Journal: Oncology Letters

Article Title: Expression of multiple immune checkpoint molecules on T cells in malignant ascites from epithelial ovarian carcinoma

doi: 10.3892/ol.2018.8101

Figure Lengend Snippet: Analysis of immune checkpoint molecules PD-1, LAG-3, TIM-3, and BTLA on CD4+ and CD8+ T cells in malignant ascites from ovarian cancer by multicolor flow cytometry. Various immune checkpoint molecules were expressed on both CD4+ and CD8+ T cells in ascites from EOC. FS, forward scatter; SS, side scatter; INT, integral; PD-1, programmed cell death protein-1; LAG-3, lymphocyte-activation gene-3; TIM-3, T-cell immunoglobulin and mucin-domain containing-3; BTLA, B and T lymphocyte attenuator.

Article Snippet: Flow cytometry analysis The following monoclonal antibodies (mAbs) were used for flow cytometry: FITC-labeled anti-human CD4 antibody (BD Biosciences Pharmingen, San Diego, CA, USA), PE-labeled anti-human CD273 (B7-DC, PD-L2; BioLegend, Inc., San Diego, CA, USA), anti-human CD274 (PD-L1, B7-H1; BioLegend, Inc.), anti-human CD279 (PD-1; BioLegend, Inc.), anti-human CD366 (TIM-3; (BioLegend, Inc.), anti-human CD272 (BTLA; BioLegend, Inc.), anti-human LAG3 (R&D Systems Inc., Minneapolis, MN, USA) and mouse IgG 1 isotype (BioLegend, Inc.) antibodies, PC5-labeled anti-CD3 (BioLegend, Inc.) antibody, APC-labeled anti-CD326 (EpCAM), anti-CD45 (Miltenyi Biotec, Bergisch Gladbach, Germany), anti-HLA-DR (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and mouse IgG 1 isotype (eBioscience, San Diego, CA, USA) antibodies, and Pacific Blue-labeled anti-CD45 (BioLegend, Inc.) and anti-CD8a (BioLegend, Inc.) antibodies.

Techniques: Flow Cytometry, Activation Assay

Journal: Oncology Letters

Article Title: Expression of multiple immune checkpoint molecules on T cells in malignant ascites from epithelial ovarian carcinoma

doi: 10.3892/ol.2018.8101

Figure Lengend Snippet: The median, quartile and range of expression rates of PD-1, LAG-3, TIM-3, and BTLA on CD4+ and CD8+ T cells in ovarian cancer ascites. PD-1, LAG-3, and BTLA exhibited higher expression levels on CD4+ T cells than on CD8+ T cells in ascites of EOC patients (P<0.001). PD-1, programmed cell death protein-1; LAG-3, lymphocyte-activation gene-3; TIM-3, T-cell immunoglobulin and mucin-domain containing-3; BTLA, B and T lymphocyte attenuator.

Article Snippet: Flow cytometry analysis The following monoclonal antibodies (mAbs) were used for flow cytometry: FITC-labeled anti-human CD4 antibody (BD Biosciences Pharmingen, San Diego, CA, USA), PE-labeled anti-human CD273 (B7-DC, PD-L2; BioLegend, Inc., San Diego, CA, USA), anti-human CD274 (PD-L1, B7-H1; BioLegend, Inc.), anti-human CD279 (PD-1; BioLegend, Inc.), anti-human CD366 (TIM-3; (BioLegend, Inc.), anti-human CD272 (BTLA; BioLegend, Inc.), anti-human LAG3 (R&D Systems Inc., Minneapolis, MN, USA) and mouse IgG 1 isotype (BioLegend, Inc.) antibodies, PC5-labeled anti-CD3 (BioLegend, Inc.) antibody, APC-labeled anti-CD326 (EpCAM), anti-CD45 (Miltenyi Biotec, Bergisch Gladbach, Germany), anti-HLA-DR (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and mouse IgG 1 isotype (eBioscience, San Diego, CA, USA) antibodies, and Pacific Blue-labeled anti-CD45 (BioLegend, Inc.) and anti-CD8a (BioLegend, Inc.) antibodies.

