Old changes (increase/decrease) in production of cytokines after 5 days in CD3+ T cells 15900046 incubated with IL-2, Exosomes, or IL-2+Exosomes. doi:10.1371/journal.pone.0049723.tpreviously described [34] [16] [20]. The size of pelleted structures was determined with dynamic light scattering (DLS) using a Zetasizer Nano. The results showed that the pellet consisted of particles with an average size of 54 nm in diameter consistent with Table 2. Human cytokine array (Chemokines).characteristic size range of exosomes (Figure 1A). The isolated exosomes stained positive for the canonical exosome markers CD81, CD63 and CD9 using flow cytometry (Figure 1B). Moreover, the presence of T cell specific KS-176 site proteins as well as otherChemokinesAlternate Name IL-2 Up-reg. get Indolactam V Down-reg. Exosomes Up-reg. 7x 0.4x 0.3x 0.2x 6x 1x 12x 9x 1x 5x 0.5x 0.2x 1x 1x 1x Down-reg. IL-2+Exosomes Up-reg. 13x 2x 15x 12x 2x 0.3x 3x 13x 1x 0 Down-reg.CCL1 CCL2 CCL3 CCL4 CCL5 CXCL1 CXCL8 CXCL10 CXCL11 CXCLI-309 MCP-1 MIP-1a MIP-1b RANTES GROa IL8 IP-10 I-TAC SDF-10x 1x 8x 1x 9xFold changes (increase/decrease) in production of chemokines after 5 days in CD3+ T cells incubated with IL-2, Exosomes, or IL-2+Exosomes. doi:10.1371/journal.pone.0049723.tProliferation of T Cells with IL2 and ExosomesTable 3. Human cytokine array (Other proteins).ProteinsAlternateName IL-2 Up-reg. Down-reg. 0.2 1x 0 1x Exosomes Up-reg. 4x 1x 0.4x 1x Down-reg. IL-2+Exosomes Up-reg. 8x 1x 0.4x 1x Down-reg.sICAM-1 C5a Serpin E1 sTREM-1 (Receptor)CD54 Complement Component 5a PAI-1 soluble Triggering Receptor Expressed on Myeloid cells-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. doi:10.1371/journal.pone.0049723.timmune associated proteins was examined on the exosomes using flow cytometry. The results showed that the vesicles were additionally positive for CD3 (Figure 1B), previously reported by Tumne et al [25], but not for CD4, CD40, ICAM-1, MHC-I, MHC-II, CD80 or CD25 (Figure 1B).Exosomes together with IL-2 Generate a Different Cytokine Production in T cells Compared to Exosomes or IL-2 aloneCultures stimulated with exosomes only. Samples stimulated with exosomes alone for five days showed significant production of macrophage migration inhibitory factor (MIF), IL16 and granulocyte-macrophage colony-stimulating factor (GMCSF), GM-CSF is also known by the alternate name CSF2 (Figure 3, Table 1). Interestingly the level of IL-16 was higher in these cultures compared to cultures stimulated with IL-2 only or exosome together with IL-2. Furthermore there was a low but significant production of the chemokine CCL1 (Figure 3). When comparing fold changes of the cytokines present at high level, GMCSF, IL-16, and MIF were increased more than eight-fold at day five (Figure 4, Table 1). The chemokine CCL1 had a seven-fold increase at day five compared to day 0 (Figure 4, Table 2). However, a notable difference was seen in exosome stimulated cells where the exosomes as such seemed to contain significant amounts of CCL5 (RANTES) i.e. immediately after the addition of exosomes (at 0 h) the supernatants showed relatively large amounts of RANTES. These levels gradually declined and, showed a more than five times decrease at day five (Figure 4 and Table 2). The two proteins tumor necrosis factor (TNF) -a and intercellular adhesion molecule 1 (ICAM1) were present but at low levels and showed a more than two-fold increase (Figure 4 and Ta.Old changes (increase/decrease) in production of cytokines after 5 days in CD3+ T cells 15900046 incubated with IL-2, Exosomes, or IL-2+Exosomes. doi:10.1371/journal.pone.0049723.tpreviously described [34] [16] [20]. The size of pelleted structures was determined with dynamic light scattering (DLS) using a Zetasizer Nano. The results showed that the pellet consisted of particles with an average size of 54 nm in diameter consistent with Table 2. Human cytokine array (Chemokines).characteristic size range of exosomes (Figure 1A). The isolated exosomes stained positive for the canonical exosome markers CD81, CD63 and CD9 using flow cytometry (Figure 1B). Moreover, the presence of T cell specific proteins as well as otherChemokinesAlternate Name IL-2 Up-reg. Down-reg. Exosomes Up-reg. 7x 0.4x 0.3x 0.2x 6x 1x 12x 9x 1x 5x 0.5x 0.2x 1x 1x 1x Down-reg. IL-2+Exosomes Up-reg. 13x 2x 15x 12x 2x 0.3x 3x 13x 1x 0 Down-reg.CCL1 CCL2 CCL3 CCL4 CCL5 CXCL1 CXCL8 CXCL10 CXCL11 CXCLI-309 MCP-1 MIP-1a MIP-1b RANTES GROa IL8 IP-10 I-TAC SDF-10x 1x 8x 1x 9xFold changes (increase/decrease) in production of chemokines after 5 days in CD3+ T cells incubated with IL-2, Exosomes, or IL-2+Exosomes. doi:10.1371/journal.pone.0049723.tProliferation of T Cells with IL2 and ExosomesTable 3. Human cytokine array (Other proteins).ProteinsAlternateName IL-2 Up-reg. Down-reg. 0.2 1x 0 1x Exosomes Up-reg. 4x 1x 0.4x 1x Down-reg. IL-2+Exosomes Up-reg. 8x 1x 0.4x 1x Down-reg.sICAM-1 C5a Serpin E1 sTREM-1 (Receptor)CD54 Complement Component 5a PAI-1 soluble Triggering Receptor Expressed on Myeloid cells-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. doi:10.1371/journal.pone.0049723.timmune associated proteins was examined on the exosomes using flow cytometry. The results showed that the vesicles were additionally positive for CD3 (Figure 1B), previously reported by Tumne et al [25], but not for CD4, CD40, ICAM-1, MHC-I, MHC-II, CD80 or CD25 (Figure 1B).Exosomes together with IL-2 Generate a Different Cytokine Production in T cells Compared to Exosomes or IL-2 aloneCultures stimulated with exosomes only. Samples stimulated with exosomes alone for five days showed significant production of macrophage migration inhibitory factor (MIF), IL16 and granulocyte-macrophage colony-stimulating factor (GMCSF), GM-CSF is also known by the alternate name CSF2 (Figure 3, Table 1). Interestingly the level of IL-16 was higher in these cultures compared to cultures stimulated with IL-2 only or exosome together with IL-2. Furthermore there was a low but significant production of the chemokine CCL1 (Figure 3). When comparing fold changes of the cytokines present at high level, GMCSF, IL-16, and MIF were increased more than eight-fold at day five (Figure 4, Table 1). The chemokine CCL1 had a seven-fold increase at day five compared to day 0 (Figure 4, Table 2). However, a notable difference was seen in exosome stimulated cells where the exosomes as such seemed to contain significant amounts of CCL5 (RANTES) i.e. immediately after the addition of exosomes (at 0 h) the supernatants showed relatively large amounts of RANTES. These levels gradually declined and, showed a more than five times decrease at day five (Figure 4 and Table 2). The two proteins tumor necrosis factor (TNF) -a and intercellular adhesion molecule 1 (ICAM1) were present but at low levels and showed a more than two-fold increase (Figure 4 and Ta.