Journal: Frontiers in Immunology

Article Title: Targeting LAG-3 and PD-1 to Enhance T Cell Activation by Antigen-Presenting Cells

doi: 10.3389/fimmu.2018.00385

Figure Lengend Snippet: Effect of programmed cell death protein 1 (PD-1) and lymphocyte activation gene 3 (LAG-3) blockade on IFN-γ secretion of different T cell subpopulations after stimulation with TLR-3-DCs. MACS-enriched CD3 + T cells of 8 healthy donor (HDs) were sorted according to CCR7 and CD45 RA expression (A) . The various T cell subpopulations were cocultured with autologous CMV, EBV, influenza, tetanus (CEFT)-pulsed TLR-3-DCs in the presence or absence of α-PD-1 (B) and α-LAG-3 (C) antibody. IFN-γ secretion was determined by cytometric bead array (CBA) assay, and the ratio between concentration with and without blocking antibody was calculated. All data are presented as box-and-whisker plots, and statistical significance was calculated against a fold change of 1.0. * p < 0.05.

Article Snippet: Immunofluorescent staining of T-cell surface antigens was performed using the following fluorescence-conjugated monoclonal antibodies: CD244 (PE, C1.7; 329507 or APC, C1.7; 329511), PD-1 (Brilliant Violet 421, EH12.7H7; 329919), CD3 (FITC, UCHT1; 300406), CD45RA (Brilliant Violet 421, HI100; 304129; all BioLegend), CD160 (APC, 688327; FAB6700A), TIM-3 (PE, 344823; FAB2365P; both R&D Systems), CD8 (PerCP-eFluor 710, SK1; 8046-0087; eBioscience), CD4 (APC-H7, RPA-T4; 560158; BD Biosciences), LAG-3 (ATTO 647N, 17B4; AG-20B-0012TS AdipoGen), CCR7 (CD197, APC, FR 11-11E8; 130-098-125; Miltenyi Biotec).

Techniques: Activation Assay, Expressing, Concentration Assay, Blocking Assay, Whisker Assay

Journal: Molecular Therapy Oncolytics

Article Title: The IAP antagonist birinapant enhances chimeric antigen receptor T cell therapy for glioblastoma by overcoming antigen heterogeneity

doi: 10.1016/j.omto.2022.11.004

Figure Lengend Snippet: Birinapant does not significantly affect CAR T cell functions in vivo NSG mice were subcutaneously implanted with 0.5 × 10 6 birinapant-resistant U87-EGFRvIII-CBG cells on the right flank. After 6 days (day 0), 5 × 10 6 2173-28ζ CAR T cells were intravenously injected. The vehicle (12.5% Captisol) or birinapant at 10 mg/kg were given by intraperitoneal injections every 3 days for a total of 10 doses since day 0. The mice were rechallenged with 3 × 10 6 U87-EGFRvIII-CBG cells subcutaneously implanted in the left flank on day 22. (A) Geometric mean of bioluminescent total flux by imaging from U87-EGFRvIII-CBG tumors in each treatment group at the indicated time points. (B) Concentration of TNF-α and IFN-γ in mouse plasma on day 7 by ELISA. (C) Geometric mean of human CAR T cell (CD45 + /CD3 + ) count in mouse blood in each treatment group at the indicated time points. (D) Percentage of CD4 (CD4 + /CD8 − ), CD8 (CD4 − /CD8 + ), naïve-like (CCR7 + /CD45RO − ), central memory (CCR7 + /CD45RO + ), effector memory (CCR7 − /CD45RO + ), and effector (CCR7 − /CD45RO − ) T cell subset among total human CAR T cells (CD45 + /CD3 + ) in mouse blood on day 21 by flow cytometry. BLI, bioluminescent imaging. p values were determined by unpaired t tests in (B). Results are presented as geometric means ± geometric SD in (A) and (C), and as means ± SD in (B). ∗p < 0.05.

Article Snippet: For flow cytometry with mouse blood samples to evaluate human CAR T cell count and phenotypes in mouse studies, the staining panel included mouse-anti-human CD45-APC (BD Biosciences; clone HI30), mouse-anti-human CD3-BV605 (BioLegend; clone OKT3), mouse-anti-human CD4-BV510 (BioLegend; clone OKT4), mouse-anti-human CD8-APC-H7 (BD Biosciences; clone SK1), mouse-anti-human CCR7-FITC (BD Biosciences; clone 150503), and mouse-anti-human CD45RO-PE (BioLegend; clone UCHL1), and a separate panel was composed of mouse-anti-human PD1-BV421 (BioLegend; clone EH12.2H7), mouse-anti-human LAG3-PerCP/Cyanine5.5 (BioLegend; clone11C3C65), rabbit-anti-human TIM3-AF700 (R&D Systems; clone 344823), and mouse-anti-human CD45-FITC (BD Biosciences; clone HI30).

Techniques: In Vivo, Injection, Imaging, Concentration Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The BCR-ABL inhibitor nilotinib influences phenotype and function of monocyte-derived human dendritic cells

doi: 10.1007/s00262-018-2129-9

Figure Lengend Snippet: Migratory capacity of DCs to CCR7 ligand. Imatinib- and nilotinib-treated and LPS-stimulated human DCs were analyzed for their migratory behavior towards CCL19/MIP-3β in transwell assays

Article Snippet: Immunostaining Cells were stained using FITC-, BB515 (brilliant blue 515) or PE-conjugated mouse monoclonal antibodies against CD14, CD80, HLA-DR (human leukocyte antigen–antigen D related), PD-1 (BD Biosciences, Heidelberg, Germany), PD-L1 (BioLegend, Koblenz, Germany), CD86, CTLA-4 (BD PharMingen, Hamburg, Germany), CD1a (DAKO Diagnostika GmbH, Hamburg, Germany), CD83 (Immunotech, Marseilles, France), CCR7, RANK (receptor activator of NF-κB, R&D Systems), and CD209 (BD Biosciences).

Techniques: