Journal: The Journal of Experimental Medicine

Article Title: Essential role of Notch signaling in effector memory CD8 + T cell–mediated airway hyperresponsiveness and inflammation

doi: 10.1084/jem.20072200

Figure Lengend Snippet: Notch receptor expression and signaling in CD8 + T EFF cells. (A) Relative gene expression of Notch family members in T EFF versus T CM cells. The expression of Notch on T EFF and T CM cells was determined by gene chip analysis. The relative gene expression difference for the hybridization signal is depicted as a ratio. #, P < 0.05 summarizing the results of three separate experiments performed in duplicate. (B) Notch signaling is activated in T EFF cells. T EFF cells were stimulated with anti-CD3 and anti-CD28 or SIINFEKL for 24 h after incubation of the cells with DMSO or GSI. Cell lysates were analyzed for the expression of Notch1 by Western blotting, and β-actin was used as a loading control. One representative of three similar experiments is shown.

Article Snippet: CD8 + T cells were positively selected using magnetic beads coated with anti-CD8 antibody (Miltenyi Biotec).

Techniques: Expressing, Gene Expression, Hybridization, Incubation, Western Blot, Control

Journal: The Journal of Experimental Medicine

Article Title: Essential role of Notch signaling in effector memory CD8 + T cell–mediated airway hyperresponsiveness and inflammation

doi: 10.1084/jem.20072200

Figure Lengend Snippet: Transfer of GSI-T EFF cells fails to enhance lung allergic responses in WT recipients. Sensitized and challenged WT mice received GSI-T EFF or DMSO-T EFF cells before secondary challenge (OVA/OVA/OVA). Control mice were those that were sensitized and challenged but received PBS at the time of secondary challenge (OVA/OVA/PBS). (A) RL values were obtained in response to increasing concentrations of inhaled MCh, as described in Materials and methods. Data represent the mean ± SEM ( n = 12 in each group). #, significant difference (P < 0.05) between DMSO-T EFF cell recipients and WT control mice or GSI-T EFF cell recipients. (B) Cellular composition of BAL fluid. #, significant difference (P < 0.05) between GSI-T EFF cell recipients and DMSO-T EFF cell recipients; ‡, significant difference (P < 0.05) between DMSO-T EFF cell recipients and WT control mice; *, significant difference (P < 0.05) between GSI-T EFF cell recipients and WT mice. (C) CD4 + and CD8 + T cells in BAL fluid. ‡, significant difference (P < 0.05) between DMSO-T EFF cell recipients and WT control mice; *, significant difference (P < 0.05) between GSI-T EFF cell recipients and WT mice. (D) Cytokine levels in BAL fluid of WT mice that received GSI-T EFF or DMSO-T EFF cells. #, significant difference (P < 0.05) between GSI-T EFF cell recipients and DMSO-T EFF cell recipients; ‡, significant difference (P < 0.05) between DMSO-T EFF cell recipients and WT control mice; *, significant difference (P < 0.05) between GSI-T EFF cell recipients and WT control mice. (E) Relative expression levels of GATA-3 and T-bet in the lung were determined by quantitative real-time PCR. Lung cells are from the same groups as described in A. They were isolated using collagenase digestion, and RNA was prepared. Results are representative of three independent experiments. The results for each group are expressed as mean ± SEM. #, significant difference (P < 0.05) between GSI-T EFF cell–transferred mice and DMSO-T EFF cell–transferred mice or WT control mice.

Article Snippet: CD8 + T cells were positively selected using magnetic beads coated with anti-CD8 antibody (Miltenyi Biotec).

Techniques: Control, Expressing, Real-time Polymerase Chain Reaction, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Essential role of Notch signaling in effector memory CD8 + T cell–mediated airway hyperresponsiveness and inflammation

doi: 10.1084/jem.20072200

Figure Lengend Snippet: Number of IFN-γ + CD3 + CD4 + or IFN-γ + CD3 + CD8 + cells in the lungs of sensitized and challenged WT mice that received DMSO-T EFF or GSI-T EFF cells

Article Snippet: CD8 + T cells were positively selected using magnetic beads coated with anti-CD8 antibody (Miltenyi Biotec).

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Essential role of Notch signaling in effector memory CD8 + T cell–mediated airway hyperresponsiveness and inflammation

doi: 10.1084/jem.20072200

Figure Lengend Snippet: Allergen-induced AHR and airway inflammation are restored in CD8 −/− mice that received DMSO-T EFF cells but not GSI-T EFF cells. Before secondary challenge, sensitized and challenged (primary) CD8 −/− mice received 5 × 10 6 GSI-T EFF or DMSO-T EFF cells via the tail vein. Control mice were sensitized and challenged and received PBS on secondary challenge. (A) AHR. #, significant difference (P < 0.05) between DMSO-T EFF cell recipients and controls or GSI-T EFF cell recipients. (B) Cell composition in BAL fluid and (C) BAL cytokine levels. The results for each group are expressed as the mean ± SEM ( n = 12). #, significant difference (P < 0.05) between GSI-T EFF cell recipients and DMSO-T EFF cell recipients; ‡, significant difference (P < 0.05) between DMSO-T EFF cell recipients and controls; *, significant difference (P < 0.05) between GSI-T EFF cell recipients and controls.

