Journal: Frontiers in Immunology

Article Title: TGF-β1 Downregulates the Expression of CX 3 CR1 by Inducing miR-27a-5p in Primary Human NK Cells

doi: 10.3389/fimmu.2017.00868

Figure Lengend Snippet: Expression profile of miR-27a-5p and CX 3 CR1 mRNA in TGF-β1-treated natural killer (NK) cells. (A) NK cells treated for 12 or 24 h with the indicated amounts of TGF-β1 were simultaneously analyzed for the expression of miR-27a-5p and CX 3 CR1. RNU44 and GAPDH were used as reference controls, respectively. Data in quadruplicate from one representative donor (donor 1), of two analyzed (donors 1 and 4), are shown. Expression relative fold changes are referred to the expression of miR-27a-5p and CX 3 CR1 mRNA in untreated NK cells (medium alone, 24 h) whose expression has been arbitrarily assigned the value 1. ** p < 0.01, *** p < 0.001, **** p < 0.0001. (B) Correlation between miR-27a-5p and CX 3 CR1 mRNA expression in TGF-β1-treated NK cells. Scatter plot showing miR-27a-5p and CX 3 CR1 mRNA expression in TGF-β1-treated NK cells from the same donor (donor 1) of ( A ). Each point represents the miR-27a-5p and CX 3 CR1 expression detected in one of the seven reported conditions, with different TGF-β1 concentrations (0, 5, and 40 ng/mL) and time of treatment (0, 12, and 24 h). The trendline, its equation and the R 2 value is reported. Significance of correlation was determined using the Pearson’s coefficient ( r = −0.765948064, p < 0.05). Data are referred to a representative donor (donor 1), of two analyzed (donors 1 and 4). (C) miR-23a-27a-24-2 cluster primary transcript expression in TGF-β1-treated NK cells. NK cells cultured for 24 h in the presence or absence of 5 ng/mL of TGF-β1 were analyzed by real-time PCR for the expression of miR-23a~27a~24-2 cluster primary transcript. GAPDH was used as reference control. Data in triplicate from one representative donor of three analyzed (donors 5, 6, and 7) are shown. Expression relative fold changes are referred to the expression of untreated NK cells (medium alone) whose expression has been arbitrarily assigned the value 1. * p < 0.05.

Article Snippet: One week after infection, CX 3 CR1 positive cells were selected using the anti-CX 3 CR1 Microbead Kit (Miltenyi Biotec).

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Control

Journal: Frontiers in Immunology

Article Title: TGF-β1 Downregulates the Expression of CX 3 CR1 by Inducing miR-27a-5p in Primary Human NK Cells

doi: 10.3389/fimmu.2017.00868

Figure Lengend Snippet: Functional interaction between miR-27a-5p and CX 3 CR1 mRNA. (A) Luciferase activity in HEK293T cells cotransfected with luciferase reporter construct containing the 3′ untranslated region (3′UTR) CX 3 CR1 and miR-27a-5p mimics. Firefly luciferase activity was normalized to the renilla luciferase activity, expressed by the same vector. Data in quadruplicate are from one experiment of three performed. Luciferase activities are referred to the activity of cells transfected with the miRNA negative control whose expression has been arbitrarily assigned the value 1. pmirGLO, parental vector; pmirCX3CR1UTRWT, pmirGLO vector containing 700 bp of the CX 3 CR1 3′UTR downstream of the firefly luciferase gene; pmirCX3CR1UTRMT, mutated version of pmirCX3CR1UTRWT containing the C>G mutation in the CX 3 CR1 3′UTR target site; miR-Neg, miRNA mimic negative control. **p < 0.01; ns, not significant. (B) CX 3 CR1 mRNA expression in NK cells transfected with miR-27a-5p inhibitor (Inh) or with a negative control miRNA Inh. Cells were cultured for 72 h in the presence of 5 ng/mL of TGF-β1. Data in triplicate are from one representative healthy donor of three analyzed (donors 8, 9, and 10). GAPDH has been used as reference control. Expression relative fold changes are referred to CX 3 CR1 mRNA expression in NK cells transfected with the negative control miRNA inhibitor, whom expression has been arbitrarily assigned the value 1. * p < 0.05.

Article Snippet: One week after infection, CX 3 CR1 positive cells were selected using the anti-CX 3 CR1 Microbead Kit (Miltenyi Biotec).

Techniques: Functional Assay, Luciferase, Activity Assay, Construct, Plasmid Preparation, Transfection, Negative Control, Expressing, Mutagenesis, Cell Culture, Control

Journal: Frontiers in Immunology

Article Title: TGF-β1 Downregulates the Expression of CX 3 CR1 by Inducing miR-27a-5p in Primary Human NK Cells

doi: 10.3389/fimmu.2017.00868

Figure Lengend Snippet: miR-27a-5p-induced downregulation of CX 3 CR1 surface expression. (A) CX 3 CR1 + HEK293T cells (clone #124) untrasfected (white bar), transfected with miR-27a-5p mimic (black bar), or miRNA negative control (stripped bar) were analyzed by flow cytometry for the expression of CX 3 CR1 at the indicated time intervals. Average of four independent experiments. 95% confidence intervals and significance (* p < 0.05) are shown. ns, non-significant. (B) Representative cytofluorimetric analysis of CX 3 CR1 surface expression. Values inside each histogram indicate the percentage (%) of positive cells. NEG: miRNA negative control.

Article Snippet: One week after infection, CX 3 CR1 positive cells were selected using the anti-CX 3 CR1 Microbead Kit (Miltenyi Biotec).

Techniques: Expressing, Transfection, Negative Control, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: TGF-β1 Downregulates the Expression of CX 3 CR1 by Inducing miR-27a-5p in Primary Human NK Cells

doi: 10.3389/fimmu.2017.00868

Figure Lengend Snippet: miR-27a-5p and CX 3 CR1 expression in NK cells cocultured under transwell conditions with SH-SY5Y neuroblastoma cell line. (A) miR-27a-5p and CX 3 CR1 mRNA expression of SH-SY5Y-conditioned NK cells from three healthy donors (donors 11, 12, and 13). Data in triplicate from each donor are shown. RNU44 and GAPDH were used as reference controls, respectively. ** p < 0.01; *** p < 0.001. (B) Cytofluorimetric analysis of CX 3 CR1 expression in NK cells from the same donors. White profiles refer to cells incubated with isotype-matched mAbs. Value inside each histogram indicates the median fluorescence intensity (MFI).

Article Snippet: One week after infection, CX 3 CR1 positive cells were selected using the anti-CX 3 CR1 Microbead Kit (Miltenyi Biotec).

Techniques: Expressing, Incubation, Fluorescence

Journal: PLoS ONE

Article Title: CX 3 CR1 Is Expressed by Human B Lymphocytes and Meditates CX 3 CL1 Driven Chemotaxis of Tonsil Centrocytes

doi: 10.1371/journal.pone.0008485

Figure Lengend Snippet: ( A ) Flow cytometric analysis of CX 3 CR1 expression in tonsil and blood B cells. Representative histograms from each B cell fractions, and THP-1 and Raji cell lines, tested as positive and negative controls, respectively, are shown. ( B ) Quantization of CX 3 CR1 by real time PCR in peripheral blood and tonsil B cells. Data are normalized to the expression of POLR2A. Values are expressed as arbitrary units calculated as fold of CX 3 CR1 expression relative to the THP-1 cell line, arbitrarily set at 1. Raji cell line was tested as negative control. Data are median, minimum and maximum values from two different experiments performed in quadruplicate. ( C ) Displacement experiments of 125 I-CX 3 CL1 in tonsil and blood B cells. Left panel . Cells were incubated for 2 h at 4°C with 1 nM 125 I-CX 3 CL1 in the absence or presence of 1, 10 and 100 nM cold CX 3 CL1. Percentage of binding inhibition by unlabeled CX 3 CL1, calculated as ratio between cell-bound cpm in the presence of unlabeled ligand and cell-bound cpm in the absence of unlabeled ligand multiplied by 100, was used as a measure for competition between 125 I-labeled and unlabelled CX 3 CL1. THP-1 and Raji cell lines were tested as positive and negative controls, respectively. Right panel . The experiments shown in the left panel were repeated using 100 nM cold CXCL8 as negative control.

Article Snippet: CD10-FITC (MEM78 clone), anti-human IgD-FITC, CD95-FITC, CD44-FITC, anti-Bcl-2-FITC, and anti-Ki-67-FITC mAbs from DAKO (Glostrup, Denmark); unconjugated CD77 and CD39 mAbs from Immunotech, Marseille, France; unconjugated rabbit anti-human CX 3 CR1 (Serotec) that detects an epitope starting from 175 to 189 amino acids; unconjugated CD3, CD56, CD68, CD10, CD27, and anti-human IgM mAbs from DAKO.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Negative Control, Incubation, Binding Assay, Inhibition, Labeling

Journal: PLoS ONE

Article Title: CX 3 CR1 Is Expressed by Human B Lymphocytes and Meditates CX 3 CL1 Driven Chemotaxis of Tonsil Centrocytes

doi: 10.1371/journal.pone.0008485

Figure Lengend Snippet: ( A ) Apoptosis evaluation in purified tonsil GC B cells. The proportion of early apoptotic GC B cells was detected by Annexin V staining at time 0 and after 1, 2, 3, and 4h culture. Results are expressed as median, minimum and maximum values from five different GC B cell suspensions. ( B ) Flow cytometric analysis of CX 3 CR1 expression on freshly purified tonsil GC, naïve, and memory B cells. Results are expressed in box plot as median percent positive cells, minimum and maximum values, and quartiles, from ten different experiments. ( C ) Chemotaxis of GC, naïve, and memory B lymphocytes to rCX 3 CL1. Results are median numbers of migrated cells, maximum and minimum values, from five different experiments for each B cell subset. * P = 0.043 for both 300 and 600 ng/ml rCX 3 CL1. ▪ = Chemotaxis of non-GC B cells to 300 ng/ml rCXCL12 tested as control. ( D ) Freshly isolated GC B cells were pre-incubated with or without PTX and subjected to chemotaxis to 300 ng/ml CX 3 CL1 or medium (nil). Results are median numbers of migrated cells, minimum and maximum values from three different experiments.

Article Snippet: CD10-FITC (MEM78 clone), anti-human IgD-FITC, CD95-FITC, CD44-FITC, anti-Bcl-2-FITC, and anti-Ki-67-FITC mAbs from DAKO (Glostrup, Denmark); unconjugated CD77 and CD39 mAbs from Immunotech, Marseille, France; unconjugated rabbit anti-human CX 3 CR1 (Serotec) that detects an epitope starting from 175 to 189 amino acids; unconjugated CD3, CD56, CD68, CD10, CD27, and anti-human IgM mAbs from DAKO.

Techniques: Purification, Staining, Expressing, Chemotaxis Assay, Control, Isolation, Incubation

Journal: PLoS ONE

Article Title: CX 3 CR1 Is Expressed by Human B Lymphocytes and Meditates CX 3 CL1 Driven Chemotaxis of Tonsil Centrocytes

doi: 10.1371/journal.pone.0008485

Figure Lengend Snippet: ( A ) CX 3 CR1 + and CX 3 CR1 − GC B cells were analyzed after staining with mAbs. Results are percent positive cells, minimum to maximum ranges from ten independent experiments. ( B ) V H 5(D)J H -μ rearrangement sequences were evaluated in CX 3 CR1 + and CX 3 CR1 − GC B cells. 104 molecular clones from five different CX 3 CR1 + GC B cell fractions were compared to 63 molecular clones from three different CX 3 CR1 − GC B lymphocyte fractions. 34 molecular clones from three different CD10 - CD27 − naive B cell fractions were tested as controls. Results are ratios between number of mutations and number of molecular clones.

Article Snippet: CD10-FITC (MEM78 clone), anti-human IgD-FITC, CD95-FITC, CD44-FITC, anti-Bcl-2-FITC, and anti-Ki-67-FITC mAbs from DAKO (Glostrup, Denmark); unconjugated CD77 and CD39 mAbs from Immunotech, Marseille, France; unconjugated rabbit anti-human CX 3 CR1 (Serotec) that detects an epitope starting from 175 to 189 amino acids; unconjugated CD3, CD56, CD68, CD10, CD27, and anti-human IgM mAbs from DAKO.

Techniques: Staining, Clone Assay

Journal: PLoS ONE

Article Title: CX 3 CR1 Is Expressed by Human B Lymphocytes and Meditates CX 3 CL1 Driven Chemotaxis of Tonsil Centrocytes

doi: 10.1371/journal.pone.0008485

Figure Lengend Snippet: ( A ) Double staining of CX 3 CR1 + GC B cells with CD27 and CD23 mAbs. Each B cell fraction was further characterized with mAbs (CD27 + cells, right lower panel; CD23 + cells, left lower panel). Results are median percent positive cells, maximum and minimum values from ten different experiments. ( B ) CX 3 CR1 + GC B cells were subjected to chemotaxis to 300 ng/ml rCX 3 CL1. Migrated cells were double stained for CD27 and CD23. One representative experiment out of seven is shown. ( C ) Purified GC B cells were subjected to CX 3 CL1-driven chemotaxis and migrated cells were collected and stained with a panel of mAbs. Results are median percent positive cells, maximum and minimum values from four different experiments.

Article Snippet: CD10-FITC (MEM78 clone), anti-human IgD-FITC, CD95-FITC, CD44-FITC, anti-Bcl-2-FITC, and anti-Ki-67-FITC mAbs from DAKO (Glostrup, Denmark); unconjugated CD77 and CD39 mAbs from Immunotech, Marseille, France; unconjugated rabbit anti-human CX 3 CR1 (Serotec) that detects an epitope starting from 175 to 189 amino acids; unconjugated CD3, CD56, CD68, CD10, CD27, and anti-human IgM mAbs from DAKO.

Techniques: Double Staining, Chemotaxis Assay, Staining, Purification

Journal: PLoS ONE

Article Title: CX 3 CR1 Is Expressed by Human B Lymphocytes and Meditates CX 3 CL1 Driven Chemotaxis of Tonsil Centrocytes

doi: 10.1371/journal.pone.0008485

Figure Lengend Snippet: ( A ) Splenocytes from WT mice double stained with anti-CX 3 CR1 and B220 mAbs were analyzed by flow cytometry. Histograms from two different WT mice are shown. ( B ) Splenocytes from WT mice were tested for chemotaxis to murine rCX 3 CL1 or rCXCL12 (control). Migrated B cells were enumerated by flow cytometry using anti-B220 mAb. Results are median number of migrated cells, minimum and maximum values from four different experiments. ( C ) CX 3 CR1 − / − , CX 3 CL1 − / − or WT mice were immunized with OVA and tested at different dilutions for specific IgG production by ELISA. Results were expressed as median optical densities (OD), maximum and minimum values, from ten different experiments. Serum from two WT mice was titrated for OVA IgG antibodies (1: 200, 1:500, 1:1000, 1:10000, 1:50000, 1:100000 dilutions) and used in each tests as standard curve, whereas serum from mice before immunization was used as negative control.

Article Snippet: CD10-FITC (MEM78 clone), anti-human IgD-FITC, CD95-FITC, CD44-FITC, anti-Bcl-2-FITC, and anti-Ki-67-FITC mAbs from DAKO (Glostrup, Denmark); unconjugated CD77 and CD39 mAbs from Immunotech, Marseille, France; unconjugated rabbit anti-human CX 3 CR1 (Serotec) that detects an epitope starting from 175 to 189 amino acids; unconjugated CD3, CD56, CD68, CD10, CD27, and anti-human IgM mAbs from DAKO.

Techniques: Staining, Flow Cytometry, Chemotaxis Assay, Control, Enzyme-linked Immunosorbent Assay, Negative Control