Journal: PLoS ONE

Article Title: HIV-Induced Type I Interferon and Tryptophan Catabolism Drive T Cell Dysfunction Despite Phenotypic Activation

doi: 10.1371/journal.pone.0002961

Figure Lengend Snippet: PBMC from HIV-uninfected donors were cultured for 24 and 48 hours in presence of control microvescicles, HIV alone or in presence of blocking antibodies against the cellular receptor for IFN-α (anti-IFNAR). CD38 and CD69 expression were analyzed by flow cytometry on gated CD3 + CD4 + and CD3 + CD8 + cells (CD4 and CD8 T cells, respectively). (A) and (C) show flow cytometry contour plots of CD69 and CD38 expression for one example experiment for CD4 and CD8 T cells, respectively. (B) and (D) show bar graphs summarizing mean fluorescence intensity (MFI) of CD38 and CD69 in CD4 and CD8 T cells, respectively (48 hours only). Mean values±standard error calculated on 5 independent experiments are shown in the bar graphs.

Article Snippet: After stimulation in culture, cells were washed and incubated for 20 min at room temperature in PBS containing 2% mouse serum (Sigma) with the following antibodies: Peridinn chlorophyll protein (PerCP)-conjugated anti-CD3 (BD Biosciences), Allophycocyanin (APC)-conjugated anti-CD4 (BD Biosciences), PE-Cy7-conjugated anti-CD8 (BD Biosciences), Phycoerythrin (PE)-conjugated anti-CD69 (BD Biosciences), fluorescein isothiocyanate (FITC)-conjugated anti-CD38 (BD Biosciences).

Techniques: Cell Culture, Blocking Assay, Expressing, Flow Cytometry, Fluorescence

Journal: PLoS ONE

Article Title: HIV-Induced Type I Interferon and Tryptophan Catabolism Drive T Cell Dysfunction Despite Phenotypic Activation

doi: 10.1371/journal.pone.0002961

Figure Lengend Snippet: PBMC from HIV-uninfected donors were cultured for 24 (upper panels) and 48 hours (lower panels) in presence or absence of recombinant IFN-α (rIFN-α). CD38 and CD69 expression were analyzed by flow cytometry on gated CD3 + CD4 + and CD3 + CD8 + cells (CD4 and CD8 T cells, respectively). Flow cytometry contour plots of CD69 and CD38 expression for one example experiment for CD4 (left panels) and CD8 T cells (right panels).

Article Snippet: After stimulation in culture, cells were washed and incubated for 20 min at room temperature in PBS containing 2% mouse serum (Sigma) with the following antibodies: Peridinn chlorophyll protein (PerCP)-conjugated anti-CD3 (BD Biosciences), Allophycocyanin (APC)-conjugated anti-CD4 (BD Biosciences), PE-Cy7-conjugated anti-CD8 (BD Biosciences), Phycoerythrin (PE)-conjugated anti-CD69 (BD Biosciences), fluorescein isothiocyanate (FITC)-conjugated anti-CD38 (BD Biosciences).

Techniques: Cell Culture, Recombinant, Expressing, Flow Cytometry

Journal: Experimental and Therapeutic Medicine

Article Title: Effects of CD4 + T lymphocytes from ovariectomized mice on bone marrow mesenchymal stem cell proliferation and osteogenic differentiation

doi: 10.3892/etm.2020.9212

Figure Lengend Snippet: CD4 + T cells are activated in OVX mice. (A) The gating strategies used for CD4 + T cells. (B) Percentage of CD4 + CD69 + cells in spleens tested using flow cytometry. (C) Expression levels of proinflammatory cytokines, IL-2, IFN-γ and TNF-α, in purified CD4 + T cells were determined using reverse transcription-quantitative-PCR. (D) TNF-α levels in the supernatant from purified CD4 + T cell cultures were measured by ELISA. * P<0.05, ** P<0.01 vs. sham. CD, cluster of differentiation; OVX, ovariectomized; IL-2, interleukin 2; IFN-γ, interferon- γ; TNF-α, tumor necrosis factor-α; IgG, immunoglobulin; APC, allophycocyanin.