Article Snippet: CD8 + T cells were positively selected using magnetic beads coated with anti-CD8 antibody (Miltenyi Biotec).

Techniques: Control

Journal: The Journal of Experimental Medicine

Article Title: Essential role of Notch signaling in effector memory CD8 + T cell–mediated airway hyperresponsiveness and inflammation

doi: 10.1084/jem.20072200

Figure Lengend Snippet: Number of IFN-γ + CD3 + CD4 + or IFN-γ + CD3 + CD8 + cells in the lungs of sensitized and challenged CD8 −/− mice that received DMSO-T EFF or GSI-T EFF cells

Article Snippet: CD8 + T cells were positively selected using magnetic beads coated with anti-CD8 antibody (Miltenyi Biotec).

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Essential role of Notch signaling in effector memory CD8 + T cell–mediated airway hyperresponsiveness and inflammation

doi: 10.1084/jem.20072200

Figure Lengend Snippet: Real-time PCR analysis of Notch ligand expression in T EFF cells. Before and after transfer of GSI-T EFF or DMSO-T EFF cells, CD8 + T EFF cells from the lungs of CD8 −/− mice were isolated using MACS beads and RNA was prepared. The relative expression of Notch ligands (Delta1, Jagged1, and Jagged2) was determined by quantitative real-time PCR. Results are from three independent experiments. The results for each group are expressed as mean ± SEM. #, significant difference (P < 0.05) between GSI-T EFF cell recipients and DMSO-T EFF cell recipients.

Article Snippet: CD8 + T cells were positively selected using magnetic beads coated with anti-CD8 antibody (Miltenyi Biotec).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Essential role of Notch signaling in effector memory CD8 + T cell–mediated airway hyperresponsiveness and inflammation

doi: 10.1084/jem.20072200

Figure Lengend Snippet: Allergen-induced AHR and airway inflammation are prevented by administration of Delta1-Fc. Secondary challenged (OVA/OVA/OVA) or control (OVA/OVA/PBS) WT mice received Delta1-Fc or human IgG. (A) Experimental protocol, (B) AHR, (C) cell composition in BAL fluid, (D) BAL cytokine levels, and (E) number of IFN-γ + –producing CD4 + or CD8 + T cells in the lung. The results for each group are expressed as the mean ± SEM ( n = 8 in each group). #, significant difference (P < 0.05) between Delta1-Fc–treated secondary challenged mice and human IgG–treated secondary challenged mice or control mice.

Article Snippet: CD8 + T cells were positively selected using magnetic beads coated with anti-CD8 antibody (Miltenyi Biotec).

Techniques: Control

Journal: Cancer Biology & Medicine

Article Title: Neutrophils as key regulators of tumor immunity that restrict immune checkpoint blockade in liver cancer

doi: 10.20892/j.issn.2095-3941.2023.0019

Figure Lengend Snippet: Driver gene-induced mouse liver cancer has a poor response to anti-PD-1 monotherapy. (A) Schematic of the experimental workflow. (B) Survival graph of pTMK/Trp53 −/− mice treated with isotype or anti-PD-1 monoclonal antibodies. The number of mice per group is shown. Log-rank test was used to calculate P -values. (C) Representative photographs of pTMK/Trp53 −/− mice treated with isotype or anti-PD-1 antibodies, showing microscopic tumors. Rulers in the photo show a minimum unit of mm. (D) The ratio of liver weight to body weight was calculated to assess tumor burden. (E) FACS analysis of populations of neutrophils, macrophages, and CD8 + T cells in the isotype and anti-PD-1 groups. (F) H&E and IHC of CD8, Ly6G, F4/80, αSMA, and CD31 in tumors or margin regions in mice treated with isotype control or anti-PD-1 antibodies. Scale bar: 20 μm.

Article Snippet: CD8 + T cells from peripheral blood mononuclear cells were purified with anti-CD8 magnetic beads (STEMCELL) and stimulated with 25 μg/mL CD3/CD28 T cell Activator (Life Technologies) and 50 U/mL rhIL-2 (STEMCELL) for 3–5 days.

Techniques: Control

Journal: Cancer Biology & Medicine

Article Title: Neutrophils as key regulators of tumor immunity that restrict immune checkpoint blockade in liver cancer

doi: 10.20892/j.issn.2095-3941.2023.0019

Figure Lengend Snippet: Tumor associated neutrophils promote CD8 + T cell exhaustion, thereby facilitating mouse liver cancer growth. (A) Schematic of the experimental approach. (B) Representative photographs of pTMK/Trp53 −/− mice treated with isotype control or anti-Ly6G antibodies, showing microscopic tumors. Ruler in the photo shows a minimum unit of mm. (C) The tumor burden was quantified by the ratio of liver weight to body weight. (D) Schematic of the therapeutic approach. (E) Bioluminescence signals showing tumor size in 2 groups at 7 and 21 days after injection of vectors. (F) Quantification of luciferase signals at 7 and 21 days after injection of vectors. (G) FACS analysis of neutrophil populations in the isotype control and anti-Ly6G groups. (H) H&E and IHC of CD3, CD8, and Ly6G in tumors or margin regions in mice treated with isotype control or anti-Ly6G antibodies. Scale bar: 50 μm. (I) FACS analysis showing dual positive PD-1 + TIM3 + CD8 + or PD-1 + LAG3 + CD8 + T cell proportions in 2 groups. (J) Dot plot showing statistical analysis of the FACS results.

