Figure 3. Vaccination with exosomes from OVA-loaded and poly(I:C)-activated DCs induces the expansion and acquisition of effector functions of endogenous OVA-specific CD4⁺ and CD8⁺ T cells with negligible OVA-specific antibody titers in vivo.

(a) Wild-type mice were vaccinated with 50 μg of the indicated Dexo formulation or saline on day 0 (prime), 14 (boost I) and 40 (boost II). On day 45, spleens and LNs were collected to analyze OVA-specific T cell responses, and blood was sampled to measure the titer of OVA-specific IgG antibodies in the serum. (b) Pentamer staining and flow cytometric analysis were used to measure the frequency of SIINFEKL-specific CD8⁺ T lymphocytes in the blood of vaccinated mice on day 19 (left) or at the end of the experimental time on day 45 in the spleen (middle) and LNs (right) in the population of viable CD3ε⁺CD8α⁺ cells. (c) Splenocytes from vaccinated mice were collected on day 45 to measure acquisition of effector functions by SIINFEKL-specific CD8⁺ T lymphocytes as detected by intracellular staining for IFNγ (left) and Granzyme-B (right) and flow cytometric analysis. (d) Splenocytes from vaccinated mice were collected on day 45 to measure acquisition of effector functions by OVA-specific CD4⁺ T lymphocytes as detected by intracellular staining for IFNγ (left) and TNFα (right) and flow cytometric analysis. (e) Splenocytes from vaccinated mice were collected on day 45 to measure IFNγ or IL-10 secreted in the cell supernatant by ELISA upon restimulation in the presence of OVA. (f) Blood from vaccinated mice was collected on day 45 and the titer of OVA-specific IgG antibodies in the serum was measured by ELISA. Data represent mean ± SEM from 2 independent experiments (N = 10). Statistical analysis was performed by one-way ANOVA and Bonferroni post-hoc test correction. ^*P < 0.05 ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001 and n.s. = not significant for comparisons of Dexo(OVA + pIC) administered i.v. or i.d. with Dexo(OVA + LPS).