Characterization of exosomes from CD3⁺ T cells stimulated with IL-2, anti-CD3 and anti-CD28.

<p>(A) Particle sizes in ultracentrifuge pellet consistent with size range of exosomes. Average exosome size was 54 nm. Measured with dynamic light scattering (B) Exosomes bound to latex beads and stained with antibodies against exosome associated proteins (CD9, CD63 andCD81) and T cell associated proteins (CD3, CD4, CD25, CD40, CD80, CD86, MHC-I, MHC-II and ICAM-1) measured with flow cytometry. Dotted line represents isotype control.</p

A comparison of cytokines and chemokines present in the supernatant of CD3⁺ T cells pulsed with IL-2, exosomes or IL-2+exosomes.

<p>Fold changes in the production of cytokines, chemokines and other proteins after five days. T cells stimulated with IL-2 or exosomes had different expression of cytokines and chemokines. Samples stimulated with “exosomes+IL-2″ generated secretion of more cytokines and chemokines compared to samples stimulated with either IL-2 or exosomes alone. A significant decrease could be noticed for CCL5 in cultures stimulated with exosomes only.</p

Proliferation of CD3⁺ T cells pulsed with autologous exosomes.

<p>(A) Number of CD3⁺ T cells measured with automatic cell counting (Sysmex) at different time points. Cells stimulated with IL-2+exosomes are increasing in numbers. (B) Proliferation of CD3⁺ T cells measured with MTT assay at day five. Cells stimulated with IL-2+exosomes showed increased proliferation. (C) Distribution of CD4⁺ and CD8⁺ cells in CD3⁺ T cells stimulated with IL-2, autologous exosomes or IL-2+exosomes measures with flow cytometry.</p

Human cytokine array (Cytokines).

<p>Fold changes (increase/decrease) in production of <b>cytokines</b> after 5 days in CD3⁺ T cells incubated with IL-2, Exosomes, or IL-2+Exosomes.</p

Human cytokine array (Other proteins).

<p>Fold changes (increase/decrease) in production of other proteins after 5 days in CD3⁺ T cells incubated with IL-2, Exosomes, or IL-2+Exosomes.</p

Human cytokine array (Chemokines).

<p>Fold changes (increase/decrease) in production of <b>chemokines</b> after 5 days in CD3⁺ T cells incubated with IL-2, Exosomes, or IL-2+Exosomes.</p

Cytokine production from exosome+IL-2 stimulated CD3⁺ T cells at day zero (0 h) and day five (120 h).

<p>Relative quantification of spot intensities was performed using Quantity One software (Bio-Rad). Each bar represents an average of the intensity from two protein spots. White bars represent 0 h and grey bars represent 120 h (day 5). The cytokines IL-5, IL-13 and GM-CSF as well as the chemokines CCL3 and CCL4 were present at higher levels at day five.</p

Cytokine production from autologous exosome stimulated CD3⁺ T cells at day zero (0 h) and day five (120 h).

<p>Relative quantification of spot intensities was performed using Quantity One software (Bio-Rad). Each bar represents an average of the intensity from two protein spots. White bars represent 0 h and grey bars represent 120 h (day 5). The exosomes appeared to contain significant amounts of CCL5 (RANTES) immediately after the addition of exosomes (at 0 h) since the supernatants showed relatively large amounts of RANTES. These levels were decreased at day five.</p

Cytokine production from IL-2 stimulated CD3⁺ T cells at day zero (0 h) and day five (120 h).

<p>Relative quantification of spot intensities was performed using Quantity One software (Bio-Rad). Each bar represents an average of the intensity from two protein spots. White bars represent 0 h and grey bars represent 120 h (day 5). Cytokines IL-5, MIF, and GM-CSF (CSF2) were present at a high level in the supernatant after five days.</p

Oxidative stress induced by H₂O₂ results in a dose related loss of viability.

<p>Dose response relationship between viability of cultured MC/9 cells (%) and concentration of H₂O₂ (50 µM-500 µM) for 24 h. The dose versus viability correlation coefficient was 0.86.</p