B-1a cells express membrane-bound IL-27

i27-Bregs and i35-Bregs are thought to suppress inflammation through secretion of their signature cytokines, IL-27 and IL-35 respectively. However, mechanisms by which the p35 and Ebi3 subunits of IL-35 or p28 and Ebi3 subunits of IL-27 associate in vivo to form the functional IL-35 or IL-27 cytokine are unknown. Recently, i35-Bregs were found to secrete i35-exosomes that suppress neuroinflammation (17, 24). This observation is akin to the finding that IL-35-producing Treg cells (iT_R35) display p35 on their cell surface and conferred regulatory functions to bystander T cells by coating them with IL-35⁺ extracellular vesicles (EVs) (24), suggesting that regulatory lymphocytes might express cell surface IL-35. In this study, we activated B-1a cells in-vitro for 72 hours by stimulation with anti-IgM/anti-CD40 and investigated whether the IL-27-producing B-1a cells express IL-27 on their cell surface or secrete exosomes that contain IL-27 (i27-exosomes). Intracellular cytokine expression analysis of the activated cells detected expansion of i27-Breg cells among CD19⁺CD5⁺CD23^-, a cell phenotype that characterizes the B-1a lymphocyte lineage ( Figure 2A ). However, it was not clear whether the IL-27 detected by intracellular cytokine staining assay was on the B-1a cell surface or derived from the coating of the activated B-1a cells with IL-27⁺ EVs or i27-exosomes. Surprisingly, when we stained the B-1a cells with p28 and Ebi3 antibodies and then performed the FACS analysis without permeabilizing the cells, we detected p28 and Ebi3 expression on CD19⁺CD5⁺CD23^- and CD19⁺CD5⁺CD23^-CD81⁺ cells, suggesting cell-surface expression of IL-27 on the B-1a cells and/or i27-exosomes ( Figure 2B ). The observation that IL-27 produced by B-1a cells might be membrane-bound is therefore surprising in view of the prevailing view that IL-27 is a soluble cytokine (4). To confirm that i27-Breg cells express membrane-bound IL-27, we cultured B-1a cells from the peritoneal cavity for 72 hours and performed immunohistochemical staining of the activated B-1a cells with antibodies specific to p28, Ebi3, as well as CD81, a tetraspanin protein which is also a BCR co-receptor (25). Visualization of IL-27 expression by confocal microscopy revealed colocalization of the p28 subunit to the plasma membrane in association with CD81 ( Figure 2C ). However, the staining pattern co-localized Ebi3 in the cytoplasm and endoplasmic reticulum (ER) with calreticulin, an ER resident protein or ER marker, suggesting that Ebi3 possibly localizes in lumen of the rough ER, with modest expression in the cytoplasm ( Figure 2D ; Supplementary Video-1 Information ).

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Figure 2

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).

B-1a cells secrete exosomes containing membrane-bound p28, Ebi3 and CD81

B-1a or B-2 cells were activated in-vitro by stimulation with LPS or anti-IgM/anti-CD40 (BCR) in exosome-depleted culture medium. EVs were isolated and purified using Exo-Quick-TC exosome precipitation solution and only EVs ranging in sizes between 50-150nm (exosomes) as determined by the nanoparticle tracking analysis (NTA) method ( Figure 3A ) were used in this study (17). Regardless of the stimulus used to activate the cells, substantial amounts of IL-27 containing EVs or i27-exosomes were detected in supernatants of B-1a cells derived from peritoneal cavity compared to similarly cultured B-2 or B-1a cells derived from the spleen ( Figure 3A ). This result was confirmed by ELISA ( Figure 3A , lower panel). To further increase amounts of exosomes for our studies, the sorted i27-Bregs from the peritoneal cavity were further expanded (>75%) by an additional 3-days cycle of stimulation with anti-IgM/anti-CD40 in exosome-depleted culture medium as previously described (17). qPCR analysis of purified i27-exosomes from 6 independent EV preparations confirmed that exosomes from each of the preparations expressed Ebi3 and IL-27p28 transcripts ( Figure 3B ). Western blot analyses also show expression of p28 and Ebi3 subunit proteins as well as the exosome-markers, CD63 and CD81 ( Figure 3C ). We also show that the exosome markers are enriched in exosomes isolated from supernatant of activated B-1a cells but absent in exosome-depleted supernatants ( Figure 3C ). Thus, the exosome-depleted supernatant that does not contain p28 or Ebi3 served as control in some experiments. This is consistent with use of exosome-depleted supernatants as controls for exosome preparation used in human clinical studies (26) or in characterizing exosomes used in rat EAU studies (27). Reciprocal co-immunoprecipitation analysis using antibodies specific to Ebi3 or p28 confirmed that the i27-exosomes express IL-27 ( Figure 3D ). Consistent with the preferential production of IL-27 by B-1a cells but not B-2 cells, B-1a cells secrete i27-exosomes but not i35-exosomes and conversely, B-2 cells secrete i35-exosomes but not i27-exosomes ( Figure 3E ). To directly demonstrate that the i27-exosomes express cell-surface IL-27, we incubated the i27-exosomes with the Tetraspanin Exo-Flow Capture Kit consisting of 9.1 µm diameter magnetic beads attached with streptavidin and tetraspanin-biotin antibodies as recommended by the manufacturer. After capture of the i27-exosomes on the magnetic beads we stained the beads with fluorescence-conjugated anti-p28, Ebi3 or CD81 antibodies and visualized specific binding and co-localization of p28, Ebi3 and CD81 on the i27-exosomes captured on Exo-Flow beads by confocal microscopy ( Figure 3F ). Interestingly, we detected CD81 in association with p28 on the surface of the i27-Breg cell while we localized CD81 expression with both p28 and Ebi3 on the i27-exosomes.

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Figure 3

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.

IL-27-Exosomes suppress experimental autoimmune uveitis

We next investigated whether i27-exosomes can be used to suppress EAU, a T cell mediated autoimmune disease in mouse (13, 20). We induced EAU in WT C57BL/6J mice by active immunization with IRBP_651-670 peptide in Complete Freund’s Adjuvant (CFA) and treated the mice with i27-exosomes. The control for our EAU studies are exosomes isolated from resting unstimulated B-1a cells (Control-Exosome). The exosomes 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 ( Figure 4A ). Mice received ~2x10¹⁰ exosomes (20ng IL-27) by retro-orbital injection 6 days after EAU induction and every day thereafter until day 10 post-immunization as described (28). Clinical symptoms of EAU is generally manifested between post-immunization (p.i.) day-12 and day-22 (29) and we monitored progression and severity of uveitis during this period by fundoscopy, a non-invasive procedure that allows visualization of fundus (retina, macula, optic disc, fovea and blood vessels) using the ophthalmoscope or fundoscopy. Fundus images obtained on day-14 p.i. showclassical features of uveitis which include papillitis, blurred optic disc margins and enlarged juxtapupillary areas (black arrows); retinal vasculitis (blue arrows); yellow-whitish retinal and choroidal infiltrates (white arrows) ( Figure 4B , left panel). On the other hand, the fundus images and bar graph of the clinical scores show a less severe disease in the eyes of the i27-exosome treated mice ( Figure 4B , right panels). Histology of the day-21 retinas of control mice exhibit hallmark EAU features characterized by infiltration of large numbers of inflammatory cells into the vitreous, destruction of retinal cells and development of retinal in-folding ( Figure 4C , top panel) while these pathological features were relatively mild in i27-exosome treated mice ( Figure 4C , bottom panel). Optical coherence tomography (OCT) is a noninvasive procedure for visualizing retina microstructure. Analysis of the retinas by OCT shows substantial accumulation of inflammatory cells in the vitreous and optic nerve head of the control mouse retina ( Figure 4D , top panel) but not the retinas of mice treated with i27-exosomes ( Figure 4D , bottom panels). Non-Treg cells have recently been shown to propagate immune tolerance and suppress inflammatory responses by coating their cell-surface with IL-35-containing EVs secreted by Treg cells (24, 30). Because retinal cells suppress uveitis through production of IL-27, we examined whether the amelioration of EAU observed in this study derived in part from passive acquisition of i27-exosomes. EAU was induced, treated with exosomes as described above and the mice were sacrificed on day 22 p.i. at the tail end of the inflammatory response that coincides with disease recovery phase. The mice were extensively perfused with saline solution to remove blood from the central retinal artery or choriocapillaris and enucleated eyes were fixed in 10% formalin, sectioned and stained with p28- or Ebi3-specific antibodies. Immunohistochemical analysis of the sections revealed that IL-27 expression localized to the photoreceptor layer of the mice treated with i27-exosomes compared the untreated mice ( Figure 4E ). This observation is of interest because the photoreceptor cells are the primary site of tissue destruction during EAU while microglia cells in the ganglion cell layer are thought to be the main producers of IL-27 in the retina (13, 15). However, recent in-vivo imaging studies have revealed that microglia cells are transiently recruited to the photoreceptor layer in response to focal injury of photoreceptor cells and presumed to play a role to limit further damage to the affected photoreceptors (31). Moreover, the detection of IL-27 in the photoreceptor layer is consistent with previous reports of expression of high levels of IL-27 receptor by photoreceptors (32). Together, these results suggest that adoptive i27-exosome immunotherapy can confer protection against severe uveitis in mice. However, future studies are required to clarify whether the IL-27 detected in the photoreceptor layer derived from passive acquisition of i27-exosomes or i27-exosome-mediated upregulation of IL-27.

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Figure 4

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 IRBP_651-670 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.

Amelioration of EAU in mice treated with i27-exosomes correlates with inhibition of pathogenic Th1/Th17 cell responses and expansion of regulatory T cells

Uveitis in mice is initiated by inflammatory cells that infiltrate the retina following the breakdown of the blood-retina-barrier and the pathogenic process is mediated primarily by Th17 and Th1 lymphocyte subsets (13, 33). We therefore investigated whether suppression of EAU observed in mice treated with i27-exosomes derived from inhibition of Th17 and/or Th1 cells by quantifying percentage of CD4⁺ T cells secreting the proinflammatory IL-17 and/or IFN-γ cytokines. Consistent with published reports, development of EAU in control mice was accompanied by significant increases of IL-17-expressing (Th17) and IFN-γ-expressing (Th1) cells in the draining lymph nodes (cervical and axillary lymph nodes) ( Figure 5A ). In contrast, we detected significantly lower percentages of IL-17-expressing and IFN-γ-expressing cells in mice treated with i27-exosomes ( Figure 5A ). In addition to its anti-inflammatory effects on proinflammatory lymphoid cells during EAU, we observed expansion of regulatory T cells in i27-exosome treated mice which correlated with amelioration of uveitis ( Figure 5B ). Although exact mechanism that induced expansion of regulatory T cells in i27-exosome treated mice is not clear, it is of note that i27-Breg immunotherapy ameliorates encephalomyelitis and uveitis in mice by propagating inhibitory signals that convert conventional B cells to regulatory lymphocytes secreting IL-10 and/or IL-35 in the eye, brain, or spinal cord (15). Moreover, in that study loss of IL-27Rα or IL-27 function abrogated capacity of i27-Bregs to suppress disease, suggesting critical role of IL-27 in the immune suppression (15). It is therefore conceivable that the IL-27 contained in i27-exosomes may promote differentiation and expansion of Tregs. Furthermore, decrease in percentages of CD11c⁺ or CD11b⁺F4/80⁺ myeloid cells in spleen of i27-exosome treated mice is also consistent with anti-inflammatory effects of i27-exosomes ( Figure 5C ).

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Figure 5

Amelioration of EAU in mice treated with i27-exosomes correlates with inhibition of pathogenic Th1/Th17 cell responses and expansion of regulatory T cells. Freshly isolated CD4⁺ T cells in the dLNs (axillary, cervical) of EAU mice were analyzed for expression of proinflammatory or immunosuppressive cytokines by the intracellular cytokine staining assay. Numbers in quadrants and the corresponding histograms indicate percentage of cells expressing IFN-γ and/or IL-17A (A) or Foxp3⁺CD25⁺ regulatory T cells (B) (n=4/group). (C) Cells isolated from the spleen were gated by FACS for myeloid cells and numbers in quadrants or the corresponding graphs indicate percentage of CD11c⁺ or CD11b⁺F4/80⁺ cells(n=4/group). The data are presented as the mean ± SD of a representative dataset from at least three determinations, *p<0.05, **p<0.01, ****p<0.0001.

Mice and ethics statement

Six- to 8-week-old C57BL/6J mice were purchased from Jackson Laboratory (Jackson Laboratory, Bar Harbor, ME). The mice were maintained and treated in accordance with National Eye Institute (NEI) and NIH Animal Care and Use Committee guidelines (Study # EY000262-19 & EY000372-14). All animal care and experimentation conformed to National Institutes of Health (NIH) guidelines and the experimental protocol was approved under NIH/NEI Animal Study Protocol (ASP) # NEI-597.