Techniques: Expressing, Activation Assay

Journal: Nature Communications

Article Title: Dysfunctional CD4 T cells in an oncovirus-specific TCR-transgenic in vivo model

doi: 10.1038/s41467-025-67588-6

Figure Lengend Snippet: A Representative dot plots showing the expression of IL-2, CD40L and TNF by splenic CD4 T cells from MolT-II (n = 5) and OT-II mice (n = 5), co-cultured with D1 dendritic cells loaded with either no antigen (unstimulated) or with specific peptide ligands envH and ovaH. B Frequency of CD40L + TNF + CD4 T cells from MolT-II and OT-II mice (n = 5 mice per strain) after stimulation with envH, ovaH or no antigen (unstimulated; MolT-II envH vs. OT-II ovaH p < 0.0001). C Representative histograms showing CFSE-labeled splenocytes from MolT-II and OT-II without stimulation or after stimulation with increasing concentrations (unstimulated, 50 nM, 500 nM, 5000 nM) of peptides envH and ovaH, respectively. D Dot plots showing the expression of CD40L, IL-2 and TNF by C57BL/6, MolT-II and OT-II CD4 T cells after in vitro stimulation with soluble αCD3e and αCD28 antibodies. E Expression of co-inhibitory checkpoint molecules PD-1, LAG-3 and CTLA-4 on CD4 T cells from C57BL/6, MolT-II and OT-II mice following either no stimulation, stimulation with suboptimal (200 ng/ml) or optimal (1 mg/ml) amounts of αCD3e (n = 5 per strain; exact p-values can be found in the Source Data file). Bar graphs show mean and SD, statistical significance (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; or ns for not significant) was determined by two-way ANOVA with Tukey’s multiple comparisons test (E) or two-way ANOVA with Šídák’s multiple comparisons test (B).

Article Snippet: MolT-II mice were injected intraperitoneally with 10 mg/kg Rat anti-mouse PD-L1 (MIH5, kindly provided by Dr. R. Arens (LUMC, Leiden, Netherlands)), Rat anti-mouse LAG-3 (clone C9B7W, BioXCell; Catalog, BE0174) and/or Syrian hamster anti-mouse CTLA-4 (clone 9H10, BioXCell; Catalog, BE0131) on days 0, 2 and 4.

Techniques: Expressing, Cell Culture, Labeling, In Vitro

Journal: bioRxiv

Article Title: Immune Checkpoint Molecules as Biomarkers of Staphylococcus aureus Bone Infection and Clinical Outcome

doi: 10.1101/2024.12.30.630837

Figure Lengend Snippet: (A) Immunofluorescent histochemistry analyses of tibia sections from uninfected and MRSA-infected humanized BLT mice 14 days post-op were performed with labeled antibodies against CD3, LAG-3, TIM-3, and PD-1 with DAPI counter stain, and representative images are shown at 4x. Note the increased numbers of T cells near the SAC (dashed yellow line) in the infected tibiae. (B) A multichromatic spectral flow cytometry analyses were performed on tibial bone marrow cells from uninfected and MRSA-infected BLT mice. Live human CD45+/CD3+/T cells and their subpopulations (CD4+, CD8+, Tregs) were analysed for immune checkpoint expression (LAG3, TIM-3, and PD-1) and proliferation (Ki67), and representative histograms are shown. Note the frequency of human CD3 + /CD4 + T cells expressing TIM-3, LAG3 & PD-1) in the cells from MRSA-infected bone marrow (n=4-8 mice, *p<0.05, t-test).

Article Snippet: Primary antibodies: The following antibodies were utilized for immunostaining: goat anti-CD3ε (clone M-20, sc-1127, RRID:AB_631128, Santa Cruz Biotechnology), mouse anti-PD-1 (10377-MM23, RRID:AB_2936309, Sino Biologicals), Rabbit anti-LAG3 (clone BLR027F, NBP2-76402, RRID:AB_3403543, Novus Biologicals), Mouse anti-TIM3/HAVCR2 (clone TIM3/4031, V8754-20UG, NSJ Bioreagents), Rabbit anti- S. aureus (PA1-7246, RRID:AB_561546, Thermo Fisher Scientific), and Mouse anti-CD66b (G10F5, NBP2-80664, RRID:AB_3096017, Novus Biologicals).

Techniques: Infection, Labeling, Staining, Flow Cytometry, Expressing