Journal: Gut

Article Title: Mucosa-associated invariant T cells link intestinal immunity with antibacterial immune defects in alcoholic liver disease

doi: 10.1136/gutjnl-2017-314458

Figure Lengend Snippet: Contact between healthy PBMC and faecal extracts of bacterial antigens, toxins and metabolites (FEB) from ALD patients and controls causes quantitative and functional impairments of MAIT. (A) FEB-induced MAIT cell depletion. Apoptosis rates are not different between the groups, illustrating that FEB-induced MAIT cell depletion is apoptosis independent. Control stools: n=12; ARC stools: n=20; SAH stools: n=7. The bar plot represents mean±SD. The black bars represent % of apoptotic VybrantFAM(+) MAIT cells; the white/shaded bars represent MAIT cell frequencies; both quantities are measured on the total CD8 T cell population, as described in ‘Materials and Methods’. (B) FEB-induced hyperactivated state on MAIT, CD69/HLA-DR. (C) FEB-induced immunoinhibitory checkpoint upregulation, PD1/TIM3/LAG3. FEB-induced suppression of MAIT cell antibacterial cytokine (D) and cytotoxic (E) responses; zebra bars represent Escherichia coli -stimulated results. In panels B–E, control stools: n=8; ARC stools: n=18; SAH stools: n=5. ALD, alcohol-related liver disease; ARC, alcohol-related cirrhosis; MAIT, mucosa-associated invariant T cells; PMBC, peripheral blood mononuclear cells; SAH, severe alcoholic hepatitis.

Article Snippet: One hour before adding E. coli , some cultures were (1) preblocked with polyclonal blocking antibody anti-PD1 (10 µg/mL, AF1086, BioTechne/R&D Systems, Abingdon, UK); (2) preincubated with ARC/SAH plasma (10%); (3) pretreated with increasing concentrations of ethanol (50–100–250 mmol/L); or (4) preincubated with ARC/SAH/HC faecal extracts (FEB, 100 BpC).

Techniques: Functional Assay

Journal: Frontiers in Immunology

Article Title: Sanguisorbae Radix Suppresses Colorectal Tumor Growth Through PD-1/PD-L1 Blockade and Synergistic Effect With Pembrolizumab in a Humanized PD-L1-Expressing Colorectal Cancer Mouse Model

doi: 10.3389/fimmu.2021.737076

Figure Lengend Snippet: SRE blockade of PD-1/PD-L1 interaction in coculture cell-based luciferase assay. (A, B) Cytotoxicity assay performed using Cell Counting Kit-8 (CCK) assay. The hPD-1/NFAT Jurkat T cells (A) and hPD-L1/TCR CHO-K1 cells (B) after treatment with SRE for 24 hours. (C, D) The PD-1/PD-L1 blockade bioassay was performed using the Bio-Glo™ luciferase assay system. After addition of hPD-1/NFAT Jurkat T cells and SRE (C) and anti-PD-1 antibodies (αPD-1) (D) , hPD-L1/TCR CHO-K1 cells were seeded for 20 hours. Data are presented as the mean ± SD. * p < 0.05 and *** p < 0.001 compared to the control.

Article Snippet: The biotinylated hPD-1 (#71109, BPS Bioscience) of 0.5 μg/mL was added to each well and incubated for 2 hours at RT.

Techniques: Luciferase, Cytotoxicity Assay, Cell Counting

Journal: Frontiers in Immunology

Article Title: Sanguisorbae Radix Suppresses Colorectal Tumor Growth Through PD-1/PD-L1 Blockade and Synergistic Effect With Pembrolizumab in a Humanized PD-L1-Expressing Colorectal Cancer Mouse Model

doi: 10.3389/fimmu.2021.737076

Figure Lengend Snippet: SRE-induced activation of T cells and cytotoxic effect of T cell-mediated cancer cells. (A, B) The cell viability was performed using the CCK-8 assay. Splenocytes were isolated from hPD-L1 MC38 cell-bearing hPD-1 knockin mice. Murine CRC hPD-L1 MC38 cells (A) and hPD-1 mice splenocytes (B) were treated with SRE for 72 hours. (C) Cocultured hPD-L1 MC38 cell viability tested by crystal violet staining; (D) Lactate dehydrogenase (LDH) released by damaged cells, detected via LDH cytotoxicity assay; (E) Relative interleukin-2 (IL-2) level, determined using the mouse IL-2 ELISA set. Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the control.