Cell sorting and i27-Breg activation

Primary mouse B cells isolated from the peritoneal cavity or spleen of C57BL/6J mice were isolated by use of kits purchased from Miltenyi Biotec including B cell Isolation kit (130–090–862), CD19 MicroBeads (130–052–201) and B-1a Cell Isolation Kit (130–097–413). Sorted CD19⁺ or B-1a cells were stimulated with LPS (5µg per ml, Sigma), anti-CD40 Abs (10µg per ml; BioXcell) and anti-IgM Abs (5µg per ml; Jackson ImmonoResearch) for 72 hours as described (15).

Experimental autoimmune uveitis

EAU was induced by active immunization of C57BL/6J mice with IRBP_651-670-peptide (300µg per mouse) in a 0.2 ml emulsion (1:1 v/v with complete Freund’s adjuvant (CFA) containing 2.5 mg per ml Mycobacterium tuberculosis strain H37RA (2.5 mg per ml) sub-cutaneous as described previously (29). Mice also received Bordetella pertussis toxin (1µg/mouse) by intraperitoneal (i.p.) injection in 100 µl of RPMI 1640 medium containing 1.0% normal mouse serum on the same day of immunization. Mice received ~2x10¹⁰ exosomes (20ng IL-27 per mouse) on day 6 post-immunization and every day until day 10 post-immunization by retro-orbital injection. For each study, 8 mice were used per group and they were matched by age and sex. Clinical disease was established and scored by fundoscopy and histology as described previously (16). Eyes were examined for disease severity using binocular microscope with coaxial illumination. Eyes for histology were enucleated 21 days post-immunization, fixed in 10% buffered formalin and serially sectioned in the vertical pupillary-optic nerve plane. All sections were stained with hematoxylin and eosin.

Fundoscopy

Funduscopic examinations were performed at day 10 to 21 after EAU induction. Briefly, following systemic administration of systemic anesthesia [intraperitoneal injection of ketamine (1.4 mg/mouse) and xylazine (0.12 mg per mouse), the pupil was dilated by topical administration of 1% tropicamide ophthalmic solution (Alcon Inc., Fort Worth, Texas). Fundus image was captured using Micron III retinal imaging microscope (Phoenix Research Labs) for small rodent or a modified Karl Storz veterinary otoendoscope coupled with a Nikon D90 digital camera, as previously described (41). To avoid subjective bias, evaluation of the fundus photographs was conducted without knowledge of the mouse identity by a masked observer. At least 6 images (2 posterior central retinal view, 4 peripheral retinal views) were taken from each eye by positioning the endoscope and viewing from superior, inferior, lateral and medial fields and each individual lesion was identified, mapped and recorded. The clinical grading system for retinal inflammation was as established (41, 42).

Imaging mouse retina by spectral-domain optical coherence tomography

SD-OCT is a noninvasive procedure that allows visualization of internal microstructure of various eye structures in living animals. An SD-OCT system with 820 nm center wavelength broadband light source (Bioptigen, NC) was used for in vivo non-contact imaging of eyes from control or EAU mice. Mice were anesthetized and the pupils dilated as described above. Mice were then immobilized using adjustable holder that could be rotated easily allowing for horizontal or vertical scan scanning. Each scan was performed at least twice, with realignment each time. The dimension of the scan (in depth and transverse extent) was adjusted until the optimal signal intensity and contrast was achieved. Retinal thickness was measured from the central retinal area of all images obtained from both horizontal and vertical scans from the same eye, using the system software, and averaged. The method used to determine the retinal thicknesses in the system software was as described (43).

Proliferation assay

B-1a and CD4+ T cells were harvested from WT C57BL/6J mice. The B-1a cells were stimulated with anti-CD40 Abs (10µg per ml) and anti-IgM Abs (5µg/ml) while the CD4⁺ T cells were stimulated with anti-CD3 (2µg/ml)/anti-CD28 (2µg/ml) Abs. Both cell types were cultured in medium containing control or i27-exosomes. Proliferative capacity of either cell type was assessed by FACS using anti-Ki67 Abs.

Flow cytometry

For intracellular cytokine detection, cells were stimulated for 4h with PMA (20ng per ml)/ionomycin (1µM). GolgiStop was added in the last hour, and intracellular cytokine staining was performed using BD Biosciences Cytofix/Cytoperm kit as recommended (BD Pharmingen, San Diego, CA, USA). FACS analysis was performed on a CytoFLEX Flow Cytometer (Beckman Coulter, Indianapolis, IN, USA) using protein-specific monoclonal antibodies and corresponding isotype control Abs (BD Pharmingen, San Diego, CA, USA) as described previously (44). As FACS analysis was performed on samples stained with mAbs conjugated with fluorescent dyes, each experiment was color-compensated. Dead cells were stained with dead cell exclusion dye (Fixable Viability Dye eFluor^® 450; eBioscience), and only live cells were analyzed. Quadrant gates were set using isotype controls with less than 0.2% background.

i27 exosome isolation

Isolation and purification of IL-27 producing B-1a cells (i27-Bregs) has previously been reported (15). Briefly, B cells isolated from mouse peritoneal cavity were activated with anti-IgM/anti-CD40 B-1a cells and non-B-1a cells were depleted by magnetic selection. The B-1a cells were then positively selected on CD5⁺ magnetic beads and we perform FACS analysis to confirm that i27-Bregs comprise >30% of the activated B-1a cells. We also routinely increase the percentage of i27-Breg cells by culturing the cells in exosome-depleted medium containing IL-27 (15). Supernatants from in vitro cultures of i27-Breg or control cells cultures are then subjected to sequential centrifugations at 300xg (10min), 3000xg (10 min) and 12000xg (10 min) and exosomes were isolated using the Exo-Quick-TC kit as previously described (17). For Western blot Analysis, the cleared supernatant was further subjected to ultracentrifugation at 100,000xg (2 h) followed by washing of the particulate fraction in PBS at 100,000xg (2 h). We measure exosome size distribution by Nanoparticle Tracking Analysis using the NanoSight system (Malvern Panalytical, MA, USA). Expression of exosome markers or IL-27 subunit proteins are then characterized and validated by Western blotting. Exosomes used in this study are isolated, purified and characterized according to recommendations of the International Society for Extracellular Vesicles (ISEV). Because B-1a cells are a relatively rare B cell population in the peritoneal cavity and each EAU study requires large amounts of exosomes, we routinely prepare several EV preparations which may contain varying amounts of i27-exosomes. For in vivo studies, we routine pool EV preparations isolated on the same day. The i27-exosomes are then characterized, quantified, aliquoted and stored frozen until use. We use the same batch of frozen i27-exosomes for each EAU experiment.

Exosome RNA quantification

For EV RNA qPCR, total RNA was isolated from i27-exosomes isolated from the culture supernatant of activated B-1a cells and control exosomes from unstimulated B1a cells using SeraMir Exosome RNA Isolation and Purification Kit (System Biosciences, Palo Alto, CA, USA). The Kit provides Spike RNA for use as internal control.

Immunofluorescence staining and confocal imaging analysis

B cells were activated as earlier described and adhered to glass slides. Briefly, the cells were fixed with BD-Fixation/permeabilization kit, adhered to glass slides by the Cytospin method or incubated for 30 min on Poly-L-lysine coated slide. Cells were blocked in 10% goat serum, 1% BSA, 0.1% Triton for 1 hour and in MOM blocking solution (Invitrogen) if the primary antibody is of mouse origin. Cells were then incubated with rabbit anti-CD81 (Cell Signaling), mouse anti-Ebi3 and rat anti-p28 (R&D system either MAB7430 or MAB18342). Cells were washed 3 time for 10 minutes and incubated with Alexa 488 (anti-rat) and Alexa 568 (anti-rabbit) and 647 (anti-mouse) secondary antibodies

ELISA

IL-35 and IL-27 cytokine in cell lysate or purified exosomes protein lysate were quantified using mouse IL-35 and IL-27-specific heterodimeric ELISA kit (BioLegend), following the protocol as recommended by manufacturer.

Immunoprecipitation and immunoblotting

Preparation of whole cell lysates and cleared lysates or cellular supernatants were immunoprecipitated with antibody that was pre-coupled to protein G-sepharose beads as described (45). Immunoprecipitates were resolved by SDS-PAGE and blots were probed with specific antibodies. The following antibodies were used for immunoprecipitation and/or Western blotting: p35(Abcam), p28 (Invitrogen), Ebi3, and β-actin (Santa Cruz, CA). Pre-immune serum was used in parallel as controls and signals were detected with HRP conjugated-secondary F(ab’)2 (Zymed Labs, San Francisco, CA) using ECL system (Amersham, Arlington Heights, IL).

We thank the NEI/NIH FLOW Cytometry Core facility for cell sorting and the NEI/NIH Imaging core facility for support with confocal microscopy.