Article Snippet: CD8 + T cells from peripheral blood mononuclear cells were purified with anti-CD8 magnetic beads (STEMCELL) and stimulated with 25 μg/mL CD3/CD28 T cell Activator (Life Technologies) and 50 U/mL rhIL-2 (STEMCELL) for 3–5 days.

Techniques: Control, Injection, Luciferase

Journal: Cancer Biology & Medicine

Article Title: Neutrophils as key regulators of tumor immunity that restrict immune checkpoint blockade in liver cancer

doi: 10.20892/j.issn.2095-3941.2023.0019

Figure Lengend Snippet: pTMK/Trp53 −/− tumor-associated neutrophils suppress CD8 + T cell activity. (A) Schematic of the experimental workflow. (B) FACS analyses and dot plot showing elevated PD-L1 expression in TANs from tumor tissues from pTMK/Trp53 −/− mice. (C-D) FACS analyses and dot plot showing decreased CD25 and CD69 expression in autologous CD8 + T cells co-cultured with TANs. (E-G) FACS analyses and dot plot showing decreased expression of cytotoxic markers (IFNγ, GZMB, and PRF1) in autologous CD8 + T cells co-cultured with TANs.

Article Snippet: CD8 + T cells from peripheral blood mononuclear cells were purified with anti-CD8 magnetic beads (STEMCELL) and stimulated with 25 μg/mL CD3/CD28 T cell Activator (Life Technologies) and 50 U/mL rhIL-2 (STEMCELL) for 3–5 days.

Techniques: Activity Assay, Expressing, Cell Culture

Journal: Cancer Biology & Medicine

Article Title: Neutrophils as key regulators of tumor immunity that restrict immune checkpoint blockade in liver cancer

doi: 10.20892/j.issn.2095-3941.2023.0019

Figure Lengend Snippet: CD8 + T and NK cells are essential for the therapeutic efficacy of anti-Ly6G blockade. (A) Schematic of the experimental approach. (B) Bioluminescence signals showing tumor sizes in all treatment groups. (C) Quantification of luciferase signals with the treatment of corresponding antibodies. (D) Representative image of microscopic tumors from the indicated groups. Ruler in the photo shows a minimum unit of mm. (E) Quantification of tumor burden according to the ratio of liver weight to body weight. (F) FACS analysis showing the depletion efficiency of neutrophils, CD8 + T and NK cells with corresponding antibodies.

Article Snippet: CD8 + T cells from peripheral blood mononuclear cells were purified with anti-CD8 magnetic beads (STEMCELL) and stimulated with 25 μg/mL CD3/CD28 T cell Activator (Life Technologies) and 50 U/mL rhIL-2 (STEMCELL) for 3–5 days.

Techniques: Luciferase

Journal: Cancer Biology & Medicine

Article Title: Neutrophils as key regulators of tumor immunity that restrict immune checkpoint blockade in liver cancer

doi: 10.20892/j.issn.2095-3941.2023.0019

Figure Lengend Snippet: Tumor associated neutrophils are the key negative regulators of ICB in mice with liver cancer. (A) Schematic of the experimental workflow. (B) Representative image of microscopic tumors from mice treated with single agents alone or the indicated combinations. Ruler in the photo shows a minimum unit of mm. (C-D) Tumor burden, quantified by nodule numbers per liver and the ratio of liver weight to body weight. (E) H&E and IHC of CD8, Ly6G staining in tumor areas or margins in mice after different treatments. Scale bars: 20 μm. (F) FACS analysis, showing the CD8 + T cell populations in 4 groups. (G-H) FACS analysis, showing the expression of IFNγ in CD8 + T cells from all groups.

Article Snippet: CD8 + T cells from peripheral blood mononuclear cells were purified with anti-CD8 magnetic beads (STEMCELL) and stimulated with 25 μg/mL CD3/CD28 T cell Activator (Life Technologies) and 50 U/mL rhIL-2 (STEMCELL) for 3–5 days.

Techniques: Staining, Expressing

Journal: Cancer Biology & Medicine

Article Title: Neutrophils as key regulators of tumor immunity that restrict immune checkpoint blockade in liver cancer

doi: 10.20892/j.issn.2095-3941.2023.0019

Figure Lengend Snippet: Schematic of the mechanism through which neutrophils, as key regulators of tumor immunity, restrict immune checkpoint blockade in liver cancer. The liver tumors with ICC and CHC displayed moderate responses to anti-Ly6G treatment but not PD-1 blockade. Depletion of neutrophils increased the infiltration of CD8 + T cells and decreased the exhausted T cells in the tumor microenvironment. Moreover, a combination of anti-Ly6G with anti-PD-L1 enhanced the infiltration of cytotoxic CD8 + T cells, thus resulting in a significantly greater decrease in tumor burden. CD8 + Tef: CD8 + effector T cell; CD8 + Tex: CD8 + exhausted T cell.

