Only indirectly loaded exosomes are able to stimulate OVA-specific T-cell proliferation in vivo. (A) BALB/c mice were adoptively transferred with a total of 5.5 × 10⁶ purified CD4⁺ T cells from DO11.10 mice and immunized either with PBS or 50 μg unloaded (Exo), directly (Pep-Exo) or indirectly (OVA-Exo) loaded exosomes. After 3 days of immunization, the percentage of (B,D) KJ1-26⁺/CD3⁺ or (C) KJ1-26⁺/CD69⁺ DO11.10 cells per spleen was determined by flow cytometry or (E) the spleens were sectioned and stained with KJ1-26 (DO11.10 T cells; red) and anti-B220 (B cells; green). The pictures shown are representative of 2 independent experiments. Bar represents 150 μm. For panel C, ***P < .001. (F) Splenocytes from DO11.10 mice were stained with CFSE and adoptively transferred to the BALB/c mice followed by OVA-Exo injection the day after. Spleens were taken 3 days later, and CFSE division was assessed in the flow cytometry. A representative experiment with 1 mouse of 4 is shown. (G) Spleen sections from mice adoptively transferred with DO11.10 CD4⁺ T cells and immunized with OVA-Exo were also stained with KJ1-26 (DO11.10 T cells; red), anti-B220 (B cells; pseudo-colored blue) and anti-CD11c (DCs; green). Original magnification: left, ×20; bar represents 150 μm; right, ×100; bar represents 15 μm.