30 research outputs found
Exosomes Released from Mycoplasma Infected Tumor Cells Activate Inhibitory B Cells
Mycoplasmas cause numerous human diseases and are common opportunistic pathogens in cancer patients and immunocompromised individuals. Mycoplasma infection elicits various host immune responses. Here we demonstrate that mycoplasma-infected tumor cells release exosomes (myco+ exosomes) that specifically activate splenic B cells and induce splenocytes cytokine production. Induction of cytokines, including the proinflammatory IFN-γ and the anti-inflammatory IL-10, was largely dependent on the presence of B cells. B cells were the major IL-10 producers. In splenocytes from B cell deficient μMT mice, induction of IFN-γ+ T cells by myco+ exosomes was greatly increased compared with wild type splenocytes. In addition, anti-CD3-stimulated T cell proliferation was greatly inhibited in the presence of myco+ exosome-treated B cells. Also, anti-CD3-stimulated T cell signaling was impaired by myco+ exosome treatment. Proteomic analysis identified mycoplasma proteins in exosomes that potentially contribute to the effects. Our results demonstrate that mycoplasma-infected tumor cells release exosomes carrying mycoplasma components that preferentially activate B cells, which in turn, are able to inhibit T cell activity. These results suggest that mycoplasmas infecting tumor cells can exploit the exosome pathway to disseminate their own components and modulate the activity of immune cells, in particular, activate B cells with inhibitory activity
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 5×10<sup>6</sup> 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 2.5×10<sup>6</sup> cells/well with or without 1 µg/ml of B16 myco+ exosomes for 24 hr, then 0.5×10<sup>6</sup> 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<sup>high</sup>CD62L<sup>low</sup> 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<sup>hi</sup>CD62L<sup>lo</sup> T cells: T cell subsets that are CD44<sup>high</sup>CD62L<sup>low</sup>. 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
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 in splenocytes by myco+ exosome treatment.
<p>(A) Splenocytes from C57BL/6 mice were cultured in a 24-well-plate at the density of 5×10<sup>6</sup> 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 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 5×10<sup>6</sup> 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