Figure 6.

Incubation of OT-II mouse OVA-Mac-EVs with anti-OVA-323 antibodies leads to their aggregation and enhances their suppressive activity. (A) OT-II mouse OVA-Mac-EVs were incubated alone or with biotinylated anti-OVA-323 IgG overnight at 4 °C. After ultracentrifugation, both OVA-Mac-EV preparations were subjected to nanoparticle tracking analysis (NTA). (B) OT-II mouse OVA-Mac-EVs were incubated alone or with biotinylated anti-OVA-323 IgG overnight at 4 °C. After ultracentrifugation, both OVA-Mac-EV preparations were absorbed onto cupper grid, negatively stained with 3% uranyl acetate, and visualized with TEM microscope. (C) OT-II mouse OVA-Mac-EVs were incubated alone or with biotinylated anti-OVA-323 IgG overnight at 4 °C. After ultracentrifugation, both OVA-Mac-EV preparations were used for 30 min treatment at 37 °C of OT-II mouse DTH effector T cells that had been pre-incubated with OVA-323 peptide for 20 min at 37 °C. Then, DTH effector T cells were adoptively transferred to naive wild type recipients (n = 5 per group) that 24 h later were challenged with OVA to elicit DTH reaction, measured as ear swelling 24 h later. (D) Scheme showing the proposed mechanism of enhancement of OVA-Mac-EVs’ suppressive activity against OVA-specific DTH effector T cells, in some instances pre-incubated with OVA-323 peptide, induced by anti-OVA-323 IgG antibodies. NTA results are shown as mean ± SD, and DTH ear swellings are expressed as delta ± SEM. One-way ANOVA with post hoc RIR Tukey test; ** p < 0.01.