TMV-associated TGF-β1 and IL-10 promote Treg expansion.

<p>(<b><u>A</u></b>) Flow cytometry analysis of TGF-β1 and IL-10 expression on TMV purified from OVCAR-3 SN and coated onto latex beads. (<b><u>B</u></b>) CD4⁺CD25^highFOXP3⁺ 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). (<b><u>C</u></b>) SMAD2/3 and STAT3 phosphorylation in Treg before and after exposure to TMV. Representative results are from one of three independent experiments for <b><u>A</u></b>, <b><u>B</u></b> and <b><u>C</u></b>. (<b><u>D</u></b>) The percentage of CD4⁺CD25^highFOXP3⁺ 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.</p

CD4⁺CD25^highFOXP3⁺ T cells and microvesicles (MV) in cancer patients and normal controls (NC).

<p>(<b><u>A</u></b>) Percentages of CD4⁺CD25^highFOXP3⁺ Treg in PBMC of cancer patients and NC. (<b><u>B</u></b>) the protein content/10 mL of serum or ascites in cancer patients and NC. The data in <b><u>A</u></b> and <b><u>B</u></b> are mean values ± SD. (<b><u>C</u></b>) Flow analyses of IL-10, TGF-β1 and FasL expression in MV purified from the ascites of OvCa patients and coated on latex beads. (<b><u>D</u></b>) Western blots of TMV isolated from OvCa SN. Molecular weights of the detected proteins are indicated. (<b><u>E</u></b>) Percentages of CD4⁺CD25⁺FOXP3⁺ cells in 8-day co-cultures of CD4⁺CD25^neg T cells with TMV obtained from various sources and used at increasing concentrations. The asterisks indicate a significant increase at p<0.05.</p

TMV promote differentiation of human Treg in culture.

<p>(<b><u>A</u></b>) Purified CD3⁺CD4⁺ T cells were labeled with CFSE and cultured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011469#s4" target="_blank">Materials and Methods</a> ± 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><u>B</u></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. (<b><u>C</u></b>) 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.</p

TMV Convert CD25^neg T cells to Treg.

<p>(<b><u>A</u></b>) Flow cytometry histograms of cultured (d5) CD4⁺CD25^neg T cells showing conversion of CD25^neg T cells into CD25⁺ T cells ± TMV or DC-derived MV (<i>left panel</i>) and expression of FOXP3 in the converted CD4⁺CD25⁺ T cells (<i>right panel</i>) in the same cultures. (<b><u>B</u></b>) A phenotypic profile of CD4⁺CD25^high T cells present in 7 day cultures of CD4⁺CD25⁺ T cells ± TMV or DC-derived MV. T cells were stained with various mAbs and evaluated by multiparameter flow cytometry. The gate is set on CD4⁺CD25^high T cells. The data are mean percentages ± SD of positive cells from three independent experiments. (<b><u>C</u></b>) MFI for FasL, IL-10, TGF-β1, granzyme B and perforin expression in CD4⁺CD25^high T cells cultured as described in (<b><u>B</u></b>) ± TMV. The data are representative of three independent experiments.</p

TMV promote expansion of human Treg in culture.

<p>The fold expansion of fresh (<i>left panel</i>) or rapamycin-expanded (<i>right panel</i>) CD4⁺CD25^high T cells to which TMV or DC-derived MV were added on day 0. Cells were stimulated with OKT3, anti-CD28 Abs and IL-2 (500 IU/mL) and cultured for 14–21 d. The data are means ± SD of six independent co-cultures. Asterisks indicate significant differences (p<0.05) between the cultures ± TMV.</p

CD4⁺CD25^high Treg are resistant to TMV-induced death.

<p>(<b><u>A</u></b>) Trypan blue positive cells after 6 h incubation ± TMV or DC-derived MV in primary T-cell subsets and CD8⁺ Jurkat cells (mag ×200) *p<0.001. (<b><u>B</u></b>) Percentages of ANXV binding to fresh CD4⁺CD25^high T cells or CD8⁺ Jurkat cells incubated ± TMV for 6 h. The data are representative dot plots from one of five independent experiments.</p