PMC

(A) FACS and ELISA analysis of MHC Class II expression on plasma-derived exosomes (Pexo) from MO5 tumor mice. 10 µg of Pexo or BSA (negative control) were used in ELISA. (B) MHC Class II+ vesicles were depleted from MO5-FLAG Pexo (21d) by MACS beads and used in the DTH experiment. Undepleted, MHC Class II-depleted or IgG-depleted MO5-FLAG Pexo (10 µg) were administered into the footpad of OVA-immunized mice. The DTH magnitudes 24 and 48 h post-challenge are shown. n=5 except the IgG depletion group (n=4). *: P<0.05; NS: not significant. MHC Class II and CD9 expression on exosomes (10 µg) before and after depletion were analyzed by ELISA. (C) Wild type, MHC Class II or Class I deficient mice were inoculated with MO5 tumor and Pexo isolated 21 days post-inoculation. OVA-immunized mice were treated with 10 µg of Pexo at the time of antigen challenge. The DTH magnitudes 24 and 48 h post-challenge are shown. n=6. Significance at: **, P<0.01; *, P<0.05; NS: not significant. MHC Class II expression on exosomes (10 µg) was analyzed by ELISA.

(A) Mice were inoculated with B16, MO5, EL4 or EG7 tumor cells and blood was collected 3 weeks post-inoculation. Exosome-like vesicles isolated from the blood plasma were examined by electron microscopy. (B) Mice pre-sensitized with OVA antigen were injected with 10 µg of plasma-derived exosomes (Pexo) into the right hind paw and challenged with 30 µg of OVA at both hind paws. Paw swelling was measured 24 and 48 h post-challenge. The mean increase of footpad thickness of the treated paws (right paws) in PBS group at each time point was set to 1, and the increases of footpad thickness in the other groups were normalized as fold increase. Data represent the pooled results of two independent experiments and are the means ± SD with 9–11 mice per group. Significance at: **, P<0.01; *, P<0.05. (C) H&E staining of the footpad tissue at 48 h post-challenge. Unchallenged footpad tissue was included as a negative control. Magnification: 20×.

(A) Infection of B16 and MO5 cells with retroviruses packaged with FLAG-Lactadherin construct generated stable cell lines (B16-FLAG and MO5-FLAG) that constantly release FLAG-tagged tumor exosomes (Texo). Left: Western blotting for FLAG on Texo isolated from tumor culture supernatant (#3 and #4, two individual stable cell lines, #4 were used in the following experiments). Exosomes derived from uninfected B16 and MO5 tumor cells were included as negative controls. The MVB marker Alix was used as a loading control for Texo. 10 µg of protein was loaded per lane. Right: ELISA detection of FLAG on MO5-FLAG Texo (2-fold dilution, 20 µg-0.16 µg) and MO5 Texo (20 µg). (B) Blood were collected from mice bearing B16, B16-FLAG, MO5 or MO5-FLAG tumor at indicated time points after tumor inoculation and plasma-derived exosomes (Pexo) were isolated respectively. Left: Western blotting for FLAG on Pexo isolated from B16 or B16-FLAG tumor mice 5, 11 or 18 days post tumor inoculation. CD9 was used as a loading control for Pexo. Right: ELISA detection of FLAG on Pexo (15 µg) isolated from MO5 or MO5-FLAG tumor mice 7, 14 or 21 days post-inoculation. MO5-FLAG Texo mixed with Pexo of naïve mice (naïve Pexo) at different percentages (10 µg, 5 µg, 2.5 µg and 1.25 µg in a total of 15 µg proteins) were used for a standard curve. Naïve Pexo was used as a negative control.