Fig 2: i27-Exosomes express membrane-bound p28, Ebi3 and CD81. (A) B-1a or CD19+ B-2 cells from mouse peritoneal cavity or spleen were stimulated with LPS or anti-IgM/anti-CD40 in exosomes-depleted medium for 72hrs (n=3-4/group). Exosomes in supernatant were purified and protein content in 5x106 exosomes was quantified by BCA protein assay (top panel) and amount of IL-27 secreted (pg/50µg exosome protein) was determined by ELISA (bottom panel) (n=3-4/group). (B-D) Exosomes isolated from supernatant of the BCR-activated B-1a cells were subjected to qPCR analysis (B) (n=6/group), Western blot analysis (C) (n=1/group) or reciprocal immunoprecipitation analysis (D). Controls in these analyses are exosome-depleted supernatant of B1a cell cultures. (E) Cell lysates and exosomes from activated B1a or B-2 cells were analyzed for IL-35 or IL-27 by ELISA(n=3/group). (F) For localization of IL-27 (p28 and Ebi3) and CD81 expression on the i27-Exosome membrane, the exosomes were captured with Tetraspanin Exo-Flow Capture Kit consisting of 9.1 µm diameter magnetic beads, stained with antibodies without fixation or permeabilization and then analyzed by confocal microscopy. The data are presented as the mean ± SD of at least three determinations, *p<0.05; **P < 0.01; ***P < 0.001; ****p<0.0001.

Fig 3: IL-27-expressing exosomes (i27-Exosomes) suppress experimental autoimmune uveitis (EAU). The exosomes used for treatment were isolated, ranged in size between 50 to 150nm and met criteria set by the 2018 International Society for Extracellular Vesicles (ISEV-18) for designation of EVs as exosomes (A) (n=3/group). Control for our EAU studies are exosomes isolated from resting unstimulated B-1a cells (Control-Exosome). EAU was induced in C57BL/6j mice by immunization with IRBP651-670 in CFA and disease progression was assessed by fundoscopy, histology and optical coherence tomography (OCT). (B) Representative fundus image of retina at day 14 after EAU induction reveal inflammation with blurred optic disc margins and enlarged juxtapupillary area (black arrows), retinal vasculitis (blue arrows) and yellow-whitish retinal and choroidal infiltrates (white arrows). Clinical scores and disease severity were based on changes at the optic nerve disc or retinal vessels and retinal and choroidal infiltrates (n=8/group). (C) Compared to mice treated with i27-exosome, histological images of day-21 eyes of mice that received the control exosome show substantial numbers of inflammatory cells in retina. H&E histological sections: Scale bar, 100µM. V, vitreous; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE/CH retinal pigmented epithelial and choroid. Blue arrows, lymphocytes; Asterisks, retinal folds(n=8/group). (D) Representative OCT images show marked increase of inflammatory cells (white arrows) in the vitreous of mice treated with control exosomes(n=8/group). (E) For the analysis of IL-27 expression in retinas of EAU mice treated with exosomes, the mice were extensively perfused with saline solution and enucleated eyes were fixed in paraffin and IL-27 expression was detected by immunohistochemical analysis (white arrow heads). Retina pigmented epithelium (RPE); Photoreceptors (R/C); outer nuclear layer (ONL); outer plexiform layer (OPL); inner plexiform layer (IPL). The data are presented as the mean ± SD of at least three determinations, **P < 0.01.

Fig 4: B-1a cells from the peritoneal cavity express membrane-bound IL-27. (A) B-1a cells from mouse peritoneal cavity were stimulated for 72 hours with anti-IgM/anti-CD40 and analyzed by intracellular cytokine staining assay. Graphs show absolute numbers of p28+, Ebi3+ or p28+Ebi3+ B cells in CD19+CD5+CD23- compartment (left panel) and IL-27-producing B-1a cells (right panel) (n=3/group). (B) B-1a cells were stimulated for 72 hours with anti-IgM/anti-CD40. The B-1a cells were stained with p28, Ebi3 or CD81 antibodies and FACS analysis was performed without permeabilizing the cells. Left panel shows absolute numbers of CD19+CD5+CD23- B-1a cells in the peritoneal cavity expressing IL-27 subunit proteins (p28, Ebi30 proteins). Right panel shows absolute numbers of B cells in CD19+CD5+CD23-CD81+ compartment that expressed p28 and/or Ebi3 on the B-1a plasma membrane(n=3/group). (C, D) Analysis and localization of cell surface expression of p28, Ebi3 or CD81 on activated i27-Breg cells by immunohistochemical staining and confocal microscopy (representative of three individual experiments).