchymal stem cells to inhibit T lymphocyte proliferation

doi: 10.1186/s13287-019-1300-3

Figure Lengend Snippet: ICAM-1, CD137L, and PDL-1 are all expressed on activated feline ASCs; however, CD137L and PDL-1 do not mediate MSC-T cell adhesion. Expression of a ICAM-1, b CD137L, and c PDL-1 ligands on activated feline ASCs. Gray histogram indicated background fluorescence of unstained samples. d Percentage of remaining fluorescence intensity from CMFDA-labeled PBMCs after removal of non-adherent cells from static adhesion assay after the addition of CD137/CD137L and PD-1/PDL-1 blocking antibodies. Data is normalized to 100% on a standard MLR condition for comparability. Fluorescent images of static adhesion assay demonstrating adherent lymphocytes to ASCs from e , h non-activated MLR, f , i stimulated MLR with ConA, and g , j stimulated MLR with ConA and addition of CD137/CD137L and PD-1/PDL-1 blocking antibodies respectively. Scale bar = 400 μm. Representative flow analysis images in a – c from 3 independent experiments. Data gathered in d from 5 independent experiments

Article Snippet: In some experiments, blocking antibodies to ICAM 1 (anti-human CD54, clone MEM-111, Thermo Fisher Scientific), LFA-1 (anti-human clone R7.1, eBioscience), CD137 (anti-mouse clone 17B5, Bio X Cell), CD137L (anti-mouse clone TKS-1, Bio X Cell), PD-1 (anti-human PD-1, polyclonal goat IgG, R&D systems), or PDL-1 (anti-human PDL-1, polyclonal goat IgG, R&D systems) were added to determine which ligands mediated PBMC-ASC adhesion.

Techniques: Expressing, Fluorescence, Labeling, Cell Adhesion Assay, Blocking Assay

Journal: Cancer immunology research

Article Title: Soluble PD-L1 as a biomarker in malignant melanoma treated with checkpoint blockade

doi: 10.1158/2326-6066.CIR-16-0329

Figure Lengend Snippet: A. Schematic diagram of identified splice variants of PD-L1. The full-length of PD-L1 consists of six exons. The transmembrane domain (TM) is located in exon 4 and marked in pink. Spliced-out regions of PD-L1-1, 3, 9, 12 are indicated with bracket symbols. Stop represents a stop codon. B. Identification of splice variants of human PD-L1. PD-L1 transcripts from A375 and M34 melanoma cell lines were generated by RT-PCR and cloned into a TA TOPO vector. Four unique PD-L1 splice variants were identified by sequencing. Three splice variants PD-L1-1, PD-L1-9, and PD-L1-12 have not been previously reported. The full-length nucleotide and amino acid sequence for membrane-bound PD-L1 is represented (top). The trans-membrane domain is shown in pink. Spliced-out regions are indicated with bracket symbols and shown in red. In each of the identified splice variants, the amino acid sequences are shown in blue. Underlines indicate additional different amino acids.

Article Snippet: The membranes were immunoblotted overnight at 4 °C with a biotinylated goat anti-human PD-L1 at 0.1 µg/ml (R&D systems), and further incubated with HRP conjugated streptavidin at 2.5 µg/ml (Jackson ImmunoResearch, West Grove, PA) at room temperature for 2 hours.

Techniques: Generated, Reverse Transcription Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Sequencing, Membrane

Journal: Cancer immunology research

Article Title: Soluble PD-L1 as a biomarker in malignant melanoma treated with checkpoint blockade

doi: 10.1158/2326-6066.CIR-16-0329

Figure Lengend Snippet: Secretion of spliced variants of PD-L1 from melanoma cell lines, suppression of T cell activation by sPD-L1, effects of BRAF inhibitor on sPD-L1 secretion in resistant melanoma cell lines, and differetial secretion of sPD-L1 variants in response to cytokine stimulations. A and B. A375 cell line was transduced with lentiviral vectors of PD-L1-1, 3, and 9 variants. sPD-L1 variants from culture medium were examined by immunoprecipitation, SDS-PAGE and immunoblotting assay. Loading samples were normalized by cell numbers. C. Effects of sPD-L1 on the proliferation of human CD4+ and CD8+ T cells. PHA-activated human CD4+ and CD8+ T cells were stimulated with anti CD3 (5µg/ml) in the absence or presence of either PD-L1-3/Fc, or PD-L1-9/Fc, or PD-L1-1/Fc (all at 10µg/ml). Proliferation of the T cells were examined by [3H]thymidine incorperation assay. Human IgG1 was used as a control. The results represent one out of two independent experiments. D. Secretion of sPD-L1 by BRAF inhibitor resistant melanoma cell lines. sPD-L1 from culture medium of either parental or PLX resistant A375 and M34 cell lines were analyzed by immunoprecipitation, SDS-PAGE and immunoblotting assay. Loading samples were normalized by cell numbers. The culture medium were from aproximately 8 × 107 cells of A375 and 1 × 107 cells of M34, respectively. PLX R respresents PLX4032 resistant. E to H. Melanoma cell lines were cultured in the absence and presence of IFNγ (200 U/ml) or IFNα (2000 U/ml), or TNFα (10 ng/ml). sPD-L1 in culture medium was analyzed by immunoprecipitation, SDS-PAGE and immunoblotting assay. Loading samples were normalized by cell numbers.

Article Snippet: The membranes were immunoblotted overnight at 4 °C with a biotinylated goat anti-human PD-L1 at 0.1 µg/ml (R&D systems), and further incubated with HRP conjugated streptavidin at 2.5 µg/ml (Jackson ImmunoResearch, West Grove, PA) at room temperature for 2 hours.

Techniques: Activation Assay, Transduction, Immunoprecipitation, SDS Page, Western Blot, Control, Cell Culture