Journal: Cell Genomics

Article Title: Proteomics of immune cells from liver tumors reveals immunotherapy targets

doi: 10.1016/j.xgen.2023.100331

Figure Lengend Snippet:

Article Snippet: Then, cells were stained with Zombie UV viability dye (Biolegend) to exclude dead cells and then surface markers were stained with the following antibodies: anti-CD8α Super Bright 702 (1/200, ThermoFisher), anti-CD45.1 Super Bright 436 (1/100, ThermoFisher), anti-CD45.2 PerCP-Cy5.5 (1/100, ThermoFisher), anti-PD1 PE (1/200, ThermoFisher), anti-Tim3 PE-Cy7 (1/200, ThermoFisher) and anti-LAG3 FITC (1/200, Thermofisher).

Techniques: Recombinant, Sequencing, Modification, Transfection, DNA Extraction, Enzyme-linked Immunosorbent Assay, Cell Isolation, Gene Expression, Mouse Assay, Software

Journal: Frontiers in Immunology

Article Title: Peripheral PD-1 + T Cells Co-expressing Inhibitory Receptors Predict SVR With Ultra Short Duration DAA Therapy in HCV Infection

doi: 10.3389/fimmu.2019.01470

Figure Lengend Snippet: Expression of inhibitory receptors on global T lymphocytes in DAA treated SVR and Relapse groups. (A) Gating strategy for identifying T cells expressing PD-1 and other inhibitory receptors using appropriate FMO controls. For B-E, left panels in each figure show change in indicated subset's frequency relative to baseline (0) after treatment initiation (week 4 and 16) in relapse and SVR groups; right panels show grouped averages in SVR, relapse (Rlps) and healthy Controls. (B) Baseline (D0) PD-1+CD160+ and PD-1+Tim-3+ CD8+ T cells, (C) D0 PD-1+CD160+, PD-1+Blimp-1+ CD4+ T cells, (D) Week 4 (W4 or EOT) PD-1hi, PD-1+2B4+ PD-1+KLRG1+, and PD-1+Blimp-1+ CD8+ T cells and (E) Week 4 (W4 or EOT) PD-1+CD39+, PD-1+Blimp-1+ CD4+ T cells in SVR, relapse and healthy controls. P- values were determined by 1 Way ANOVA with Bonferroni's-Dunn's post-hoc test for pairwise multiple comparisons, p ≤ 0.05 is significant. Controls, healthy control; D0, baseline; W4, week 4/End of treatment (EOT); W16, 12 weeks after EOT. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Phenotyping was performed to assess the expression of multiple T cell lineage, activation and inhibitory receptors by using the following monoclonal antibodies in two different panels containing: Fixable Aqua Live/dead (Invitrogen), anti-CD3 Alexa Fluor 700 (BD Biosciences), anti-CD4 APC-Cy7 (BD Biosciences), anti-CD8 Brilliant Violet 510 (Biolegend), anti-CD244 PercP-Cy5.5 (Biolegend), anti-CD39 FITC (Biolegend), anti-Tim-3 PE-CY7 (eBioscience), anti-PD-1 Brilliant Violet 421 (Biolegend), anti- Blimp-1 APC (eBioscience), anti-CCR7 FITC (Biolegend), anti-CD45RO APC-ef780 (Biolegend), anti-CD160 PE-Cy7 (Biolegend), anti-CTLA-4 PE-Cy5 (Biolegend).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Peripheral PD-1 + T Cells Co-expressing Inhibitory Receptors Predict SVR With Ultra Short Duration DAA Therapy in HCV Infection

doi: 10.3389/fimmu.2019.01470

Figure Lengend Snippet: HCV specific CD8+ T cells in PD-1+ CD8 T cell subsets. (A) Gating strategy to detect HCV tetramer (tet+) CD8+ T cells. PD-1 and 2B4 expression among global (gray) and HCV tet+ (red) CD8+ T cells show most tet+ cells in PD-1+2B4+ subset. (B) Example of CD8 T cell subsets distinguished by PD-1 and 2B4 expression (2B4+PD-1-, 2B4-PD-1-, 2B4-PD-1+, 2B4+PD-1+) show relative abundance of HCV tet+ cells in four subsets. (C) Frequency of HCV tet+ T cells in CD8 T cells subsets distinguished by PD-1/2B4, PD-1/CD160 and PD-1/Tim-3 show average tet+ cells in different subsets ( N = 5 HCV HLA-A*02 patients). Statistical significances determined by 1 Way ANOVA with Turkey's Multiple Comparison Test, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Phenotyping was performed to assess the expression of multiple T cell lineage, activation and inhibitory receptors by using the following monoclonal antibodies in two different panels containing: Fixable Aqua Live/dead (Invitrogen), anti-CD3 Alexa Fluor 700 (BD Biosciences), anti-CD4 APC-Cy7 (BD Biosciences), anti-CD8 Brilliant Violet 510 (Biolegend), anti-CD244 PercP-Cy5.5 (Biolegend), anti-CD39 FITC (Biolegend), anti-Tim-3 PE-CY7 (eBioscience), anti-PD-1 Brilliant Violet 421 (Biolegend), anti- Blimp-1 APC (eBioscience), anti-CCR7 FITC (Biolegend), anti-CD45RO APC-ef780 (Biolegend), anti-CD160 PE-Cy7 (Biolegend), anti-CTLA-4 PE-Cy5 (Biolegend).

