Calibration experiments with CD34⁺ Kasumi-1 exosomes isolated by ultracentrifugation.

<p>A) Isolated exosomes were loaded at increasing protein concentrations onto gels and blotted using anti-CD34 Ab. Intensity of each band was measured in pixels. The graph illustrates a linear relationship between exosomal protein levels and pixel intensity in Western blots. B) Isolated exosomes (20 µg protein) were added to increasing volumes of CD34 microbeads <i>(left</i>). Five or 10 µL of microbeads were sufficient to capture 20 µg of exosomes. Next, a 10 µL aliquot of beads was used to capture increasing concentrations of Kasumi-1 exosomes (<i>right</i>). The graph shows that the capacity of CD34 microbeads to capture up to 80 µg of input exosomes increased linearly. However, at 80 µg only about 40% of exosomal proteins were captured, suggesting that additional microbeads are necessary for capture of all Kasumi-1 exosomes.</p

Capture of CD34⁺ blast-derived exosomes directly from AML patients' plasma.

<p>A) Negatively stained electron microscope images of exosomes captured on CD34 microbeads (* shows a vacant microbead). B) Increasing AML plasma volumes were used for capture of CD34⁺ exosomes by microbeads and recovered exosomes were studied by Western blots. The graph shows a linear relationship between the input plasma volumes and pixel densities of captured and blotted CD34⁺ exosomes. C) Exosomes were captured from plasma samples obtained from five CD34⁺ AML patients and were analyzed by Western blots. The percentage of leukemic blasts in the peripheral circulation of each of the patients is shown. CD81 serves as the exosome marker. D) Removal of platelet-derived exosomes from plasma using anti-CD61 Ab-coated microbeads prior to capture of CD34⁺ exosomes. Exosomes captured with CD61 microbeads (<i>left</i>: CD61⁺) and CD34⁺ exosomes captured after removing CD61⁺ exosomes (<i>right</i>: CD61neg/CD34⁺) are shown in a representative Western blot of three evaluated.</p

Characteristics of captured exosomes.

<p>A) A representative Western blot profile of the captured CD34⁺ exosomes and of non-captured CD34^neg exosomes which were isolated by ultracentrifugation from the same AML plasma sample. B) After co-incubation of NK cells with CD34⁺ exosomes captured directly from AML plasma or CD34^neg exosomes as described in materials and Methods, NKG2D expression (in MFI) was found to be down-regulated only with CD34+ exosomes (<i>red</i>) compared to CD34^neg exosomes (<i>green</i>) or control without exosomes (<i>blue</i>). The <i>gray</i> peak denotes isotype control. In contrast, NKp46 expression was down-regulated by co-incubation with CD34^neg exosomes.</p

Capture of CD34+ exosomes from total exosomal fractions by CD34+ microbeads.

<p>Isolated Kasumi-1 exosomes (Kas) and total exosome fractions isolated by ultracentrifugation from normal donor plasma (NC) or from CD34⁺ AML patient plasma (AML) were used for capture with CD34 microbeads. After the 1^st capture and removal of beads, 2^nd capture was performed with a fresh aliquot of CD34 microbeads. The final unbound fractions were ultracentrifuged to collect remaining exosomes. While the 2^nd capture was necessary to recover all CD34⁺ Kasumi-1 exosomes, a single capture was sufficient to recover all CD34⁺ exosomes from the bulk of exosomes isolated from the AML plasma. There were no CD34⁺ exosomes captured from the bulk exosomal fraction isolated from a normal donor's plasma. CD81 expression indicates that the unbound fractions still contain CD34^neg exosomes.</p

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

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 increase suppressor activity of Treg.

<p>The FLOCA was used to simultaneously measure suppression proliferation of CFSE-labeled autologous CD4⁺CD25^neg RC and their apoptosis upon co-incubation with CFSE-negative Treg. RC cells stimulated with OKT3, anti-CD28 mAb and IL-2 (150 IU/mL) were co-cultured for 5 d with Treg pre-incubated or not with TMV. At harvest, cells were stained with 7-AAD and examined by flow cytometry. The suppressor assays were performed at the S:RC ratio of 1∶1. Treg pre-incubated with TMV induced higher levels of apoptosis (<b><u>A</u></b>) and greater inhibition of RC proliferation (<b><u>B</u></b>). The data are from one experiment of five performed. When Treg were pretreated with Concanamycin A or GrB inhibitor I and then incubated with TMV, the frequency of 7-AAD⁺ RC was lower (<b><u>C</u></b>) as was RC proliferation inhibition (<b><u>D</u></b>). Treg pretreated with FasL Ab and then incubated with TMV induced RC death (<b><u>C</u></b>) and inhibited RC proliferation (<b><u>D</u></b>).</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

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

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