Article Snippet: CD8 + T cells from peripheral blood mononuclear cells were purified with anti-CD8 magnetic beads (STEMCELL) and stimulated with 25 μg/mL CD3/CD28 T cell Activator (Life Technologies) and 50 U/mL rhIL-2 (STEMCELL) for 3–5 days.

Techniques:

Journal: Frontiers in Immunology

Article Title: PD-1-Mediated PI3K/Akt/mTOR, Caspase 9/Caspase 3 and ERK Pathways Are Involved in Regulating the Apoptosis and Proliferation of CD4 + and CD8 + T Cells During BVDV Infection in vitro

doi: 10.3389/fimmu.2020.00467

Figure Lengend Snippet: Western blot analysis of PD-1 protein expression after CP BVDV infection. Shown are the representative western blot results and densitometric analyses of five independent experiments showing the expression of PD-1 protein on CD4 + T cells (A) , CD8 + T cells (B) , and CD21 + B cells (C) . The uninfected CD4 + T cells, CD8 + T cells, and CD21 + B cells were used as the controls. N.S., not significant, *** p < 0.001, ** p < 0.01. Data are presented as mean ± SD ( n = 5 per group).

Article Snippet: The PBMC were then individually incubated with monoclonal antibodies against either bovine CD3 (ab16669, Abcam, Cambridge, UK), CD4 (MCA834GA, Bio-Rad, CA, USA), CD8 (MCA837GA, Bio-Rad, CA, USA), CD21 (MCA1424GA, Bio-Rad, CA, USA) or CD14 (MCA2678GA, Bio-Rad, CA, USA) for 30 min at 4°C followed by the addition of magnetic beads conjugated with rabbit anti-IgG, mouse anti-IgG1 or IgG2a+b (Miltenyi Biotech, Auburn, CA).

Techniques: Western Blot, Expressing, Infection

Journal: Frontiers in Immunology

Article Title: PD-1-Mediated PI3K/Akt/mTOR, Caspase 9/Caspase 3 and ERK Pathways Are Involved in Regulating the Apoptosis and Proliferation of CD4 + and CD8 + T Cells During BVDV Infection in vitro

doi: 10.3389/fimmu.2020.00467

Figure Lengend Snippet: Western blot analysis of PD-1 protein expression after NCP BVDV infection. Shown are the representative western blot results and densitometric analyses of five independent experiments showing the expression of the PD-1 protein on CD4 + T cells (A) , CD8 + T cells (B) , and CD21 + B cells (C) . The uninfected CD4 + T cells, CD8 + T cells, and CD21 + B cells were used as the controls. N.S., not significant, *** p < 0.001, * p < 0.05. Data are presented as mean ± SD ( n = 5 per group).

Article Snippet: The PBMC were then individually incubated with monoclonal antibodies against either bovine CD3 (ab16669, Abcam, Cambridge, UK), CD4 (MCA834GA, Bio-Rad, CA, USA), CD8 (MCA837GA, Bio-Rad, CA, USA), CD21 (MCA1424GA, Bio-Rad, CA, USA) or CD14 (MCA2678GA, Bio-Rad, CA, USA) for 30 min at 4°C followed by the addition of magnetic beads conjugated with rabbit anti-IgG, mouse anti-IgG1 or IgG2a+b (Miltenyi Biotech, Auburn, CA).

Techniques: Western Blot, Expressing, Infection

Journal: Frontiers in Immunology

Article Title: PD-1-Mediated PI3K/Akt/mTOR, Caspase 9/Caspase 3 and ERK Pathways Are Involved in Regulating the Apoptosis and Proliferation of CD4 + and CD8 + T Cells During BVDV Infection in vitro

doi: 10.3389/fimmu.2020.00467

Figure Lengend Snippet: Effect of the PD-1 blockade on the apoptosis of BVDV-infected CD4 + and CD8 + T cells. Shown are the results of flow cytometry analyses of apoptosis of CP BVDV-infected CD4 + T cells (A) and CD8 + T cells (B) , as well as NCP BVDV-infected CD4 + T cells (C) , and CD8 + T cells (D) . The infected CD4 + and CD8 + T cells without antibody were used as the controls. ** p < 0.01, * p < 0.05. Data are presented as mean ± SD ( n = 5 per group).

Article Snippet: The PBMC were then individually incubated with monoclonal antibodies against either bovine CD3 (ab16669, Abcam, Cambridge, UK), CD4 (MCA834GA, Bio-Rad, CA, USA), CD8 (MCA837GA, Bio-Rad, CA, USA), CD21 (MCA1424GA, Bio-Rad, CA, USA) or CD14 (MCA2678GA, Bio-Rad, CA, USA) for 30 min at 4°C followed by the addition of magnetic beads conjugated with rabbit anti-IgG, mouse anti-IgG1 or IgG2a+b (Miltenyi Biotech, Auburn, CA).

Techniques: Infection, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: PD-1-Mediated PI3K/Akt/mTOR, Caspase 9/Caspase 3 and ERK Pathways Are Involved in Regulating the Apoptosis and Proliferation of CD4 + and CD8 + T Cells During BVDV Infection in vitro

doi: 10.3389/fimmu.2020.00467

Figure Lengend Snippet: Effect of PD-1 blockade on the proliferation of BVDV-infected CD4 + and CD8 + T cells. Shown are the results of proliferation assay of CP BVDV-infected CD4 + T cells (A) and CD8 + T cells (B) as well as NCP BVDV-infected CD4 + T cells (C) and CD8 + T cells (D) . The infected CD4 + and CD8 + T cells without antibody were used as the controls. *** p < 0.001, ** p < 0.01, * p < 0.05. Data are presented as mean ± SD ( n = 5 per group).

Article Snippet: The PBMC were then individually incubated with monoclonal antibodies against either bovine CD3 (ab16669, Abcam, Cambridge, UK), CD4 (MCA834GA, Bio-Rad, CA, USA), CD8 (MCA837GA, Bio-Rad, CA, USA), CD21 (MCA1424GA, Bio-Rad, CA, USA) or CD14 (MCA2678GA, Bio-Rad, CA, USA) for 30 min at 4°C followed by the addition of magnetic beads conjugated with rabbit anti-IgG, mouse anti-IgG1 or IgG2a+b (Miltenyi Biotech, Auburn, CA).

Techniques: Infection, Proliferation Assay

Journal: Frontiers in Immunology

Article Title: PD-1-Mediated PI3K/Akt/mTOR, Caspase 9/Caspase 3 and ERK Pathways Are Involved in Regulating the Apoptosis and Proliferation of CD4 + and CD8 + T Cells During BVDV Infection in vitro

doi: 10.3389/fimmu.2020.00467

Figure Lengend Snippet: Effect of PD-1 blockade on viral replication in CD4 + and CD8 + T cells. The time course of virus replication in CP BVDV-infected CD4 + T cells (A) . The time course of virus replication in CP BVDV-infected CD8 + T cells (B) . The time course of virus replication in NCP BVDV-infected CD4 + T cells (C) . The time course of virus replication in NCP BVDV-infected CD8 + T cells (D) . The BVDV-infected cells without antibody were used as control groups. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group. Data are presented as mean ± SD ( n = 5 per group).

Article Snippet: The PBMC were then individually incubated with monoclonal antibodies against either bovine CD3 (ab16669, Abcam, Cambridge, UK), CD4 (MCA834GA, Bio-Rad, CA, USA), CD8 (MCA837GA, Bio-Rad, CA, USA), CD21 (MCA1424GA, Bio-Rad, CA, USA) or CD14 (MCA2678GA, Bio-Rad, CA, USA) for 30 min at 4°C followed by the addition of magnetic beads conjugated with rabbit anti-IgG, mouse anti-IgG1 or IgG2a+b (Miltenyi Biotech, Auburn, CA).

Techniques: Virus, Infection, Control

Journal: Frontiers in Immunology

Article Title: PD-1-Mediated PI3K/Akt/mTOR, Caspase 9/Caspase 3 and ERK Pathways Are Involved in Regulating the Apoptosis and Proliferation of CD4 + and CD8 + T Cells During BVDV Infection in vitro

doi: 10.3389/fimmu.2020.00467

Figure Lengend Snippet: CLSM analysis of virus-infected CD4 + and CD8 + T cells at 96 hpi. CP BVDV-infected CD4 + T cells (A) . CP BVDV-infected CD8 + T cells (B) . NCP BVDV-infected CD4 + T cells (C) . NCP BVDV-infected CD8 + T cells (D) . The red color denotes BVDV Npro, the green color denotes ATP1A1 and the blue color denotes DAPI.

Article Snippet: The PBMC were then individually incubated with monoclonal antibodies against either bovine CD3 (ab16669, Abcam, Cambridge, UK), CD4 (MCA834GA, Bio-Rad, CA, USA), CD8 (MCA837GA, Bio-Rad, CA, USA), CD21 (MCA1424GA, Bio-Rad, CA, USA) or CD14 (MCA2678GA, Bio-Rad, CA, USA) for 30 min at 4°C followed by the addition of magnetic beads conjugated with rabbit anti-IgG, mouse anti-IgG1 or IgG2a+b (Miltenyi Biotech, Auburn, CA).

