Journal: PLoS ONE

Article Title: Identification of Mycobacterium tuberculosis -Specific Th1, Th17 and Th22 Cells Using the Expression of CD40L in Tuberculous Pleurisy

doi: 10.1371/journal.pone.0020165

Figure Lengend Snippet: PFCs from TBP and PBMCs from healthy donors were incubated for eight hours in the presence of medium alone, ESAT-6/CFP-10 peptides with anti-CD28 plus anti-CD49d or anti-CD3 plus anti-CD28 monoclonal antibodies (mAbs).(A) Expression of CD40L on CD4 + and CD8 + T cells in PFCs from TBP and (B) PBMCs from healthy donors were detected by FACS. Data shown are representative of five independent experiments.

Article Snippet: Purified anti-CD28 (clone CD28.2) and anti-CD49d (clone 9F10) mAbs were purchased from BD Biosciences (San Jose, CA).

Techniques: Incubation, Expressing

Journal: PLoS ONE

Article Title: Identification of Mycobacterium tuberculosis -Specific Th1, Th17 and Th22 Cells Using the Expression of CD40L in Tuberculous Pleurisy

doi: 10.1371/journal.pone.0020165

Figure Lengend Snippet: PFCs were stimulated with ESAT-6/CFP10 peptides, anti-CD28 and anti-CD49d mAbs. CD4 + CD40L + and CD4 + CD40L − cells were gated.(A) CD4 + CD40L + but not CD4 + CD40L − T cells express IFN-γ, IL-2, TNF-α, IL-17 or IL-22. The numbers in the quadrants are the percentages of cells. (B) Ratio of IFN-γ, IL-2, TNF-α, (C) IL-17 and IL-22 expression by CD4 + CD40L + or CD4 + CD40L − cells. Results shown are the mean ± SEM from 12 to 21 independent experiments. * P <0.05; ** P <0.01; *** P <0.001. (D) Histogram graph of T-bet expression by CD4 + CD40L + T cells. Filled histogram represents isotype control staining and open histogram represents T-bet staining. Data shown are representative of five independent experiments.

Article Snippet: Purified anti-CD28 (clone CD28.2) and anti-CD49d (clone 9F10) mAbs were purchased from BD Biosciences (San Jose, CA).

Techniques: Expressing, Staining

Journal: PLoS ONE

Article Title: Identification of Mycobacterium tuberculosis -Specific Th1, Th17 and Th22 Cells Using the Expression of CD40L in Tuberculous Pleurisy

doi: 10.1371/journal.pone.0020165

Figure Lengend Snippet: PFCs were stimulated with ESAT-6/CFP-10 peptides, anti-CD28 and anti-CD49d mAbs for eight hours. For detection of intracellular cytokines, anti-IFN-γ, anti-IL-2 and anti-TNF-α monoclonal antibodies were labeled with the same fluorescence. The staining of CD40L and cytokines of IFN-γ/IL-2/TNF-α were conducted in one FACS tube.(A) Th1 cytokine (IFN-γ, IL-2 or TNF-α) producing and nonproducing cells were gated. The expression of CD40L was evaluated. (B) Summary of the CD40L expression data within Th1 cytokine producing and nonproducing cells. Horizontal lines represent mean ± SEM. (C) CD4 + IFN-γ + and CD4 + IFN-γ − cells within CD4 + CD40L + T cells were further gated, and the expression of IL-2 and TNF-α are shown. (D) The total antigen response within CD4 + CD40L + T cells was defined as the number of cells expressing any combination of IFN-γ, IL-2 or TNF-α. The average percentages for each subset are shown. (E) The percentages of cells producing three cytokines (triple positive), two cytokines (double positive) or only one cytokine (single positive) within the total CD4 + CD40L + T cell response. Data shown are the mean values from five independent experiments.

Article Snippet: Purified anti-CD28 (clone CD28.2) and anti-CD49d (clone 9F10) mAbs were purchased from BD Biosciences (San Jose, CA).

Techniques: Labeling, Fluorescence, Staining, Expressing

Journal: PLoS ONE

Article Title: Identification of Mycobacterium tuberculosis -Specific Th1, Th17 and Th22 Cells Using the Expression of CD40L in Tuberculous Pleurisy

doi: 10.1371/journal.pone.0020165

Figure Lengend Snippet: PFCs were stimulated with ESAT-6/CFP-10 peptides, anti-CD28 and anti-CD49d mAbs. CD4 + CD40L + and CD4 + CD40L − T cells were gated. (A) The expression of CD45RA, CD45RO, CD62L, CCR7 and (B) the expression of activation markers CD69, CD25, HLA-DR and ICOS by CD4 + CD40L + and CD4 + CD40L − T cells. One representative datapoint from five independent experiments is shown.

Article Snippet: Purified anti-CD28 (clone CD28.2) and anti-CD49d (clone 9F10) mAbs were purchased from BD Biosciences (San Jose, CA).

Techniques: Expressing, Activation Assay

Journal: PLoS ONE

Article Title: Identification of Mycobacterium tuberculosis -Specific Th1, Th17 and Th22 Cells Using the Expression of CD40L in Tuberculous Pleurisy

doi: 10.1371/journal.pone.0020165

Figure Lengend Snippet: PFCs were stimulated with ESAT-6/CFP-10 peptides, anti-CD28 and anti-CD49d mAbs in the presence of a fluorescently labeled anti-CD40L monoclonal antibody and monensin. CD4 + T cells were first isolated with magnetic-beads. (A) The expression of CD40L within purified CD4 + T cells was demonstrated, and (B) CD4 + CD40L + and CD4 + CD40L − T cells were further sorted by flow cytometry. (C) Sorted CD4 + CD40L + or (D) CD4 + CD40L − T cells were co-cultured with purified CD14 cells independently in the presence of ESAT-6/CFP-10 peptides. The intracellular expression of IFN-γ, IL-2 or TNF-α by sorted CD4 + CD40L + and CD4 + CD40L − T cells was detected by flow cytometry. (E) The amounts of IFN-γ, IL-2, TNF-α, IL-17 and IL-22 were measured supernatants were measured using ELISA.

Article Snippet: Purified anti-CD28 (clone CD28.2) and anti-CD49d (clone 9F10) mAbs were purchased from BD Biosciences (San Jose, CA).

Techniques: Labeling, Isolation, Magnetic Beads, Expressing, Purification, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay