Journal: Biomedicines

Article Title: Esterification of p -Coumaric Acid Improves the Control over Melanoma Cell Growth

doi: 10.3390/biomedicines11010196

Figure Lengend Snippet: N -butyl p -coumarate stimulates the NK and CD8 T cell activation and controls the tumor growth. ( A , B ) splenocytes from C57BL/6 were incubated with p -coumaric acid ( p -CA, white), ethyl p -coumarate ( 1 , red) or n-butyl p -coumarate ( 2 , blue) at 0.1 mM for 6 h and the CD69 expression was analyzed by flow cytometry in the NK ( A ) and CD8 T ( B ) cells. The figure represents mean ± SD of one single experiment (n = 5/group). C-D: C57Bl/6 mice were injected i.p. with a saline solution containing p -CA or compound 2 (2 mg/animal) for 5 consecutive days. Control animals received the corresponding DMSO concentration. At day three of the treatment, 2 × 10 5 B16-F10 melanoma cells were inoculated i.v., and the number of tumor nodules was assessed fifteen days later. ( C ) representative figure from one animal/group. ( D ) The figure represents the mean ± SEM of the lung nodule counts from two individual experiments (n = 4–5/group). * p < 0.05. **** p < 0.0001.

Article Snippet: The total splenic cells were cultured with p -CA or compounds 1 or 2 (0.1 mM) for 6 h. Then, the cells were incubated with LIVE/DEAD Fixable Aqua Dead Stain Kit (ThermoFischer, Eugene, OR, USA) in the presence of Fc blocking (anti-CD16/32), washed, and labeled with anti-CD3 (APC-Cy7), anti-CD8 (PE-Cy7), anti-NK1.1 (FITC), and anti-CD69 (eFluor TM 450) (All antibodies are from eBioscience, San Diego, CA, USA).

Techniques: Activation Assay, Incubation, Expressing, Flow Cytometry, Injection, Saline, Control, Concentration Assay

Journal:

Article Title: T Cells with a CD4 + CD25 + Regulatory Phenotype Suppress In Vitro Proliferation of Virus-Specific CD8 + T Cells during Chronic Hepatitis C Virus Infection

doi: 10.1128/JVI.79.12.7860-7867.2005

Figure Lengend Snippet: Inhibition of HCV and influenza virus-specific CD8+ T-cell proliferation by Treg cells in chronically HCV-infected patients and healthy blood donors. (A) T-cell lines from HCV-infected patients were tested for Treg-mediated suppression (ratio 3:1) of HCV-specific (patients C1 [two epitopes], C2, C3, and C4) and influenza virus-specific (patients C1, C2, C3, C4, and C5) CD8+ T-cell proliferation after 7 days of antigen-specific stimulation by tetramer staining. To calculate the percentage of CD4+CD25+ cell-mediated inhibition after 7 days in culture the following formula was used: 100 − (percentage of tetramer-positive CD8+ T cells in the presence of CD4+CD25+ cells divided by the percentage of tetramer-positive CD8+ T cells in the presence of CD4+CD25− cells) × 100. Patients with resolved HCV infection and healthy blood donors were tested in the same way for Treg-mediated suppression of HCV-specific (patients R1, R2 [two epitopes], R3, R7, and R8 [two epitopes]) and influenza virus-specific (patients R2, R3, R4, R5, and R6 and healthy blood donors) CD8+ T-cell proliferation. Importantly, a significant difference was observed between the inhibition of HCV-specific and influenza virus-specific CD8+ T cells in chronically HCV-infected patients compared to patients with resolved HCV infection and healthy blood donors (only influenza virus). (B) Frequency of CD4+CD25+ T cells in the peripheral blood of chronically HCV-infected patients, patients with resolved HCV infection, and healthy blood donors.

Article Snippet: We blocked 0.2 to 0.3 × 10 6 cells per well with immunoglobulin G1 pure (BD PharMingen, San Jose, CA) for 20 min and stained with anti-CD8-phycoerythrin (CD8 PE, Miltenyi Biotec, Auburn, CA or CD8 Cy7-PE, BD PharMingen, San Jose, CA) antibody for 20 min on a 96-well plate at 4°C.

Techniques: Inhibition, Virus, Infection, Staining

Journal:

Article Title: T Cells with a CD4 + CD25 + Regulatory Phenotype Suppress In Vitro Proliferation of Virus-Specific CD8 + T Cells during Chronic Hepatitis C Virus Infection

doi: 10.1128/JVI.79.12.7860-7867.2005

Figure Lengend Snippet: Depletion of CD4+ cells enhances HCV-specific CD8+ T-cell responses. Tetramer staining of a T-cell line after 7 days in culture. PBMC (white bars) and PBMC depleted of CD4+ cells (black bars) were stimulated with HCV peptides and cultured for 7 days. In all cases, in vitro stimulated T-cell lines showed an enhanced expansion after depletion of CD4+ cells.

Article Snippet: We blocked 0.2 to 0.3 × 10 6 cells per well with immunoglobulin G1 pure (BD PharMingen, San Jose, CA) for 20 min and stained with anti-CD8-phycoerythrin (CD8 PE, Miltenyi Biotec, Auburn, CA or CD8 Cy7-PE, BD PharMingen, San Jose, CA) antibody for 20 min on a 96-well plate at 4°C.

Techniques: Staining, Cell Culture, In Vitro

Journal:

Article Title: T Cells with a CD4 + CD25 + Regulatory Phenotype Suppress In Vitro Proliferation of Virus-Specific CD8 + T Cells during Chronic Hepatitis C Virus Infection

doi: 10.1128/JVI.79.12.7860-7867.2005

Figure Lengend Snippet: CD4+CD25+ regulatory T cells suppress HCV-specific CD8+ T-cell response. (A) PBMC depleted of CD4+ cells were cultured alone (grey bars), in the presence of CD4+CD25− (black bars) or CD4+CD25+ (white bars) for 7 days. Cells were stained with HLA-A2 tetramers. (B) PBMC depleted of CD4+ cells were cultured alone, in the presence of CD4+CD25− or CD4+CD25+ for 7 days. Representative density plots of cells stained with tetramer and anti-CD8 are shown.

Article Snippet: We blocked 0.2 to 0.3 × 10 6 cells per well with immunoglobulin G1 pure (BD PharMingen, San Jose, CA) for 20 min and stained with anti-CD8-phycoerythrin (CD8 PE, Miltenyi Biotec, Auburn, CA or CD8 Cy7-PE, BD PharMingen, San Jose, CA) antibody for 20 min on a 96-well plate at 4°C.

Techniques: Cell Culture, Staining

Journal:

Article Title: T Cells with a CD4 + CD25 + Regulatory Phenotype Suppress In Vitro Proliferation of Virus-Specific CD8 + T Cells during Chronic Hepatitis C Virus Infection

doi: 10.1128/JVI.79.12.7860-7867.2005

Figure Lengend Snippet: Treg cells inhibit proliferation of HCV specific tetramer-positive CD8+ T cells. (A) CD4-depleted PBMC were stimulated with HCV peptide 3 prior to culture with decreasing numbers of CD4+CD25+ regulatory T cells (CD4-depleted PBMC: Treg cells). After 7 days in culture, cells were tested for virus-specific responses by tetramer staining. (B) CD4-depleted PBMC were labeled with CFSE- and stimulated with HCV peptide 3 prior to culture with high (ratio 3:1) or low (ratio 100:1) numbers of CD4+CD25+ regulatory T cells. Representative histograms are shown. The cells are gated on CD8+ HCV tetramer positive cells.

Article Snippet: We blocked 0.2 to 0.3 × 10 6 cells per well with immunoglobulin G1 pure (BD PharMingen, San Jose, CA) for 20 min and stained with anti-CD8-phycoerythrin (CD8 PE, Miltenyi Biotec, Auburn, CA or CD8 Cy7-PE, BD PharMingen, San Jose, CA) antibody for 20 min on a 96-well plate at 4°C.

Techniques: Virus, Staining, Labeling

Journal: Cell Death & Disease

Article Title: NK cell intrinsic regulation of MIP-1 α by granzyme M

doi: 10.1038/cddis.2014.74

Figure Lengend Snippet: Recruitment and function of GrzM −/− NK cells is altered in the liver after L. monocytogenes infection. ( a and b ) Age-matched female WT and GrzM −/− mice were infected i.v. with 10 4 CFU of L. monocytogenes. At indicated time points after infection, mice were killed, livers ( a ) and spleens ( b ) were harvested, lymphocytes isolated and stained with mAbs reactive with NK1.1 and CD3 (shown are absolute numbers of NK cells), with ( c ) NK1.1, CD3, CD27 and CD11b, with Ly6G − /CD11b + /F4/80 + ( d ), CD4 ( e ), CD8 ( f ) or NK1.1, CD3 and IFN- γ ( g ) and analysed by flow cytometry. Results are pooled from six independent experiments, total n =5–8

Article Snippet: The following antibodies from e-Bioscience (San Diego, CA, USA) were used: CD8-PE-CY7 (clone 53–6.7), CD4- APC-Cy7 (clone RM4-5), NK1.1-PE (PK 136), CD3-Pacific Blue (17A2), CD69-FITC (H1.2F3), CD11b-APC-Cy7 (M1/70), Ly6G-PE (1A8), F4/80-APC (BM8), IFN- γ -FITC (XMG1.2), Mip-1 α -APC (PFFM3), CD27-PeCy7 (LG.7F9).

Techniques: Infection, Isolation, Staining, Flow Cytometry