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: Medicine

Article Title: Immune checkpoint proteins PD-1 and TIM-3 are both highly expressed in liver tissues and correlate with their gene polymorphisms in patients with HBV-related hepatocellular carcinoma

doi: 10.1097/MD.0000000000005749

Figure Lengend Snippet: PD-1 expression in liver tissues (×200). (A) Tumor tissues of HCC; (B) tumor adjacent tissues of HCC; (C) liver tissues of cirrhosis; (D) IHC score of PD-1 expression in (a) tumor tissues of HCC, (b) tumor adjacent tissues of HCC, and (c) liver tissues of cirrhosis. Black arrows indicate examples of positive staining cells. HCC = hepatocellular carcinoma, IHC = immunohistochemistry, PD-1 = programmed death-1.

Article Snippet: The sections were incubated with primary antibodies mouse monoclonal anti-PD-1 antibody (Abcam, Cambridge, MA) at a dilution of 1:50 or rabbit polyclonal anti-TIM-3 antibody (BioVision, Milpitas, CA) at a dilution of 1:150 at 37 °C for 30 minutes.

Techniques: Expressing, Staining, Immunohistochemistry

Journal: Medicine

Article Title: Immune checkpoint proteins PD-1 and TIM-3 are both highly expressed in liver tissues and correlate with their gene polymorphisms in patients with HBV-related hepatocellular carcinoma

doi: 10.1097/MD.0000000000005749

Figure Lengend Snippet: PD-1 expression in tumor tissues of HBV-related HCC (n = 171), tumor adjacent tissues of HCC (AT, n = 171), and cirrhotic liver tissues (LC, n = 34) according to the genotypes of PD1 polymorphism. AA, AG, and GG = genotype AA, genotype AG, and genotype GG of PD1 +8669 G/A (rs10204525) polymorphism, HBV = hepatitis B virus, HCC = hepatocellular carcinoma, IHC = immunohistochemistry, AT = adjacent tissues of HCC, LC = liver cirrhosis, PD-1 = programmed death-1.

Article Snippet: The sections were incubated with primary antibodies mouse monoclonal anti-PD-1 antibody (Abcam, Cambridge, MA) at a dilution of 1:50 or rabbit polyclonal anti-TIM-3 antibody (BioVision, Milpitas, CA) at a dilution of 1:150 at 37 °C for 30 minutes.

Techniques: Expressing, Immunohistochemistry

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: 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