Article Snippet: Inc.) and magnetic beads coated with anti-mouse CD4 antibodies (Miltenyi Biotec., Inc.), CD3 and CD28 (both, BD Biosciences), a volumetric microscope, an inverted phase contrast microscope and camera system (Olympus Corporation), a flow cytometer (Beckman Coulter, Inc.), an Alkaline Phosphatase Staining kit (cat. no. P0321; Beyotime Institute of Biotechnology), a mouse TNF-α ELISA kit (cat. no. MTA00B; R&D Systems, Inc.), anti-TNF-α antibodies (cat. no. AF-410-NA; R&D Systems, Inc.), FITC-conjugated anti-CD3 (cat. no. 11-0032-82; eBioscience; Thermo Fisher Scientific, Inc.), allophycocyanin (APC)-conjugated anti-CD4 (cat. no. 17-0042-82; eBioscience; Thermo Fisher Scientific, Inc.), phycoerythrin (PE)-conjugated anti-CD69 (cat. no. 12-0691-82; eBioscience; Thermo Fisher Scientific, Inc.) and Armenian hamster IgG isotype control (cat. no. 12-4888-81; eBioscience; Thermo Fisher Scientific, Inc.).

Techniques: Flow Cytometry, Expressing, Purification, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: PTPN3 expressed in activated T lymphocytes is a candidate for a non-antibody-type immune checkpoint inhibitor

doi: 10.1007/s00262-019-02403-y

Figure Lengend Snippet: Lymphocyte activation by PTPN3 inhibition was observed only in CD3+ T cells, not in CD3− cells. a An FACS analysis of activated lymphocytes before selection. CD3+CD4+NKG2D− cells (b) and CD3+CD8+NKG2D+ cells (c) were separately collected using microbeads. CD3− (NK) cells (d) were collected using FACS sorting. e The migration ability of lymphocytes was analyzed by time-lapse imaging. The graph shows the moving distance of lymphocytes analyzed by the Image-Pro Analyzer software program. f The proliferation of FAR red-labeled lymphocytes was estimated by FACS. g The PTPN3 mRNA expression in CD3− cells and CD3+ cells after activation was estimated by real-time RT-PCR. h The migration ability of non-activated lymphocytes was analyzed by the chamber method. i The cytotoxicity of non-activated lymphocytes was investigated. After co-culture of SCC cells and lymphocytes for 72 h, the absorbance of viable cancer cells was measured. j The PTPN3 mRNA expression in lymphocytes stimulated by allo-pancreatic cancer (SUIT-2) cell-pulsed mature DCs without using anti CD3 mAb was estimated by real-time RT-PCR. Similar results were obtained in two different healthy volunteers in all experiments. Data are presented as the mean ± SD. Lymphocytes and DCs from healthy volunteers were used in all experiments. *p < 0.05

Article Snippet: Fluorescence-activated cell sorting (FACS) Cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD3, CD4, and monoclonal antibodies (mAbs), or phycoerythrin (PE)-conjugated anti-NKG2D, CD8 and CD69 mAbs (BD Pharmingen, San Diego, CA, USA).

Techniques: Activation Assay, Inhibition, Selection, Migration, Imaging, Software, Labeling, Expressing, Quantitative RT-PCR, Co-Culture Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: PTPN3 expressed in activated T lymphocytes is a candidate for a non-antibody-type immune checkpoint inhibitor

doi: 10.1007/s00262-019-02403-y

Figure Lengend Snippet: PTPN3 inhibition increases the cytotoxicity against cancer cells in vivo. a–f Therapy experiments using allo-activated lymphocytes targeting SUIT-2 cells from healthy volunteers are shown. Allo-activated lymphocytes transfected with control shRNA or PTPN3#2 shRNA were administered at 6, 9, 13, and 16 days after tumor injection (arrows in b). a Representative photos of formed tumors. b The tumor volume at the indicated days and c Kaplan–Meier plot of the tumor growth are shown. d The tumor weight at 22 days after tumor injection was assessed. e Representative photos of immunohistochemical staining of CD8 in formed tumors. Original magnification ×200. f The numbers of labeled tumor-infiltrating-administrated lymphocytes (TILs) were counted. g–l Therapy experiments using tumor and autologous lymphocytes from a patient with ovarian cancer are shown. Autologous-activated lymphocytes transfected with control shRNA or PTPN3#2 shRNA were administered at 11, 14, 18, and 21 days after tumor injection (arrows in h). g Representative photos of formed tumors. h The tumor volume at the indicated days and i Kaplan–Meier plot of the tumor growth are shown. j Representative photos of immunohistochemical staining of CD3 in formed tumors. Original magnification ×200. k The numbers of non-tumor-tissue-infiltrating-administrated lymphocytes (ntTILs) were counted. l The numbers of ntTILs in the spleen, liver and lung were counted. Data are presented as the mean ± SD. *p < 0.05