Techniques: Expressing

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: miRNA-5119 regulates immune checkpoints in dendritic cells to enhance breast cancer immunotherapy

doi: 10.1007/s00262-020-02507-w

Figure Lengend Snippet: Increased mRNAs and proteins from PD-L1, galectin-9 and HVEM genes in spleen DCs from breast cancer-bearing mice. a Detection of miRNAs encoding PD-L1, galectin-9 and HVEM by RT-qPCR. DCs were isolated from spleens of wild type and breast tumor-bearing mice using CD11c magnetically-labeled beads (Miltenyi Biotec, USA). CD11c cDNA was used to perform RT-qPCR and normalized to β-action expression. b Detection of inhibitory receptors ligand on CD11c+ DCs by flow cytometry. Spleen cells were collected from naive mice and breast tumor-bearing mice. Flow cytometry was performed with FITC-labeled CD11c, PerCP-eFluor 710-labeled PD-L1, PE-CY7-labeled galectin-9, APC-labeled HVEM antibody staining. Data are representative of three mice from at least three independent experiments. Two-tailed unpaired Student’s t tests were performed to determine statistical significance. Error bars represent standard deviation (*p < 0.05; n.s., p > 0.05)

Article Snippet: To measure in vivo IR levels, secreted cytokine levels, and T reg detection, lymphocytes isolated from tumor-bearing mice treated with miR-5119 mimic/inhibitor or control miRNA-engineered DCs were stained with anti-CD8-FITC, anti-PD-1-PerCP-eFluor 710, anti-TIM-3-PE-CY7, anti-BTLA-APC, anti-IL-2-PE, anti-TNF-α-PE, anti-IFN-γ-PE, anti-CD4-PE-CY5, anti-CD25-PE, and anti-Foxp3-FITC (eBioscience, San Diego, CA).

Techniques: Quantitative RT-PCR, Isolation, Labeling, Expressing, Flow Cytometry, Staining, Two Tailed Test, Standard Deviation

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: miRNA-5119 regulates immune checkpoints in dendritic cells to enhance breast cancer immunotherapy

doi: 10.1007/s00262-020-02507-w

Figure Lengend Snippet: miR-5119 mimic-transfected DC vaccines suppress T cell exhaustion in vivo. a miR-5119 mimic-engineered DC vaccines increased exhausted CD8+ T cell proliferation in vivo. Spleen cells were isolated from miR-5119 mimic/inhibitor DC vaccine-treated or control miRNA engineered DC vaccine-treated breast tumor-bearing mice. Isolated cells were labeled with CFSE and seeded in 96 well plate at 1 × 106 cells/well. Wild type BALB/c bone marrow DCs were pulsed with 4T1 freeze–thaw antigen (50 ng/ml) and co-cultured with spleen cells from three groups (DC: T cells = 1:10) at 37 °C in a humidified atmosphere and 5% CO2 for 3 days. T-Cell proliferation was detected by flow cytometry. b miR-5119 mimic-engineered DC vaccines reduced apoptosis in exhausted CD8+ T cells in vivo. Spleen cells were isolated from miR-5119 mimic/inhibitor DC vaccine-treated or control miRNA DC vaccine-treated breast tumor-bearing mice. CD8+ T cells were purified from the collected splenocytes using magnetically-labeled beads. Apoptosis was detected by flow cytometry using FITC-Annexin V/PI detection. c miR-5119 mimic-engineered DC vaccines inhibited immune checkpoint molecule levels in exhausted CD8+ T cells in vivo. Spleen cells were collected from miR-5119 mimic/inhibitor DC vaccine-treated or control miRNA DC vaccine-treated breast tumor-bearing mice. Flow cytometry was performed after FITC-labeled CD8, PerCP-eFluor 710-labeled PD-1, PE-CY7-labeled TIM-3, APC-labeled BTLA antibody staining. Data are representative of three mice per group. One-way ANOVA analysis was performed to determine statistical significance. Error bars represent standard deviation (*p < 0.05; n.s., p > 0.05)

Article Snippet: To measure in vivo IR levels, secreted cytokine levels, and T reg detection, lymphocytes isolated from tumor-bearing mice treated with miR-5119 mimic/inhibitor or control miRNA-engineered DCs were stained with anti-CD8-FITC, anti-PD-1-PerCP-eFluor 710, anti-TIM-3-PE-CY7, anti-BTLA-APC, anti-IL-2-PE, anti-TNF-α-PE, anti-IFN-γ-PE, anti-CD4-PE-CY5, anti-CD25-PE, and anti-Foxp3-FITC (eBioscience, San Diego, CA).

Techniques: Transfection, Vaccines, In Vivo, Isolation, Labeling, Cell Culture, Flow Cytometry, Purification, Staining, Standard Deviation