Journal: Cancer immunology research

Article Title: KIR3DL3 is an inhibitory receptor for HHLA2 that mediates an alternative immunoinhibitory pathway to PD1

doi: 10.1158/2326-6066.CIR-20-0315

Figure Lengend Snippet: (A) T cells were purified from whole blood of 4 normal donors and activated with CD3/CD28 antibody tetramers and flow cytometric analysis was performed in duplicate at the indicated days to assess KIR3DL3 expression in gated CD3+CD4+ and CD3+CD8+ T cells. Plots show all values measured with the center of the whiskers plot representing the median. Representative plots showing KIR3DL3 expression at (B) day 0 (unactivated) and (C) day 21 post activation. N=2 experiments with similar results. (D) KIR3DL3 expression on NK-92 MI (left panel) but minimally on NK-92 cells (right panel). N>3 replicates.

Article Snippet: The CHOK1 cells expressing anti-CD3 scFV (BPS Bioscience 60539), Jurkat cells expressing the NFAT reporter (Luc) (BPS Bioscience 60621) and Jurkat cells expressing IL2 reporter (Luc) (BPS Bioscience 60481) were purchased from BPS Bioscience (BPS Bioscience, San Diego, CA, USA).

Techniques: Purification, Expressing, Activation Assay

Journal: Cancer immunology research

Article Title: KIR3DL3 is an inhibitory receptor for HHLA2 that mediates an alternative immunoinhibitory pathway to PD1

doi: 10.1158/2326-6066.CIR-20-0315

Figure Lengend Snippet: (A) Jurkat IL2-reporter T cells expressing KIR3DL3 were co-cultured with CHO cells expressing anti-CD3 scFV, CHO cells coexpressing anti-CD3 scFV and HHLA2, or untransfected CHO cells in the presence or absence of CD28 mAb as indicated. Luciferase activity represented as relative light units (RLU). (B, C) Jurkat IL2-reporter T cells expressing KIR3DL3 were co-cultured with CHO cells coexpressing anti-CD3 scFV and HHLA2 in the presence of CD28 mAb and (B) HHLA2 mAbs or (C) KIR3DL3 mAbs. Fold activation of IL2 reporter luciferase activity is presented as mean ± S.D. (n≥3; **** P≤0.0001).

Article Snippet: The CHOK1 cells expressing anti-CD3 scFV (BPS Bioscience 60539), Jurkat cells expressing the NFAT reporter (Luc) (BPS Bioscience 60621) and Jurkat cells expressing IL2 reporter (Luc) (BPS Bioscience 60481) were purchased from BPS Bioscience (BPS Bioscience, San Diego, CA, USA).

Techniques: Expressing, Cell Culture, Luciferase, Activity Assay, Activation Assay

Journal: Frontiers in Immunology

Article Title: A Small Molecule Antagonist of PD-1/PD-L1 Interactions Acts as an Immune Checkpoint Inhibitor for NSCLC and Melanoma Immunotherapy

doi: 10.3389/fimmu.2021.654463

Figure Lengend Snippet: PDI-1 restores T cell cytotoxicity and cytokine production. (A, B) Cytotoxicity assay of activated CD3+ T cells cultured for 18 h at an effector to target ratio of 5 to 1 with CFSE-labeled H1975, A549 (A) A375, or SK-MEL-2 (B) cells alone or in the presence of either PDI-1 (4 μM), nivolumab (1 μg/ml), or an isotype control Ab. At the end of the incubation, cells were labeled with PI and dead or dying tumor cells (CFSE+ PI+) were quantified by flow cytometry. (C, D) Cytokine production by activated CD3+ T cells incubated for 18 h, as described for (A, B) . Culture supernatants were collected and levels of IFN-γ, TNF-α, perforin, and granzyme B were measured by ELISA assay. (E) Activated CD3+ T cells were cultured for 18 h with hPD-L1-TCR-HEK293T cells or TCR-HEK293 T cells in the presence or absence of PDI-1 (4 μM) or nivolumab (1 μg/ml). Dead/dying HEK293 cells were quantified by flow cytometry as described for (A, B) . Data represent the mean ± SEM of three replicates. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant.

