Myco+ exosomes induce B cell activation and expansion.

<p>Splenocytes were treated with 1 µg/ml of B16 myco+ exosomes or B16 myco− exosomes, or cultured untreated for 72 hr. Cells were harvested and analyzed by FACS. (A) Expression of CD25, CD40, CD86, CD80, CD23, IgD, IgM, CD1d, CD5 and CD43 in the B cell gate (CD19+B220+). (B) Percentage of B cells in total splenocytes within the live cell gate after exosome treatment. Data represents the mean ± SD of four independent experiments. Significance at: *, P<0.05. (C) Expression of CD25, CD69, CD44, CD62L, CD80 and CD86 in the CD4+ T cell gate and the CD8+ T cell gate. Black line: B16 myco+ exosome treatment; grey line: B16 myco− exosome treatment; grey solid: untreated.</p

Cytokine induction by myco+ exosomes after exosome membrane disruption or mycoplasma removal reagent treatment of parental cells.

<p>(A) Mycoplasma-infected cells were treated with Plasmocin for 2 wk and tested to be mycoplasma-free. (B) B16 and EL4 myco+ exosomes were subjected to 5 cycles of freeze/thaw (F/T) or sonication (sonic). Splenocytes were treated with 1 µg/ml of myco+ exosomes, F/T exosomes, sonic exosomes or exosomes derived from Plasmocin-treated cells (plasmo) for 72 hr. IL-10 production was measured by ELISA. (C) IFN-γ production measured by ELISA. Induction of IL-10 and IFN-γ by plasmo exosomes was significantly reduced compared with intact, F/T and sonic exosomes. Significance at: *, P<0.05.</p

Intracellular cytokine staining of myco+ exosome-treated splenocytes.

<p>WT splenocytes were cultured with or without 1 µg/ml of B16 myco+ exosome for 48 hr in 24-well-plate at 5×10⁶ cells/1.5 ml media/well with 30 U/ml of rmIL-2. Brefeldin A was added to the culture for the last 6 hr before cells were harvested. Cells were first surface stained for CD19, B220, CD4 and CD8, and then stained for intracellular IL-10 and IFN-γ. (A) Percentage of IL-10+ cells in the B cell, CD4+ T cell and CD8+ T cell gates. Numbers in each plot represent % cells in each cell gate. Figures show the data of one representative experiment of three with similar results. (B) Fold increase of % IL-10+ cell in the B cell, CD4+ cell and CD8+ cell gate. Data represents the mean ± SD of three independent experiments. Significance at: *, P<0.05. (C) Percentage of IL-10+ B cells, IL-10+ CD4+ cells and IL-10+ CD8+ cells in total splenocytes in untreated or B16 myco+ exosome-treated splenocytes. Data represents the mean ± SD of three independent experiments. Significance at: *, P<0.05. (D) Percentage of IFN-γ+ cells in the B cell, CD4+ T cell and CD8+ T cell gates. Numbers in each plot represent % cells in each cell gate. Figures show the data of one representative experiment of three with similar results. (E) Fold increase of % IFN-γ+ cell in the B cell, CD4+ cell and CD8+ cell gate. Data represents the mean ± SD of three independent experiments. (F) Percentage of IFN-γ+ B cells, IFN-γ+ CD4+ cells and IFN-γ+ CD8+ cells in total splenocytes in untreated or B16 myco+ exosome-treated splenocytes. Data represents the mean ± SD of three independent experiments. Significance at: *, P<0.05.</p

Cytokine induction in splenocytes by myco+ exosome treatment.

<p>(A) Splenocytes from C57BL/6 mice were cultured in a 24-well-plate at the density of 5×10⁶ cells/1.5 ml media/well in the presence of 30 U/ml rmIL-2 and were treated with either myco+ exosomes or myco− exosomes (1 µg/ml), or left untreated for 72 hr. The IL-10 and IFN-γ levels (pg/ml) in the culture supernatants were measured by ELISA. Treatments were conducted in duplicates or triplicates in each experiment. Data represent the averaged cytokine levels ± SD of three independent experiments. (B) Dose-dependent cytokine induction by myco+ exosomes. Splenocytes were treated with an increasing dose of myco+ exsosomes (0.1, 1 and 10 µg/ml) for 72 hr, and the cytokine levels were measured by ELISA. Treatments were conducted in duplicates. Data represent the averaged cytokine levels ± SD of three independent experiments. Significance at: *, P<0.05.</p

The induction of IFN-γ-producing T cells by myco+ exosomes increases in the absence of B cells.