Article Snippet: Fluorescence-activated cell sorting (FACS) Cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD3, CD4, and monoclonal antibodies (mAbs), or phycoerythrin (PE)-conjugated anti-NKG2D, CD8 and CD69 mAbs (BD Pharmingen, San Diego, CA, USA).

Techniques: Inhibition, In Vivo, Transfection, shRNA, Injection, Immunohistochemical staining, Staining, Labeling

Journal: The EMBO Journal

Article Title: Triggering MSR1 promotes JNK‐mediated inflammation in IL‐4‐activated macrophages

doi: 10.15252/embj.2018100299

Figure Lengend Snippet: Immunoblot against MSR1 of TUBE pull‐downs of MSR1 WT and KO BMDMs shows increasing amounts of ubiquitylated MSR1 upon alternative activation and further increases upon MSR1 ligation with fucoidan or oxLDL. Msr1 +/+ (WT) and Msr1 −/− (KO) M0 macrophages and M2 (IL‐4) macrophages untreated or stimulated with the MSR1 ligands fucoidan or oxLDL (50 μg/ml, 30 min) were analysed for the phosphorylated and the total forms of JNK1/2 and MSR1. ITGAM (CD11b) serves as a loading control. Both fucoidan and oxLDL activate JNK in a MSR1‐dependent manner. IL4‐activated MSR1 knock‐out BMDMs were transfected with WT or K27R MSR1 and treated with 50 μg/ml fucoidan for 1 h. Mutation of the ubiquitylation site K27 abolishes MSR1 signalling. qPCR data of Tnfa, Il1b and Ccl2 mRNA levels in WT and MSR1 KO M0 and M2 (IL‐4) BMDMs show an MSR1‐dependent increase in pro‐inflammatory cytokines in response to MSR1 ligation by fucoidan. Inhibition of JNK by JNK‐IN8 reduces expression of Tnfa and Ccl2 upon MSR1 ligation, showing that it is JNK‐dependent. Flow cytometry analysis of cell surface markers in WT and MSR1 −/− M0 macrophages and M2 (IL‐4) macrophages untreated or stimulated with the MSR1 ligands fucoidan (50 μg/ml, 24 h). Data show MSR1‐dependent increase in the early activation markers CD54, CD69 and CD86 and a decrease in the M2 marker CD301b/Mgl2. CD11b serves as a control. Data information: Error bars represent SEM. *** P < 0.0001; **** P < 10 −5 (Student's t ‐test). (A, B) Data are representative of three independent experiments. (D, E, F) Three biological replicates.

Article Snippet: Following 15‐min incubation in ice, cells were washed once in PBS/FBS and then incubated with 100 μl PBS/FBS containing 1–100 dilution of phycoerythrin (PE)‐conjugated CD54, CD69, CD86, CD301b or CD36 antibodies (eBioscience) for 30 min on ice, respectively.

Techniques: Western Blot, Activation Assay, Ligation, Control, Knock-Out, Transfection, Mutagenesis, Inhibition, Expressing, Flow Cytometry, Marker

Journal: eLife

Article Title: Calcium-mediated shaping of naive CD4 T-cell phenotype and function

doi: 10.7554/eLife.27215

Figure Lengend Snippet:

Article Snippet: Antibody , Phycoerythrin (PE)-conjugated anti-CD69 (H1.2F3) , BD Biosciences , Cat# 553237.

Techniques: Recombinant, Software, Expressing