Article Snippet: The next day, transfect 1 μl TCR activator and hPD-L1 (Cat#79455, BPS Bioscience) or the only TCR activator (Cat#79455, Cat#79455) into cells following the manufacturer’s protocol.

Techniques: Cytotoxicity Assay, Cell Culture, Labeling, Incubation, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: A Small Molecule Antagonist of PD-1/PD-L1 Interactions Acts as an Immune Checkpoint Inhibitor for NSCLC and Melanoma Immunotherapy

doi: 10.3389/fimmu.2021.654463

Figure Lengend Snippet: PDI-1 reverses inhibition of NFAT activity by PD-1/PD-L1. (A) Schematic of the principle underlying the luciferase reporter assay, in which luciferase expression in Jurkat cells was driven by the NFAT promoter. (B, C) hPD-1-NFAT-Jurkat cells were cultured alone or with hPD-L1-TCR-HEK293T cells (B) or TCR-HEK293T cells (C) overnight. PDI-1 or nivolumab was then added to the cells and luciferase activity was measured after 6 h. (D) hPD-1-NFAT-Jurkat cells were cultured with PDI-1 alone for 6 h and luciferase activity was measured. Data represent the mean ± SEM of three replicates. * p < 0.05; ** p < 0.01; ns, not significant.

Article Snippet: The next day, transfect 1 μl TCR activator and hPD-L1 (Cat#79455, BPS Bioscience) or the only TCR activator (Cat#79455, Cat#79455) into cells following the manufacturer’s protocol.

Techniques: Inhibition, Activity Assay, Luciferase, Reporter Assay, Expressing, Cell Culture

Journal: Nature Communications

Article Title: Antisense oligonucleotide modulation of non-productive alternative splicing upregulates gene expression

doi: 10.1038/s41467-020-17093-9

Figure Lengend Snippet: Candidate genes for TANGO proof of concept.

Article Snippet: PD-L1 antibody was validated via overexpression using cDNA from the PD-L1/TCR Activator Mammalian Expression Kit purchased from BPS Bioscience (Cat# 60610). cDNA and TCR activator were specially requested not to be mixed.

Techniques:

Journal: Nature Communications

Article Title: Antisense oligonucleotide modulation of non-productive alternative splicing upregulates gene expression

doi: 10.1038/s41467-020-17093-9

Figure Lengend Snippet: a RT-PCR (left panel) and qPCR (right panel) of a selected PCCA ASO (ASO-29) targeting the exon inclusion event transfected in HEK293 cells at increasing concentrations for 24 h. Exact p -values for bars with asterisks are 0.0008 and 0.0001, respectively. b RT-PCR (left panel) and qPCR (right panel) of a selected SYNGAP1 ASO (ASO-71) targeting the alternative 3′ss transfected in HEK293 cells at increasing concentrations for 24 h. Exact p -values for bars with asterisks are 0.0001 and 0.0003, 6.99e-5 and 4.25e-5, respectively. c RT-PCR (left panel) and qPCR (right panel) of a selected CD274 ASO (ASO-125) targeting the alternative intron transfected in Huh7 cells at increasing concentrations for 24 h. Cell were treated with 50 μg/mL of CHX for 3 h prior to harvesting to visualize and quantify the non-productive mRNA. Exact p -values for bars with asterisks are 6.38e-6 and 9.21e-5, respectively. d RT-PCR (left panel) and qPCR (right panel) of two selected SCN1A ASOs (ASO-135 and ASO-136) targeting the exon inclusion event delivered by free uptake into ReNCell VM cells at increasing concentrations for 72 h. RT-PCR results (bar graphs on the left) show dose-dependent reductions of the non-productive mRNA and qPCR results (bar graphs on the right) show dose-dependent increases of productive mRNA. Exact p-values for bars with asterisks are 1.69e-10, 2.50e-11, and 1.49e-11; 2.91e-8, 2.05e-10, and 2.59e-11; 4.21e-7, 1.10e-8, and 1.35e-10; and 7.26e-5, 4.01e-8, and 9.08e-12, respectively. No-ASO (−), scramble (SC), and mismatch (MM) controls were included in each experiment at the same increasing concentrations as the respective targeting ASOs. All experiments were performed in three biological replicates. Data are presented as mean values ±SD. P -values were calculated using two-sided t -test. Asterisks denote ASOs that are statistically significant ( p < 0.001) in both RT-PCR and qPCR analyses. Red and gray rectangles denote NMD-inducing event and protein-coding exons, respectively. Source data are provided as a Source Data .

