Journal: PLoS ONE

Article Title: Tumor-Derived Microvesicles Induce, Expand and Up-Regulate Biological Activities of Human Regulatory T Cells (Treg)

doi: 10.1371/journal.pone.0011469

Figure Lengend Snippet: ( A ) Purified CD3 + CD4 + T cells were labeled with CFSE and cultured as described in ± TMV or DC-derived MV (5 µg/mL). On days 3, 5 and 8, the frequency of CD4 + CD25 + FOXP3 + Treg among proliferating T cells was determined by flow cytometry. The data (means ± SD) represent three independent experiments (*p<0.01). ( B ) Proliferating CD3 + CD4 + T cells (squares) were tested for co-expression of CD25 in a representative co-culture ± TMV. A higher proportion of proliferating CD4 + T cells expressed CD25 in the co-culture with TMV than without TMV. ( C ) The proliferating CD4 + CD25 + T cells in the co-cultures with TMV were evaluated for the frequency of FOXP3 + T cells upon gating on the CD4 + CD25 high subset (see box). Over 90% of these cells also expressed intracellular FOXP3. Data are representative for one out of 6 cultures tested.

Article Snippet: Total CD3 + CD4 + T cell fractions or isolated CD4 + CD25 + cells were cultured in AIMV medium with plate-bound OKT3 (1 µg/mL; American Type Culture Collection), soluble anti-CD28 Abs (1 µg/mL) and IL-2 (150 IU/mL) at 37°C/5%CO 2 in wells of 96-wells plates.

Techniques: Purification, Labeling, Cell Culture, Derivative Assay, Flow Cytometry, Expressing, Co-Culture Assay

Journal: PLoS ONE

Article Title: Tumor-Derived Microvesicles Induce, Expand and Up-Regulate Biological Activities of Human Regulatory T Cells (Treg)

doi: 10.1371/journal.pone.0011469

Figure Lengend Snippet: ( A ) Flow cytometry analysis of TGF-β1 and IL-10 expression on TMV purified from OVCAR-3 SN and coated onto latex beads. ( B ) CD4 + CD25 high FOXP3 + T cells were cultured with OKT3, anti-CD28 and IL-2 (150 IU/mL) +/− TMV for 72 h at 37°C in the presence of Golgistop and then stained for CD4, CD3, CD25 and intracellular TGF-β1 and IL-10. Expression of both cytokines was up-regulated in the presence of TMV (p<0.05). ( C ) SMAD2/3 and STAT3 phosphorylation in Treg before and after exposure to TMV. Representative results are from one of three independent experiments for A , B and C . ( D ) The percentage of CD4 + CD25 high FOXP3 + T cells increased among CD4 + CD25 + T cells cultured in the presence of TMV but not DC-derived MV. Neutralizing anti-TGF-β1 and/or anti-IL-10 Abs inhibited the induction of Treg by TMV. Non-blocking IgG isotype control Abs were used as controls. Asterisks indicate decreases (p<0.05) in Treg percentages in the presence of neutralizing Abs. Results are means ± SD of three independent experiments.

Article Snippet: Total CD3 + CD4 + T cell fractions or isolated CD4 + CD25 + cells were cultured in AIMV medium with plate-bound OKT3 (1 µg/mL; American Type Culture Collection), soluble anti-CD28 Abs (1 µg/mL) and IL-2 (150 IU/mL) at 37°C/5%CO 2 in wells of 96-wells plates.

Techniques: Flow Cytometry, Expressing, Purification, Cell Culture, Staining, Derivative Assay, Blocking Assay

Journal: PLOS Neglected Tropical Diseases

Article Title: A comparative analysis of the dendritic cell response upon exposure to different rabies virus strains

doi: 10.1371/journal.pntd.0012994

Figure Lengend Snippet: MoDCs were exposed to RABV strains dogRV, SHBRV, and SAD P5, at MOIs of 1 and 5, or stimulated with 10 ng/mL of LPS, for 48 h. CFSE-labeled autologous T cells were added at a ratio of 10:1 for 2.5 days. As negative (neg) and positive (pos) controls, T cells were cultured without moDCs, with or without CD3/CD28 activation beads. Dashed lines separate the results for co-cultures with moDCs exposed to the different conditions (left) and cultures of only T cells as controls (right). A Proliferated and non-proliferated T cell populations of one representative donor (donor 2), as determined by measurement of sideward scatter (SSC) and CFSE fluorescence intensities. B Percentage of proliferated T cells for all donors. Colors represent the different donors and triangles show mean values. C Cytokine concentrations in co-culture supernatants. Tiles represent mean values of all donors. T cell only conditions were excluded from statistical analysis. n = 6 individual donors. Only statistical comparisons with mock controls are shown. *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05.

Article Snippet: The CD14 + and CD3 + cell fractions were isolated from PBMCs using magnetic activated cell sorting according to manufacturer’s guidelines (Miltenyi Biotec) to obtain monocytes and T cells, respectively.

Techniques: Labeling, Cell Culture, Activation Assay, Fluorescence, Co-Culture Assay