Fig 2: IFN-? promoted CCL5 expression on macrophages, promoting leukocytes infiltration into the plaque area. (a) Female Apoe-/- mice (n = 15) were put on a Western diet for eight weeks, then the aortic cells were pooled to prepared as described in the methods and stained with an antibody against CD45. Then, CD45+ leukocytes and CD45- non-leukocytes were sorted by FACS, and qPCR was used to analyze Ccl5 mRNA expression. (b and c) Immunofluorescence of the aortic root. The Apoe-/- mice (n = 8) and the Batf3-/- Apoe-/- mice (n = 8) were fed a Western diet for 12 weeks. The cryosections of the aortic root were stained with an antibody against CD45 (red), MAC3 (red), and CCL5 (green). The cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (blue). Images were viewed and captured with a Laser Scanning Confocal Microscope. Scale bars: 100 µm, dashed lines indicate the internal elastic lamina, arrows pointing to representative colocalized cells. (d) Primary splenic macrophages were sorted as described in supplementary data, and then treated with100 ng/mL IFN-? for 6 h. Ccl5 mRNA expression was analyzed by qPCR. The Apoe-/- mice (n = 8) and the Batf3-/- Apoe-/- mice (n = 8) were fed a Western diet for 6 weeks, and cryosections of the aortic root were performed. (e) H&E staining. Scale bars: 100 µm. (f) Immunohistochemistry. Representative images of leukocytes (CD45), T cells (CD3), DCs (CD11c), and macrophages (Mac3) in the aortic are shown. Scale bars: 200 µm. Data are presented as mean ± SD. Differences of a P < 0.05 were considered to be statistically significant. **P < 0.01; ***P < 0.001; ns, not significant. Data are representative of three independent experiments.

Fig 3: Thymic exosomes induce maturation of thymocytes.After three days in culture with thymic CD11c+ DCs and/or thymic exosomes (a), spleen exosomes (b), 100 nm latex beads (c), RNase treated thymic exosomes (d) or Proteinase K treated thymic exosomes (e); thymocytes were stained for surface expression of CD4+S1P1+Qa2+. After three days in culture with C57BL/6 or Balb/c thymic CD11c+ DCs, or C57BL/6 or Balb/c thymic exosomes, the C57BL/6 thymocytes were stained for surface expression of CD4+S1P1+Qa2+ (f). Left figure shows representative flow cytometry plots with indicated stimulations and surface markers. Right figure shows normalized flow cytometry data from at least three individual experiments. Data from exosome experiments are representative from at least three to five individual experiments.

Fig 4: Exosomes are taken up by thymic DCs and CD4+ thymocytes.SP CD4+ thymocytes and thymic DCs were isolated and stained with CD4 PE-Cy7 and CD11c APC, respectively. After addition of FITC-stained thymic exosomes, cells were harvested after 15, 30, 60 and 240 min, and analyzed on an ImageStream X Mark II, imaging flow cytometry. (a) Representative images of SP CD4+ thymocytes (red, left) and thymic CD11c+ DCs (red, right) at indicated time points (min) after addition of FITC-stained thymic exosomes (green). (b) Representative cytometry plots of SP CD4+ thymocytes (top) or CD11c+ thymic DCs (bottom) at indicated time points after addition of thymic exosomes. (c) Kinetics of exosome uptake by SP CD4+ thymocytes (dashed line) and thymic DCs (solid line). (d) Representative images of SP CD4+ thymocytes (red) 240 min after addition of untreated FITC-stained thymic exosomes (green, top left), FITC stained 100 nm latex beads (green, top right), FITC stained thymic exosomes treated with Proteinase K (green, bottom left) or FITC stained thymic exosomes treated with RNase (green, bottom right). (e) Representative cytometry plots of SP CD4+ thymocytes (top) or CD11c+ thymic DCs (bottom) at indicated time points after addition of FITC stained 100 nm latex beads. (f) Amounts of cell associated thymic exosomes, normalized to untreated CD11c+ DCs or SP CD4+ thymocytes, 240 min after addition to RNase or Proteinase K treated SP CD4+ thymocytes and CD11c+ DCs. FITC-dye centrifuged in the same way as FITC stained exosomes was added to SP CD4+ thymocytes or tymic DCs, without generating any detectable staining of the cells.

Fig 5: Thymic exosomes suppress formation of CD4+CD25+FoxP3+ thymocytes.After three days in culture with thymic CD11c+ DCs and/or thymic exosomes, the thymocytes were stained for surface expression of CD4+CD25+FoxP3+. (a) Representative flow cytometry plot with indicated stimulations (left) and normalized flow cytometry data from 6 individual experiments with the indicated stimulations (right). Data is displayed as means ± SEM. (b) Representative diagram of CD4+CD25- thymocytes cultured for three days with CD11c+ DCs and indicated concentrations of thymic exosomes followed by staining for surface expression of CD4+CD25+FoxP3+. Data is representative for two individual experiments. (c) After three days in culture with thymic CD11c+ DCs and/or 100 nm latex beads thymocytes were stained for surface expression of CD4+CD25+FoxP3+. After three days in culture with thymic CD11c+ DCs and/or RNase (d) or Proteinase K (e) treated thymic exosomes, the thymocytes were stained for surface expression of CD4+CD25+FoxP3+. Data is representative of three individual experiments. After three days in culture with C57BL/6 or Balb/c thymic CD11c+ DCs, or C57BL/6 or Balb/c thymic exosomes, the C57BL/6 thymocytes were stained for surface expression of CD4+CD25+FoxP3+ (f). Data is pooled from two independent experiments and displayed as means ± SEM.