Article Snippet: PD-L1 antibody was validated via overexpression using cDNA from the PD-L1/TCR Activator Mammalian Expression Kit purchased from BPS Bioscience (Cat# 60610). cDNA and TCR activator were specially requested not to be mixed.

Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection

Journal: Nature Communications

Article Title: Antisense oligonucleotide modulation of non-productive alternative splicing upregulates gene expression

doi: 10.1038/s41467-020-17093-9

Figure Lengend Snippet: a Bar graph plotting the quantification of PCCA western blot analysis from HEK293 cells treated with hit ASO at increasing concentrations for 48 h. Equal protein loading was confirmed with Ponceau staining (Supplementary Fig. ). b Same for SynGAP ( p = 4.62e-6). c Bar graph plotting flow cytometry derived fold change of the Mean Fluorescent Intensity of PD-L1 from Huh7 cells treated with the hit ASO at increasing concentrations for 120 h. No-ASO (−), scramble (SC), and mismatch (MM) controls were included in each experiment at the same increasing concentrations as the respective targeting ASOs ( p = 7.70e-5). All experiments were performed in three biological replicates. Data are presented as mean values ±SD. P -values were calculated based on two-sided t -test. Asterisks indicate p < 0.001. Source data are provided as a Source Data .

Article Snippet: PD-L1 antibody was validated via overexpression using cDNA from the PD-L1/TCR Activator Mammalian Expression Kit purchased from BPS Bioscience (Cat# 60610). cDNA and TCR activator were specially requested not to be mixed.

Techniques: Western Blot, Staining, Flow Cytometry, Derivative Assay

Journal: Genetic Epidemiology

Article Title: A Method for Gene-Based Pathway Analysis Using Genomewide Association Study Summary Statistics Reveals Nine New Type 1 Diabetes Associations

doi: 10.1002/gepi.21853

Figure Lengend Snippet: Pathways with FDR P -values of FM-(MIN) S method less than 0.05

Article Snippet: Four of the enriched pathways of Table , the BioCarta pathways “Adhesion molecules on Lymphocyte,” “Antigen dependent B cell activation,” “B lymphocyte cell surface molecules,” and “Lck and Fyn tyrosine kinases in initiation of TCR activation” were previously identified by Peng et al. as enriched with T1D by analysing only the WTCCC GWAS dataset.

Techniques: Activation Assay, Expressing, Gene Expression

Journal: PLoS Computational Biology

Article Title: Consensus-Phenotype Integration of Transcriptomic and Metabolomic Data Implies a Role for Metabolism in the Chemosensitivity of Tumour Cells

doi: 10.1371/journal.pcbi.1001113

Figure Lengend Snippet: Pathways significant by over representation analysis with respect to platinum drug sensitivity.

Article Snippet: lck and fyn tyrosine kinases in initiation of tcr activation BioCarta , 18 , 0 , 3 , 3 , N/A.

Techniques: Activation Assay, Binding Assay