Confirmation That Ag-Specific Exosomes Mediate miRNA Delivered Suppression in Two Different DTH Systems Various findings of Ab-dependent, exosome-mediated suppression in hapten CS, were now repeated in protein Ag-induced DTH. exosome-coating FLC target APCs by binding to peptide-Ag-major histocompatibility complexes. This renders APCs capable of inhibiting DTH effector T cells. Thus, our studies describe a novel immune tolerance mechanism mediated by FLC-coated, Ag-specific, miRNA-150-carrying exosomes that act on the APC and are particularly effective after oral administration. = 5 mice in each group or three samples in each experimental repetition. In addition to biological activity, the descriptive characteristics and propertieslike phenotype of the assayed OVA Ag-specific suppressive exosomesresembled the hapten-specific exosome-like nanovesicles isolated from mice tolerized to hapten and also were produced by CD3+ and CD8+ T cells . OVA-specific exosomes highly expressed CD9 and CD81 tetraspanins with significantly lower expression of CD63 (Figure 1C). Noteworthy for the current OVA system, all of the exosomes expressing CD9 tetraspanin also co-expressed surface Ig FLC (Figure 1C, bottom center and right), but here were obvious high (about 10%) and low expressing (about 90%) EV and even exosomes of non-immune animals were slightly positive for the Ig FLC. In addition, transmission electron microscopy revealed the presence of vesicles with billamelar membrane (Figure 1D), characterized by average size around 150 nm, as determined by nanoparticle tracking analysis (Figure 1E). Importantly, these Ts cell-derived exosomes were determined to be biologically active by isolation and then testing for suppressive function of the Benserazide HCl (Serazide) subpopulation eluted from anti-CD9-linked affinity columns that was strongly Mouse monoclonal to BID suppressive compared to the flow through fraction that did not bind to the anti-CD9 columns (Figure 1F, Group D vs. C). Nanoparticle tracking analysis of OVA Ts cell exosomes eluted from the anti-CD9 affinity column showed the presence of small EV in the Benserazide HCl (Serazide) column eluate (Figure 1G). Thus, special phenotypic characteristics of small EVs, most likely exosomes, were surely linked to their function. The linkage of phenotype and function is not often tested elsewhere. In addition, when OVA-specific Ts cell-derived exosomes were separated on OVA-linked affinity column, they also strongly suppressed adoptively transferred DTH response (Figure 2A). Open in a separate window Figure 2 Ag specificity of suppressive exosomes shown by OVA Ag affinity chromatography and criss-cross Ag-testing, and the essential role of antigen-primed macrophages in tolerance induction. (A) OVA-induced DTH is suppressed only by exosomes present in the eluate of OVA Ag-affinity chromatography column separating the exosomes (Group D column eluate vs. Group C column flow Benserazide HCl (Serazide) through, 90% suppression vs. 0% suppression). (B) Ag specificity of suppressive exosome treatment employing analogous anti-KLH protein suppressor T cell exosomes from KLH Ag tolerized donors, that do not inhibit OVA DTH when given IP at the 24-h peak of the OVA-elicited DTH ear swelling response. (C) OVA-induced DTH cannot be suppressed by intravenous tolerization with systemic administration of high doses of OVA-linked syngeneic red blood cells in mice depleted of antigen-presenting macrophages (Group D vs. C). Results are shown as delta standard error (SE), one-way ANOVA with post hoc RIR Tukey test. = 5 mice in each group. In summary, all of these findings concerning phenotypic and descriptive properties of the OVA Ts exosome EV, fit with the recent most thorough description of small EVs of endosomal origin, i.e., exosomes . We also have linked this phenotype to the suppressive function of EVs by testing of their biological activity after separation on OVA Ag and anti-CD9 affinity columns (Figure 1F and Figure 2A). Therefore, we have classified the OVA-specific, Ts cell-derived, functionally active, suppressive nanovesicles as exosomes, a small subtype of the vast family of EVs. 2.4. In Vivo Testing of Ag-Specificity Benserazide HCl (Serazide) of the Ts Cell-Derived Exosomes Expression of FLC and binding to OVA in Ag-affinity chromatography suggested the Ag-specificity of Ts cell-derived exosome action. By employing of Ts cell-derived exosomes induced by the antigenically non-cross reactive protein keyhole limpet hemocyanin (KLH), we have shown that the exosome suppression Benserazide HCl (Serazide) is Ag-specific. In this case, KLH-specific exosomes harvested from lymphoid cells of mice identically tolerized to KLH, and similarly IP injected into actively OVA-immunized mice just.