Techniques: Virus, Infection

Journal: Frontiers in Immunology

Article Title: PD-1-Mediated PI3K/Akt/mTOR, Caspase 9/Caspase 3 and ERK Pathways Are Involved in Regulating the Apoptosis and Proliferation of CD4 + and CD8 + T Cells During BVDV Infection in vitro

doi: 10.3389/fimmu.2020.00467

Figure Lengend Snippet: Effect of PD-1 blockade on the PD-1 downstream signaling molecules in CP BVDV-infected CD4 + T cells. Shown are the results of densitometric analyses of the levels of p-PI3K (A) , p-Akt (B) , cleaved-caspase 9 (C) , cleaved-caspase 3 (D) , p-mTOR (E) , p-ERK (F) in bar graph format as well as the representative results (G) of Western blot analysis of PI3K, p-PI3K, Akt, p-Akt, cleaved-caspase 9, cleaved-caspase 3, mTOR, p-mTOR, ERK, p-ERK, and β-actin. The infected CD4 + and CD8 + T cells without antibody were used as control groups. *** p < 0.001, ** p < 0.01, * p < 0.05. Data are presented as mean ± SD ( n = 5 per group).

Article Snippet: The PBMC were then individually incubated with monoclonal antibodies against either bovine CD3 (ab16669, Abcam, Cambridge, UK), CD4 (MCA834GA, Bio-Rad, CA, USA), CD8 (MCA837GA, Bio-Rad, CA, USA), CD21 (MCA1424GA, Bio-Rad, CA, USA) or CD14 (MCA2678GA, Bio-Rad, CA, USA) for 30 min at 4°C followed by the addition of magnetic beads conjugated with rabbit anti-IgG, mouse anti-IgG1 or IgG2a+b (Miltenyi Biotech, Auburn, CA).

Techniques: Infection, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: PD-1-Mediated PI3K/Akt/mTOR, Caspase 9/Caspase 3 and ERK Pathways Are Involved in Regulating the Apoptosis and Proliferation of CD4 + and CD8 + T Cells During BVDV Infection in vitro

doi: 10.3389/fimmu.2020.00467

Figure Lengend Snippet: Effect of PD-1 blockade on downstream signaling molecules in CP BVDV-infected CD8 + T cells. Shown are the results of densitometric analyses of the levels of p-PI3K (A) , p-Akt (B) , cleaved-caspase 9 (C) , cleaved-caspase 3 (D) , p-mTOR (E) , p-ERK (F) in bar graph format as well as the representative results (G) of Western blot analysis of PI3K, p-PI3K, Akt, p-Akt, cleaved-caspase 9, cleaved-caspase 3, mTOR, p-mTOR, ERK, p-ERK, and β-actin. The infected CD4 + and CD8 + T cells without antibody were used as control groups. *** p < 0.001, ** p < 0.01. Data are presented as mean ± SD ( n = 5 per group).

Article Snippet: The PBMC were then individually incubated with monoclonal antibodies against either bovine CD3 (ab16669, Abcam, Cambridge, UK), CD4 (MCA834GA, Bio-Rad, CA, USA), CD8 (MCA837GA, Bio-Rad, CA, USA), CD21 (MCA1424GA, Bio-Rad, CA, USA) or CD14 (MCA2678GA, Bio-Rad, CA, USA) for 30 min at 4°C followed by the addition of magnetic beads conjugated with rabbit anti-IgG, mouse anti-IgG1 or IgG2a+b (Miltenyi Biotech, Auburn, CA).

Techniques: Infection, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: PD-1-Mediated PI3K/Akt/mTOR, Caspase 9/Caspase 3 and ERK Pathways Are Involved in Regulating the Apoptosis and Proliferation of CD4 + and CD8 + T Cells During BVDV Infection in vitro

doi: 10.3389/fimmu.2020.00467

Figure Lengend Snippet: Effect of PD-1 blockade on downstream signaling molecules in NCP BVDV-infected CD4 + T cells. Shown are the results of densitometric analyses of the levels of p-PI3K (A) , p-Akt (B) , cleaved-caspase 9 (C) , cleaved-caspase 3 (D) , p-mTOR (E) , p-ERK (F) in bar graph format as well as the representative results (G) of Western blot analysis of PI3K, p-PI3K, Akt, p-Akt, cleaved-caspase 9, cleaved-caspase 3, mTOR, p-mTOR, ERK, p-ERK, and β-actin. The infected CD4 + and CD8 + T cells without antibody were used as control groups. N.S., not significant, *** p < 0.001, ** p < 0.01. Data are presented as mean ± SD ( n = 5 per group).

Article Snippet: The PBMC were then individually incubated with monoclonal antibodies against either bovine CD3 (ab16669, Abcam, Cambridge, UK), CD4 (MCA834GA, Bio-Rad, CA, USA), CD8 (MCA837GA, Bio-Rad, CA, USA), CD21 (MCA1424GA, Bio-Rad, CA, USA) or CD14 (MCA2678GA, Bio-Rad, CA, USA) for 30 min at 4°C followed by the addition of magnetic beads conjugated with rabbit anti-IgG, mouse anti-IgG1 or IgG2a+b (Miltenyi Biotech, Auburn, CA).

Techniques: Infection, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: PD-1-Mediated PI3K/Akt/mTOR, Caspase 9/Caspase 3 and ERK Pathways Are Involved in Regulating the Apoptosis and Proliferation of CD4 + and CD8 + T Cells During BVDV Infection in vitro

doi: 10.3389/fimmu.2020.00467

Figure Lengend Snippet: Effect of PD-1 blockade on the PD-1 downstream signaling molecules in NCP BVDV-infected CD8 + T cells. Shown are the results of densitometric analyses of the levels of p-PI3K (A) , p-Akt (B) , cleaved-caspase 9 (C) , cleaved-caspase 3 (D) , p-mTOR (E) , p-ERK (F) in bar graph format as well as the representative results (G) of Western blot analysis of PI3K, p-PI3K, Akt, p-Akt, cleaved-caspase 9, cleaved-caspase 3, mTOR, p-mTOR, ERK, p-ERK, and β-actin. The infected CD4 + and CD8 + T cells without antibody were used as control groups. N.S., not significant, *** p < 0.001, ** p < 0.01. Data are presented as mean ± SD ( n = 5 per group).

Article Snippet: The PBMC were then individually incubated with monoclonal antibodies against either bovine CD3 (ab16669, Abcam, Cambridge, UK), CD4 (MCA834GA, Bio-Rad, CA, USA), CD8 (MCA837GA, Bio-Rad, CA, USA), CD21 (MCA1424GA, Bio-Rad, CA, USA) or CD14 (MCA2678GA, Bio-Rad, CA, USA) for 30 min at 4°C followed by the addition of magnetic beads conjugated with rabbit anti-IgG, mouse anti-IgG1 or IgG2a+b (Miltenyi Biotech, Auburn, CA).

Techniques: Infection, Western Blot, Control

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Targeting Nuclear Receptor Coactivator SRC-1 Prevents Colorectal Cancer Immune Escape by Reducing Transcription and Protein Stability of PD-L1.

doi: 10.1002/advs.202310037

Figure Lengend Snippet: Figure 2. SRC-1 promotes CRC cell immunosuppression by reducing tumor infiltration and antitumor activity of CD8+ T cells. A) Knockdown of SRC-1 increased the number of infiltrated CD3+ and CD8+ T cells by immunohistochemical analysis in CMT93 tumors (n = 5 per group). B) The mRNA levels of IFN𝛾, GZMB, and TNF𝛼were elevated in SRC-1-deficient CMT93 tumors (n = 4 per group). C) Representative FACS analysis plots and quantification of CD8+ T cells gated from CD45+ TILs in CMT93 tumors (n = 4 per group). D) Representative FACS analysis plots and quantification of the percentage

Article Snippet: Splenic OT-I cells were magnetically purified by anti-mouse CD8 microbeads (130-117-044, Miltenyi).

Techniques: Activity Assay, Knockdown, Immunohistochemical staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Targeting Nuclear Receptor Coactivator SRC-1 Prevents Colorectal Cancer Immune Escape by Reducing Transcription and Protein Stability of PD-L1.

doi: 10.1002/advs.202310037

Figure Lengend Snippet: Figure 6. Genetic depletion or pharmacological inhibition of SRC-1 enhances the antitumor effect of PD-L1 antibody. A, B) The tumor growth (A) and weight (B) of CMT93 bearing mice were attenuated in the combination group compared with SRC-1-deficient or PD-L1 antibody monotherapy at day 22 of treatment (n = 6 per group). Male C57BL/6 mice were subcutaneously inoculated with Ctrl or KOSRC-1 CMT93 cells and treated with IgG control, PD-L1 antibody (100 μg per mouse) and combination group. C) The number of tumor-infiltrating CD3+ and CD8+ T cells in CMT93 tumors with indicated treatments (n = 6 per group) as described in (A, B). D) Quantification of IFN𝛾+ cells among CD8+ T cells in CMT93 tumors treated with SRC-1 knockout and PD-L1 antibody alone or in combination (n = 5 per group). E) SRC-1 inhibitor bufalin suppressed JAK1-STAT3-IRF1 signaling and PD-L1 expression

Article Snippet: Splenic OT-I cells were magnetically purified by anti-mouse CD8 microbeads (130-117-044, Miltenyi).

Techniques: Inhibition, Control, Knock-Out, Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Targeting Nuclear Receptor Coactivator SRC-1 Prevents Colorectal Cancer Immune Escape by Reducing Transcription and Protein Stability of PD-L1.

doi: 10.1002/advs.202310037

Figure Lengend Snippet: Figure 7. SRC-1 is positively correlated with PD-L1 in human CRC specimens. A) Protein levels of SRC-1, IRF1, and PD-L1 were measured in 12 out of 36 pairs of human CRC tumor tissues and their surrounding non-tumorous tissues. The asterisk indicates SRC-1-positive CRC specimens. B) Relative protein levels of SRC-1, IRF1, and PD-L1 in human CRC samples (n = 36). Band density was quantified using ImageJ software and normalized to 𝛽-actin levels. C) The protein level of SRC-1 was positively correlated with IRF1 and PD-L1 in human CRC specimens (n = 36). D) The mRNA level of SRC-1 was positively correlated with IRF1 and PD-L1 in human CRC specimens (n = 36). E) SRC-1 was positively correlated with PD-L1, but negatively correlated with the infiltration of CD8+ T cells in CRC tissue array specimens (n = 75). F) The positive correlation was found between SRC-1 and PD-L1 expression in COAD patients. G) The JAK-STAT pathway was found to be associated with SRC-1. The GSEA plot shows significant enrichment for the JAK-STAT signaling pathway (normalized enrichment score (NES) = 2.02, and nominal p value = 1.88e-09).

