Journal: Oncology Letters

Article Title: Natural killer cells inhibit pulmonary metastasis of hepatocellular carcinoma in nude mice

doi: 10.3892/ol.2016.4170

Figure Lengend Snippet: Isolation and culture of the NK cells. (A) CD56 + CD3 − cells were identified as the marker of the NK cells. The percentage of the NK cells in peripheral blood mononuclear cells was 10–30%. The NK cell percentage of certain samples was >30%. (B) The purity of the NK cells was >95%. (C) NK cell culture suspensions formed aggregates or clusters (magnification, ×100). NK, natural killer; PE, phycoerythrin; APC, allophycocyanin; CD, cluster of differentiation.

Article Snippet: Flow cytometry was used to examine the expression of the following cell surface proteins: Cluster of differentiation (CD)56 [phycoerythrin (PE)-labeled mouse anti-human CD56 clone B159; BD Biosciences, San Diego, CA, USA], CD3 [allophycocyanin (APC)-labeled mouse anti-human CD3 clone UCHTI; BD Pharmingen, San Diego, CA, USA], NKG2D [fluorescein isothiocyanate (FITC)-labeled anti-human NKG2D clone 1D11; eBioscience, Inc., San Diego, CA, USA], NKB1 (FITC-labeled anti-human NKB1 clone DX9; BD Pharmingen), perforin (FITC Perforin Reagent set containing FITC-labeled mouse anti-human perforin clone δG9 and FITC-mouse immunoglobulin (Ig)G2bκ clone 27–35; BD Pharmingen) and granzyme (FITC-labeled mouse anti-human granzyme clone GB11; BD Pharmingen).

Techniques: Isolation, Marker, Cell Culture

Journal: BMC Immunology

Article Title: Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides

doi: 10.1186/1471-2172-6-24

Figure Lengend Snippet: HLA-DR*1101-Ig and HLA-DR*1101-Bir molecules can be loaded with synthetic peptides . Purified soluble HLA-DR*1101-Ig and HLA-DR*1101-Bir molecules were loaded with equal amount of p2 peptide. Stability of the peptide-loaded HLA-DR*1101 αβ heterodimers was determined by running complexes in reducing condition on SDS gels without boiling, or by testing their capacity to elicit IFN-γ production in specific CD4 + T cell clones. (a) SDS-resistance assay of HLA-DR*1101-Bir molecules. Indicated are the protein bands corresponding to the migration of either the HLA-DR*1101-Bir αβ heterodimer, or the single HLA-DRα-Bir or HLA-DR*1101β chains. Lane nil: unloaded HLA-DR*1101-Bir molecules. Lane p2: p2-loaded HLA-DR*1101-Bir molecules. (b) SDS-resistance assay of HLA-DR*1101-Ig molecules. Indicated are the protein bands corresponding to the migration of either the HLA-DR*1101-Ig αβ heterodimer, or the single HLA-DRα-Ig or HLA-DR*1101β chains. Lane nil and p2 are the same as per HLA-DR*1101-bir. (c) IFN-γ production by the p2-specific CD4 + T cell clone (TCC) 162 in response to p2-loaded HLA-DR*1101-Bir or HLA-DR*1101-Ig molecules, attached to plastic. The response of the MAGE-3 p39-specific CD4 + TCC 2C3.35 is shown as control. T cells were cultured in the presence of the indicated peptide-pulsed HLA-DR*1101 recombinant molecules, in the presence of a costimulatory dose of PMA. As control, T cells were cultured in the presence only of sub-optimal doses of PMA (PMA), or activated by plastic-bound anti-CD3 mAb (Anti-CD3). The release of IFN-γ in the culture supernatant was determined by ELISA 48 h later.

Article Snippet: The following antibodies have been used: FITC- and APC-labelled mouse anti human CD3 (Becton Dickinson, San Jose, CA, USA), Quantum Red-conjugated mouse anti-human CD4 (SIGMA), PE-labelled rat anti-human IFN-γ (Becton Dickinson).

Techniques: Purification, Clone Assay, Migration, Control, Cell Culture, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: BMC Immunology

Article Title: Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides

doi: 10.1186/1471-2172-6-24

Figure Lengend Snippet: HLA-DR*1101-Bir, but not HLA-DR*1101-Ig, stain specific CD4 + T cells, and display a temperature-dependence . CD4 + TCC specific for p2, endowed with different affinity of recognition, and an irrelevant CD4 + TCC were stained with p2-loaded HLA-DR*1101-Bir tetramers at different temperature. (a) The relative affinity for the p2-HLA-DR*1101 displayed by the two TCC clones 162 and 51 is determined in an IFN-γ-releasing assay following production in response to different doses of p2 peptide. 10 4 T cells are incubated with 5 × 10 4 HLA-DR*1101 LCL cells and the indicated doses of p2 peptide. After 48 hours, the concentration of IFN-γ in the culture supernatant is measured by ELISA. Indicated are the concentration of p2 peptide required to elicit half-maximum release of IFN-γ in the two TCC. (b) Staining of TCC162, TCC51 and the irrelevant TCC with either p2-loaded HLA-DR*1101-Ig or p2-loaded HLA-DR*1101-Bir tetramers. Staining is performed for 2 hours at the indicated temperatures with 10 μg of tetramer per sample. The tetramer staining on CD3 + CD4 + gated cells is shown. The amount of surface TCR, obtained by staining with anti-CD3 mAb, expressed by the TCC in the different conditions is shown by the mean fluorescent intensity (CD3 mfi).

Article Snippet: The following antibodies have been used: FITC- and APC-labelled mouse anti human CD3 (Becton Dickinson, San Jose, CA, USA), Quantum Red-conjugated mouse anti-human CD4 (SIGMA), PE-labelled rat anti-human IFN-γ (Becton Dickinson).

Techniques: Staining, Clone Assay, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: BMC Immunology

Article Title: Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides

doi: 10.1186/1471-2172-6-24

Figure Lengend Snippet: HLA-DR*1101-Bir tetramer staining correlates with the activation state of the target CD4 + T cells . The p2-specific CD4 + TCC 162 was stained with p2-loaded HLA-DR*1101-Bir tetramers at different time after in vitro restimulation with p2 and APCs. The histograms in the upper row display the amount of surface TCR (mfi) expressed by the T cells at the time of tetramer staining, as determined by anti-CD3 staining. The histograms in the lower row show the staining with the p2-loaded HLA-DR*1101-Bir tetramers (mfi) performed at the indicated time after restimulation.

Article Snippet: The following antibodies have been used: FITC- and APC-labelled mouse anti human CD3 (Becton Dickinson, San Jose, CA, USA), Quantum Red-conjugated mouse anti-human CD4 (SIGMA), PE-labelled rat anti-human IFN-γ (Becton Dickinson).

Techniques: Staining, Activation Assay, In Vitro