Article Snippet: The biotinylated hPD-1 (#71109, BPS Bioscience) of 0.5 μg/mL was added to each well and incubated for 2 hours at RT.

Techniques: Activation Assay, CCK-8 Assay, Isolation, Knock-In, Staining, LDH Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Sanguisorbae Radix Suppresses Colorectal Tumor Growth Through PD-1/PD-L1 Blockade and Synergistic Effect With Pembrolizumab in a Humanized PD-L1-Expressing Colorectal Cancer Mouse Model

doi: 10.3389/fimmu.2021.737076

Figure Lengend Snippet: SRE elevated the activation of hPD-1 + CD8 + T cells and the CD8 + T cell-mediated killing effect on hPD-L1 MC38 cancer. (A) Cocultured hPD-L1 MC38 cell viability, tested by crystal violet staining. Cocultured hPD-L1 MC38 cells detected with fluorescence microscopy (× 200) (B) and determined by fluorescent-activated cell sorting analysis (C) . (D) LDH released from damaged cells; (E) Relative perforin 1 (PRF1) level, determined with use of the mouse PRF1 ELISA kit. Data are presented as the mean ± SD. ** p < 0.01 and *** p < 0.001 compared to the vehicle group.

Article Snippet: The biotinylated hPD-1 (#71109, BPS Bioscience) of 0.5 μg/mL was added to each well and incubated for 2 hours at RT.

Techniques: Activation Assay, Staining, Fluorescence, Microscopy, FACS, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Sanguisorbae Radix Suppresses Colorectal Tumor Growth Through PD-1/PD-L1 Blockade and Synergistic Effect With Pembrolizumab in a Humanized PD-L1-Expressing Colorectal Cancer Mouse Model

doi: 10.3389/fimmu.2021.737076

Figure Lengend Snippet: Sanguisorbae Radix extract reduced tumor growth in the hPD-L1 MC38 cell allograft hPD-1 mouse model. (A) Body weight (grams); (B) Tumor volume after 18 days; (C) Tumor weight after 18 days; (D) Images of tumor tissues (bar indicates 5 mm); (E) hPD-L1 MC38 tumor-bearing mice 18 days after treatment; (F) Representative microscopic images (×400) of CD8 and PRF1-positive area of tumor tissues calculated using immunohistochemical analysis. Data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the vehicle group.

Article Snippet: The biotinylated hPD-1 (#71109, BPS Bioscience) of 0.5 μg/mL was added to each well and incubated for 2 hours at RT.

Techniques: Immunohistochemical staining, Standard Deviation

Journal: bioRxiv

Article Title: The natural stilbenoid (–)-hopeaphenol inhibits cellular entry of SARS-CoV-2 USA-WA1/2020, B.1.1.7 and B.1.351 variants

doi: 10.1101/2021.04.29.442010

Figure Lengend Snippet: Identification of stilbenoids as Spike-ACE2 inhibitors by AlphaScreen. A, Demonstration of AlphaScreen-based fluorescence due to interactions of His-tagged Spike RBD (from USA-WA1/2020) and Fc-tagged ACE2 peptides. B, Dose-response curves of stilbenoids and resveratrol on fluorescence inhibition due to disruption of RBD/ACE2 interactions. C, Demonstration of AlphaScreen-based fluorescence due to interactions of His-tagged PD-1 and Fc-tagged PD-L1 peptides. D, Dose-response curves of stilbenoids and control inhibitor BMS-1166 on fluorescence inhibition due to disruption of PD-1/PD-L1 interactions.

Article Snippet: 0.5 nM of human PD-L1-Fc (Sino Biological) was incubated with 5 nM HIS-tagged human PD-1 (Sino Biological) in the presence of 5 μg/mL protein A AlphaScreen acceptor bead and 5 μg/mL nickel chelate donor bead in a total volume of 10 μL of 20 mM HEPES (pH 7.4), 150 mM NaCl, and 0.005% Tween in white, opaque low-volume 384-well plates.