Article Snippet: Splenic OT-I cells were magnetically purified by anti-mouse CD8 microbeads (130-117-044, Miltenyi).

Techniques: Software, Expressing

Journal:

Article Title: T cell ignorance in mice to Borna disease virus can be overcome by peripheral expression of the viral nucleoprotein

doi:

Figure Lengend Snippet: In vivo T cell depletion delays Borna disease in mice

Article Snippet: The cell suspension was incubated with R-phycoerythrin (R-PE)-labeled anti-CD4 and fluorescein-labeled anti-CD8 antibodies (1:200; Life Technologies, Gaithersburg, MD) for 20 min at room temperature, washed, and incubated with FACS lysing solution (Becton-Dickinson) as described by the manufacturer to eliminate red blood cells.

Techniques: In Vivo

Journal:

Article Title: T cell ignorance in mice to Borna disease virus can be overcome by peripheral expression of the viral nucleoprotein

doi:

Figure Lengend Snippet: Expression of activation markers on brain-derived CD8+ T cells. Neonatally infected MRL mice were sacrificed at the peak of neurological disease, and brain lymphocytes were isolated. Uninfected age-matched MRL mice served as donors for spleen lymphocytes. Lymphocytes were stained with anti-CD8-FITC or anti-CD4-FITC and biotinylated mAb specific for CD25 (IL-2 receptor) (A), CD62L (l-selectin) (B), or CD69 (very early activation antigen) (C) followed by streptavidin-R-PE detection. Three independent experiments were performed and dot plots are shown from one representative experiment. (Left) Analysis of spleen lymphocytes from a naive mouse. (Right) Brain-derived lymphocytes. Percentages of CD4+ and CD8+ T cells positive for the indicated marker are given in the upper quadrants.

Article Snippet: The cell suspension was incubated with R-phycoerythrin (R-PE)-labeled anti-CD4 and fluorescein-labeled anti-CD8 antibodies (1:200; Life Technologies, Gaithersburg, MD) for 20 min at room temperature, washed, and incubated with FACS lysing solution (Becton-Dickinson) as described by the manufacturer to eliminate red blood cells.

Techniques: Expressing, Activation Assay, Derivative Assay, Infection, Isolation, Staining, Marker

Journal:

Article Title: T cell ignorance in mice to Borna disease virus can be overcome by peripheral expression of the viral nucleoprotein

doi:

Figure Lengend Snippet: Ex vivo CTL activity of brain lymphocytes from BDV-infected mice is directed mainly against the viral nucleoprotein p40. Brain lymphocytes from severely diseased MRL mice (A) or VV-specific CTLs (B) were used as effectors on VV-infected L929 target cells expressing the indicated recombinant BDV proteins or irrelevant control proteins. (C) CTL assay with lymphocytes from the brain of a severely diseased CBA/J (H-2k) mouse. (D) Spleen lymphocytes of MRL mice lack BDV-specific CTLs. Effector cells from brains or spleens of either severely diseased BDV-infected or healthy mock-infected MRL mice were incubated with L929 target cells expressing BDV p40. Brain lymphocyte preparations from uninfected MRL mice usually contained less than 105 lymphocytes. They were resuspended in the same volume of medium as lymphocytes from brains of diseased animals, and identical volumes of both cell preparations were used in the CTL assays. (E) MHC restriction of CTL activity. Target cells were either MC57G cells (H-2b) or L929 cells (H-2k) infected with the indicated vaccinia viruses. Effector cells were from brains of MRL (H-2k) mice with severe Borna disease. (F) Specific depletion of CD8+ T cells from brain lymphocyte preparations abolishes CTL activity. Brain lymphocyte preparations containing 2 × 106 lymphocytes/ml were incubated for 30 min at 4°C with 107 magnetic beads coated with anti-CD4 or anti-CD8 antibodies (Dynal, Great Neck, NY) to deplete the respective T cell subset. Lymphocytes complexed to magnetic beads were removed, and the remaining cells were used as effectors in standard cytotoxicity assays. Spontaneous lysis of target cells usually was less than 20% except in F, where it was 40%. Specific lysis is plotted against the indicated effector-to-target-cell ratios.

Article Snippet: The cell suspension was incubated with R-phycoerythrin (R-PE)-labeled anti-CD4 and fluorescein-labeled anti-CD8 antibodies (1:200; Life Technologies, Gaithersburg, MD) for 20 min at room temperature, washed, and incubated with FACS lysing solution (Becton-Dickinson) as described by the manufacturer to eliminate red blood cells.

Techniques: Ex Vivo, Activity Assay, Infection, Expressing, Recombinant, CTL Assay, Incubation, Magnetic Beads, Lysis