<p>WT or μMT spleen cells were cultured with or without 1 µg/ml of B16 myco+ exosome for 48 hr and stained for intracellular IFN-γ. (A) Induction of IFN-γ+CD8+ T cells in WT and μMT splenocyte cultures. Data shows one representative experiment of three with similar results. Numbers in each plot represent % cells in CD8+ cell gate. (B) Fold increase of % IFN-γ+ cells in the CD8+ cell gate in WT and μMT splenocytes upon B16 myco+ exosome treatment. Data shows the mean ± SD of three independent experiments. Significance at: *, P<0.05. (C) Induction of IFN-γ+CD4+ T cells in WT and μMT splenocyte cultures. Data shows one representative experiment of three with similar results. Numbers in each plot represent % cells in the CD4+ cell gate. (D) Fold increase of % IFN-γ+ cells in the CD4+ cell gate in WT and μMT splenocytes upon B16 myco+ exosome treatment. Data shows the mean ± SD of three independent experiments. Significance at: *, P<0.05.</p

Cytokine induction by myco+ exosomes in WT and μMT splenocytes.

<p>Splenocytes from either WT mice or μMT mice were cultured in 24-well-plate at 5×10⁶ cells/1.5 ml media/well with 30 U/ml of rmIL-2 and treated with 1 µg/ml of B16 myco+ exosomes or left untreated for 72 hr. IL-10 and IFN-γ levels in the culture supernatants were measured by ELISA. Treatments were conducted in triplicates in each experiment. Data represents the averaged cytokine levels ± SD of three independent experiments. Significance at: *, P<0.05.</p

T cell proliferation is inhibited when co-cultured with myco+ exosome-treated splenocytes or purified B cells.

<p>Splenocytes (T cell-depleted) or purified splenic B cells were cultured in 24-well-plate at 2.5×10⁶ cells/well with or without 1 µg/ml of B16 myco+ exosomes for 24 hr, then 0.5×10⁶ of CFSE-labeled T cells (CD45.1+) were added to the culture and stimulated with 10 µg/ml of anti-CD3e. Cells were co-cultured for another 3 days and T cell proliferation was analyzed by CFSE dilution. (A) Gating of CD45.1+CD8+ T cells and CD45.1+CD4+ T cells. Expression of CD44 and CD62L were shown within each T cell gate in non-treated and B16 myco+ exosome treated co-cultures. Non-treated cells without anti-CD3e were included as an unstimulated control. T cells that are CD44^highCD62L^low represent the activated T cell subset. (B) Proliferation of CD8+ T cells and CD4+ T cells in myco+ exosome-treated splenocytes shown by CFSE dilution. Total T cells: total CD8+ or CD4+ T cells. CD44^hiCD62L^lo T cells: T cell subsets that are CD44^highCD62L^low. Unstimulated: Non-treated T cells without anti-CD3 stimulation. (C) Proliferation of CD8+ T cells and CD4+ T cells when co-cultured with myco+ exosome-treated B cells, shown by CFSE dilution.</p

Suppression of OVA-specific DTH response by local administration of MO5 exosomes.

<p>Mice pre-sensitized with OVA were injected with 10 µg of B16 exosomes, 10 µg of MO5 exosomes or PBS alone in their right hind paws and were challenged with OVA at both hind paws. Paw swellings were measured 24 h and 48 h post-challenge. (A) Representative results showing the increase in footpad thickness (×0.01 mm) of treated and contralateral paws. n = 5. (B) Pooled results of two independent experiments showing the fold increase in footpad thickness as compared to the treated paws in PBS group. n = 10. **: P<0.01; *: P<0.05; NS: not significant.</p

Characterization of tumor exosomes.

<p>(A) EM micrographs of exosomes isolated from EL4, EG7, B16 and MO5 cell culture supernatants. (B) Western blot analysis of exosomes and cell lysates. 10 µg of proteins were loaded per lane. (C) IP detection of OVA protein (40∼45 kD) in both cell lysates and exosomes. (D) FACS analysis of MHC class I, MHC class II and CD81 expression on cells and exosomes.</p

Tumor exosomes inhibit BMDC maturation and induce TGF-β1 production.

<p>(A) Day 8 BMDCs (purity >90%) were treated with 10 µg/ml of tumor exosomes or cultured untreated for 3 days. The expression of I-A^b and CD86 were analyzed by FACS. LPS treatment (1 µg/ml) for 24 h was used as a DC maturation control. (B) TGF-β1 protein levels (pg/ml) in DC culture supernatants after exosome treatment. Data show the mean values of two independent experiments ± SD. (C) TGF-β1 contents in exosome preparations (pg/10 µg of exosomes). For each exosome sample, the data shown represent the mean value of three preparations ± SD.</p