Techniques: Amplified Luminescent Proximity Homogenous Assay, Fluorescence, Inhibition

Journal: Cell Reports Medicine

Article Title: Discovery of galectin-8 as an LILRB4 ligand driving M-MDSCs defines a class of antibodies to fight solid tumors

doi: 10.1016/j.xcrm.2023.101374

Figure Lengend Snippet:

Article Snippet: The plate was then washed with PBST (PBS, 0.05% Tween 20) (MA0015, Meilunbio) (T8220, Solarbio) and blocked with 3% BSA (97061-420, VWR) at 37°C for 90 min. After repeated washing, hFc-tagged recombinant proteins, including CD3ε (10977-H02H; Sino Biological), CTLA-4 (CT4-H5255; Acro Biosystems), CD28 (CD8-H525a; Acro Biosystems), CD96 (TAE-H5252; Acro Biosystems), LAG-3 (LA3-H5255; Acro Biosystems), TIM-3 (TM3-H5258; Acro Biosystems), CD40 (CD0-5253; Acro Biosystems), ICOS (ICS-H5258; Acro biosystems), OX40 (OX0-H5255; Acro Biosystems), TIGHT (TIT-H5254; Acro Biosystems), LY86 (10242-H02H; Sino Biological), LILRB4 (16742-H02H; Sino Biological), CD27 (CD7-H5254; Acro biosystems), PD-1 (10377-H02H; Sino Biological), and CD8b (11031-HCCH; Sino Biological), were added into each well.

Techniques: Recombinant, Derivative Assay, Microarray, Enzyme-linked Immunosorbent Assay, Lysis, Blocking Assay, Staining, Transfection, Marker, Flow Cytometry, shRNA, Plasmid Preparation, Negative Control, Silver Staining, Bicinchoninic Acid Protein Assay, Selection, Extraction, Labeling, RNA Expression, Cell Culture, Software

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Increased antitumor activities of glypican-3-specific chimeric antigen receptor-modified T cells by coexpression of a soluble PD1–CH3 fusion protein

doi: 10.1007/s00262-018-2221-1

Figure Lengend Snippet: In vitro behavior of CAR-T cells after repetitive stimulation with SK-HEP-1-GPC3 cells. a CAR expression on the T-cell surface after three rounds of antigen-specific stimulation with SK-HEP-1-GPC3 cells. b Each data set is pooled from four independent experiments with individual donors (n = 4, mean ± SEM; ns not significant, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001); c The ratio of CD8+ versus CD4+ T cells after three rounds of antigen-specific stimulation (n = 3, mean ± SEM; ns not significant, P > 0.05, *P < 0.05, **P < 0.01); d Proliferation of both CAR-T cells after three rounds of antigen-specific stimulation. The two groups of CAR-T cells were prestained with CFSE before the third stimulation, and then the stained CAR-T cells were cocultured with SK-HEP-1-GPC3 at a 1:1 effector-to-target ratio for 96 h. The intensity of CFSE in each group was measured by flow cytometry. e After three rounds of antigen-specific stimulation with SK-HEP-1-GPC3 cells, the expression of exhaustion biomarkers on the T-cell surface, including PD-1, TIM-3 and LAG-3, was determined using flow cytometry with the indicated antibodies; f Each data set is pooled from three independent experiments with individual donors (n = 3, mean ± SEM; ns not significant, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001); g Cytokine release of the engineered T cells after three rounds of antigen-specific stimulation with SK-HEP-1-GPC3 cells. A total of 1 × 106 engineered T cells were cocultured with 1 × 106 tumor cells for 24 h. The levels of IL-2, IFN-γ and TNF-α in the supernatants were evaluated by ELISA (n = 6, mean ± SEM; ns not significant, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001)

Article Snippet: The level of sPD1 secreted into the culture medium of the transduced T cells was detected using human PD1/PDCD1/CD279 ELISA kits (Sino Biological) according to the manufacturer’s instructions.

Techniques: In Vitro, Expressing, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay