Journal: PLoS ONE

Article Title: Clonal Analysis of the T-Cell Response to In Vivo Expressed Mycobacterium tuberculosis Protein Rv2034, Using a CD154 Expression Based T-Cell Cloning Method

doi: 10.1371/journal.pone.0099203

Figure Lengend Snippet: The shown CD4 + T-cell clone that had been expanded was restimulated with the Rv2034 peptide pool and analyzed for the expression of CD154 expression, IFN-γ, TNF-α and IL-2 (black dots). Data is representative of over three independent experiments. CD154 and Th1 cytokine expression of non-activated T cells is indicated in grey dots. Dot blots show single live CD14 − CD19 − CD3 + CD4 + T cells. The frequency of all CD3 + CD4 + T cell subsets identified upon stimulation are indicated in the corners of each plot.

Article Snippet: All included, surface staining ; anti-CD3 PE-Cy5 or anti-CD3 PE-Cy7, (both BD Biosciences), anti-CD4 Texas Red (Caltag), anti-CD8 HorizonV500 (BD Biosciences), anti-CD14 PB (Invitrogen) and anti-CD19 PB (Invitrogen), ICS; anti-CD154 APC-Alexa eFluor 780 (eBioscience).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Clonal Analysis of the T-Cell Response to In Vivo Expressed Mycobacterium tuberculosis Protein Rv2034, Using a CD154 Expression Based T-Cell Cloning Method

doi: 10.1371/journal.pone.0099203

Figure Lengend Snippet: To identify the immunogenic epitope(s) in Rv2034, the CD4 + T-cell clone was stimulated with all individual Rv2034 20-mer peptides with 10 aa overlap; Rv2034 recombinant protein; Rv2034 peptide pool; control ESAT-6/CFP10 fusion protein; an Ag85B/ESAT-6/Rv2034 trimeric fusion protein; and negative and positive control conditions. Autologous irradiated PBMC were used as APCs. Both IFN-γ (open bars) and T-cell proliferation (black bars) were determined. CPM bars represent median ranging the highest and lowest value (n = 3) (A). To determine the Rv2034 p81–100 specific response by flow cytometry, the CD4 + T-cell clone was stimulated with Rv2034 p81–100 (B), Rv2034 protein (C) and Rv2034 p11–30 (D) using autologous irradiated PBMC, in the presence of BFA. Intracellular CD154 and Th1 cytokine expression was determined. Data is representative of three independent experiments. Flow cytometry plots show single live CD14 − CD19 − CD3 + CD4 + T cells, the frequency of all subsets of CD3 + CD4 + T cells are indicated in the corners of each plot.

Article Snippet: All included, surface staining ; anti-CD3 PE-Cy5 or anti-CD3 PE-Cy7, (both BD Biosciences), anti-CD4 Texas Red (Caltag), anti-CD8 HorizonV500 (BD Biosciences), anti-CD14 PB (Invitrogen) and anti-CD19 PB (Invitrogen), ICS; anti-CD154 APC-Alexa eFluor 780 (eBioscience).

Techniques: Recombinant, Positive Control, Irradiation, Flow Cytometry, Expressing

Journal: PLoS ONE

Article Title: Clonal Analysis of the T-Cell Response to In Vivo Expressed Mycobacterium tuberculosis Protein Rv2034, Using a CD154 Expression Based T-Cell Cloning Method

doi: 10.1371/journal.pone.0099203

Figure Lengend Snippet: The CD4 + T-cell clone was stimulated with Rv2034 p81–100 in presence of BFA and analyzed the expression of CD154 (A-D), IFN-γ and IL-17 (A) and additional markers: the transcriptional factor markers T-bet (Th1), GATA-3 (Th2), RORγt (Th17) and FoxP3 (Treg) (B); T-cell regulatory markers: CD39, IL-10 and CD25 (C); T-cell cytotoxicity markers: Granzyme B, CD107a and Perforin (D). Markers were analyzed using the mAb subset panels described in the materials and methods section. Data is representative for two independent experiments. Stimulated T cells are indicated in black and unstimulated T cells in grey. Dot blots show single live CD14 − CD19 − CD3 + CD4 + T cells. The frequency of all CD3 + CD4 + T cell subsets identified upon stimulation are indicated in the corners of each plot.

Article Snippet: All included, surface staining ; anti-CD3 PE-Cy5 or anti-CD3 PE-Cy7, (both BD Biosciences), anti-CD4 Texas Red (Caltag), anti-CD8 HorizonV500 (BD Biosciences), anti-CD14 PB (Invitrogen) and anti-CD19 PB (Invitrogen), ICS; anti-CD154 APC-Alexa eFluor 780 (eBioscience).

Techniques: Expressing