Insulinoma-Released Exosomes or Microparticles Are Immunostimulatory and Can Activate Autoreactive T Cells Spontaneously Developed in Nonobese This information is current as Diabetic Mice of September 28, 2021. Huiming Sheng, Saleema Hassanali, Courtney Nugent, Li Wen, Emma Hamilton-Williams, Peter Dias and Yang D. Dai J Immunol 2011; 187:1591-1600; Prepublished online 6 July Downloaded from 2011; doi: 10.4049/jimmunol.1100231 http://www.jimmunol.org/content/187/4/1591 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Insulinoma-Released Exosomes or Microparticles Are Immunostimulatory and Can Activate Autoreactive T Cells Spontaneously Developed in Nonobese Diabetic Mice

Huiming Sheng,* Saleema Hassanali,* Courtney Nugent,* Li Wen,† Emma Hamilton-Williams,‡ Peter Dias,* and Yang D. Dai*

Exosomes (EXO) are secreted intracellular microparticles that can trigger inflammation and induce Ag-specific immune responses. To test possible roles of EXO in autoimmunity, we isolated small microparticles, mainly EXO, from mouse insulinoma and examined their activities to stimulate the autoimmune responses in NOD mice, a model for human type 1 diabetes. We demonstrate that the EXO contains strong innate stimuli and expresses candidate diabetes autoantigens. They can induce secretion of inflammatory

cytokines through a MyD88-dependent pathway, and activate purified APC and result in T cell proliferation. To address whether Downloaded from EXO or the secreted microparticles are possible autoimmune targets causing islet-specific inflammation, we monitored the T cell responses spontaneously developed in prediabetic NOD mice for their reactivity to the EXO, and compared this reactivity between diabetes-susceptible and -resistant congenic mouse strains. We found that older NOD females, which have advanced islet de- struction, accumulated more EXO-reactive, IFN-g–producing lymphocytes than younger females or age-matched males, and that pancreatic lymph nodes from the prediabetic NOD, but not from the resistant mice, were also enriched with EXO-reactive Th1 cells. In vivo, immunization with the EXO accelerates insulitis development in nonobese diabetes-resistant mice. Thus, EXO or http://www.jimmunol.org/ small microparticles can be recognized by the diabetes-associated autoreactive T cells, supporting that EXO might be a possible autoimmune target and/or insulitis trigger in NOD or congenic mouse strains. The Journal of Immunology, 2011, 187: 1591–1600.

tissue-specific autoimmune response is thought to occur alterations (8). This indicates that a triggering event may be as- following a triggering event, which could be from an sociated with initiating the early insulitis, and that progression A exogenous infectious pathogen, an endogenous super- from insulitis to complete destruction of the pancreatic islets and antigen, a physiological stress event, or a noninfectious environ- subsequent diabetes may then be due to a dysfunctional immune mental agent (1). For most autoimmune diseases, such events have regulatory network (9, 10). not been well defined or are too diverse to be confirmed. In the It has previously been suggested that a wave of pancreatic islet by guest on September 28, 2021 NOD mice, what has been an excellent model for studying genetic cell apoptosis during islet development and remodeling in early life susceptibility to human type 1 diabetes (T1D) (2), the existence might be the triggering event (11). However, such physiological and/or characteristics of such triggering agents or events likewise cell death must also occur frequently in resistant mice and whose remain unclear. One interesting feature using the NOD model of islets are free from insulitis. Alternatively, increased apoptosis disease is that many NOD congenic mouse strains develop insu- may rather promote immune tolerance (12). Because islet-specific litis while remaining partially or completely resistant to diabetes autoimmune attack starts from peri-insulitis, Winer et al. (13) (3–5), whereas other resistant strains such as B10 and C57BL/6 proposed that peri-islet Schwann-like glial cells, but not the are free from insulitis (6, 7). Also, NOD.scid mice, which lack insulin-producing b cells, could be the initial targets of the au- both T and B cells, have been shown to exhibit abnormal pan- toimmune response. The mechanism that leads to this early peri- creatic physiology, including vascular pathology and metabolic insulitis and targeting of the glial cells is unknown. Nevertheless, autoreactive T cells specific for candidate Ags expressed in the glial cells were detectible (14). *Division of Immune Regulation, Torrey Pines Institute for Molecular Studies, San Exosomes (EXO) are small (50–100 nm) microparticles/vesicles † Diego, CA 92121; School of Endocrinology, Yale University School of Medicine, that originate from fusion of late endosomal compartments with New Haven, CT 06520; and ‡Department of Immunology and Microbial Science, The Scripps Research Institute, San Diego, CA 92037 the plasma membrane (15). Under normal physiological condi- Received for publication January 24, 2011. Accepted for publication June 3, 2011. tions or in responding to stress or tissue damage, various cell types with hematopoietic or nonhematopoietic origins can secrete This work was supported by Career Grant DK076741 from the National Institutes of Health (to Y.D.D.) and a research grant from the Diabetes National Research Group this type of microparticle to transport membrane proteins (15) or (to Y.D.D.). genetic materials such as microRNA (16) on purposes of intra- Address correspondence and reprint requests to Dr. Yang D. Dai, Torrey Pines In- cellular communications or tissue repair. Recent evidence sug- stitute for Molecular Studies, 3550 General Atomics Court, San Diego, CA 92121. gests that secreted membrane microparticles, particularly EXO, E-mail address: [email protected] can stimulate immune responses (17). Interestingly, many tumor The online version of this article contains supplemental material. cells also actively release EXO, and some of them may contain Abbreviations used in this article: CBA, cytometric bead array; EXO, exosome; GAD65, glutamic acid decarboxylase 65 kDa; HSC70, heat shock protein cognate strong proinflammatory stimuli capable of inducing tumor-specific 70; Ing-LN, inguinal lymph node cells; KO, knockout; NOR, nonobese diabetes immunity (18–20). However, immunization with tumor-derived resistant; Pan-LN, pancreatic lymph node cells; SI, stimulation index; T1D, type 1 EXO frequently causes immune suppression and even promotes diabetes. tumor growth (21, 22), possibly due to induction of myeloid im- Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 mune suppressor cells (23) or regulatory T cells (24). This led us www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100231 1592 EXOSOMES IN TISSUE-SPECIFIC AUTOIMMUNITY to study EXO-induced responses in an autoimmune scenario, at a round-bottom 96-well plate at room temperature for 2 h, with protection a stage of disease where pathogenic effectors, rather than regu- from light. After two washes, the incubated beads were resuspended in 200 latory T cells, may be preferentially activated in genetically sus- ml washing buffer and then subjected to FACS analysis. Because each type of the beads is coated with a different capture Ab specific for one of the six ceptible individuals. cytokines and has a distinct fluorescent intensity in the red channel when We have successfully isolated EXO from mouse insulinoma. We analyzed in a flow cytometer, the six individual cytokines can be simul- have shown that the EXO preparation has strong adjuvant effect to taneously measured. The concentration of each cytokines was extrapolated induce proinflammatory cytokines, including IL-6 and TNF-a.We from the standard curves by testing with the respective recombinant pro- teins of the cytokines. have confirmed that MyD88 is required for this adjuvant effect (25), suggesting an involvement of TLR-mediated sensing of Proliferation assay the innate stimuli enclosed in the insulinoma-derived EXO. We To assess T cell activation by EXO, total splenocytes (5 3 105 cells/200 ml/ detected candidate islet Ags, particularly glutamic acid decar- well) were cultured with EXO, Con A, or medium only in 96-well flat- boxylase 65 kDa (GAD65), expressed in the EXO. Thus, EXO bottom plates for 72 h, followed by pulsing with 0.5 mCi/well [3H]thy- may act as both an endogenous adjuvant and a unique Ag carrier midine (Amersham Biosciences, Pittsburgh, PA) for 18 h. Cells were harvested with a Micro Cell Harvester (Skatron Instruments, Sterling, VA), to trigger immune responses. Based on our observations that EXO 3 and incorporation of [ H]thymidine was measured on a Wallac MicroBeta can cause insulitis in resistant mice and EXO-reactive Th1 cells Trilux counter (Perkin Elmer, Boston, MA). Stimulation indexes (SI) were are enriched in prediabetic NOD mice, we propose that abnormal calculated as cpm of EXO or Con A/cpm of medium control. EXO or a dysregulated EXO-releasing pathway may trigger islet- ELISPOT specific autoimmunity in diabetes-susceptible individuals.

The number of EXO-reactive, IFN-g–secreting cells was evaluated using Downloaded from an ELISPOT assay. Briefly, ELISPOT plates (MAHA S4510; Millipore) Materials and Methods were precoated overnight at 4˚C with 5 mg/ml anti–IFN-g Ab (BD Bio- Mice sciences) and blocked with complete 10% FCS RPMI 1640 culture me- dium at room temperature for 2 h. Splenocytes were added at 106/well in NOD/LtJ (NOD), NOR/LtJ (nonobese diabetes resistant [NOR]), NOD. the presence of EXO or Con A, or medium. After culture for 48 h, the scid, and C57BL/6 (B6) mice were purchased from The Jackson Labora- plates were washed, and IFN-g–positive spots were detected using 2 mg/ml tory (Bar Harbor, ME) and maintained as inbred strains at the animal fa- biotinylated anti–IFN-g Ab (BD Biosciences), followed by addition of cility of the Torrey Pines Institute for Molecular Studies. NOD/MrkTac HRP-conjugated streptavidin (Sigma-Aldrich). The spots were developed http://www.jimmunol.org/ strain, NOD.B10-H-2b (NOD.H-2b) (7), NOD.B10 Idd5 congenic mice, using 3-amino-9-ethyl-carbazole substrate (BD Biosciences). line 6146 carrying B10-derived Idd5.2 locus (26), were obtained from Taconic (Hudson, NY) and maintained as inbred strain at Torrey Pines IFN-g secretion assay Institute for Molecular Studies. NOD.Idd3/5 mice, line 1591, which carry IFN-g–secreting cells were identified and quantified by flow cytometry both a B6-derived Idd3 and a B10-derived Idd5 T1D-protective loci (27), using a mouse cytokine secretion-capture assay kit (Miltenyi Biotec, Au- were housed at the animal facility of The Scripps Research Institute (San burn, CA), which could capture the secreting cytokines and retain on cell Diego, CA). Splenocytes from NOD.MyD88 knockout (MyD88.KO) mice surface. Briefly, splenocytes or lymph node cells (106/sample/well) were were prepared by L. Wen (Yale University, New Haven, CT) and shipped first activated by EXO for 48 h, and washed twice with ice-cold buffer. To to us overnight. Experimental protocols were conducted with approvals capture the secreted IFN-g, the preactivated cells were resuspended in

from the Ethical Review Committee of Torrey Pines Institute for Molec- by guest on September 28, 2021 40 ml ice-cold buffer and 10 ml IFN-g capture reagent (provided in the kit), ular Studies. followed by incubation on ice for 5 min. After adding 1 ml/sample of 37˚C Preparation of EXO warm medium, the cells were incubated again at 37˚C for 45 min with slow rotation. The cells were then washed once and resuspended in 40 ml ice- MIN6 insulinoma cell line was used for EXO preparation. This cell line was cold buffer with 10 ml IFN-g detection Ab (provided in the kit), together originally derived from a transgenic C57BL/6 strain that overexpressed with additional fluorescent-labeled Abs to identify subsets of lymphocytes. SV40 T Ag under an insulin promoter (28). A second insulinoma cell line, After staining for another 15 min on ice, the cells were washed and re- NIT-1, was also used for EXO preparation, which was, however, estab- suspended in the ice-cold buffer for flow cytometry analysis. lished from a SV40 T Ag transgenic NOD strain (29). To prepare EXO, insulinoma cells were cultured in 150-cm2 culture flasks using high glu- SDS-PAGE and Western blotting analysis cose DMEM, supplied with 10% FCS that was precentrifuged at 100,000 MIN6 or NIT-1 cell lysates were prepared by resuspending cell pellets (5 3 3 g for 90 min to remove serum EXO. Culture supernatants were har- 106 cells) in 200 ml cell-lysing buffer (containing 20 mM Tris.HCl, 5 mM vested every 2 or 3 d, depending on the density of the cells in the flasks, EDTA, 1% Nonidet P-40, 1 mM NaVO4, 1% SDS, and 10 mg/ml aprotinin/ and stored in 4˚C, and when the cells reached confluence, trypsin–EDTA leupeptin), and then incubated on ice for 15 min, followed by centrifu- treatment was used to release the adherent cells and one-third of the cells gation at 15,000 rpm for 20 min at 4˚C. To perform SDS-PAGE, cell were inoculated for subculture. Once collected, a large volume of culture lysates or EXO samples were denatured by incubation with sample buffer supernatant (1 liter), EXO isolation was performed following a proto- at 95˚C for 10 min. SDS-PAGE–separated protein samples were electro- col provided by C. Thery (Curie Institute, Paris, France). Briefly, the transferred to nitrocellulose membrane (Amersham, GE Healthcare Life culture supernatants were centrifuged at 3000 rpm for 15 min, followed by Sciences, Piscataway, NJ) using a semidry trans-blot (Bio-Rad Labora- filtration using 200-nm pore-size membrane to remove cell debris and tories, Hercules, CA). The membrane was immunoblotted with 1 mg/ml large particles. The filtered supernatants were concentrated in an Amicon primary Abs, followed by a respective secondary HRP-labeled anti-IgG Stirred Cells unit (Millipore, Billerica, MA) using an Ultracel membrane (Amersham). The protein bands were visualized with an ECL detection disc of 300-kDa nominal m.w. limit (Millipore). EXO in the concentrated system (Amersham). Clones 4-17 or 144 (provided by C. Hampe, Uni- supernatant was harvested by spinning in an ultracentrifuge (Sorvall Dis- versity of Washington, Seattle, WA) were used for detecting GAD65. Anti- 3 covery 90SE; Hitachi) at 100,000 g for 90 min. The EXO pellets were heat shock protein cognate 70 (HSC70) was purchased from Stressgen then washed once with PBS and resuspended in PBS. Protein concentration (Enzo Life Sciences, Plymouth Meeting, PA). Anti-chromagranin A was was determined by Bradford protein assay (Bio-Rad, Hercules, CA). purchased from Abcam (Cambridge, MA). Normally, 1 liter MIN6 culture supernatant yields 0.5–1.0 mg EXO. Cytokine detection assay Results Mouse MIN6 insulinoma can release EXO that expresses A cytometric bead array (CBA)-based flow cytometry method (BD Bio- sciences) was used to analyze for six different inflammatory cytokines, as candidate islet Ags follows: IL-6, IL-10, MCP-1, IFN-g, TNF-a, and IL-12p70, according to EXO was isolated from the culture supernatant of MIN6 insuli- the manufacturer’s protocol. Briefly, 50 ml culture supernatant of EXO- stimulated splenocytes that were collected from young (8- to 12-wk-old) noma cells. As demonstrated by electron microscopy, the majo- NOD or congenic mouse strains was incubated with 50 ml mixture of six rity of the microparticles retain a round, vesicular shape, with a types of cytokine-capturing beads and 50 ml PE-detection reagent in diameter ,100 nm (Fig. 1A). Initial mass spectrometry analysis The Journal of Immunology 1593

FIGURE 1. MIN6 insulinoma cells release EXO that expresses GAD65 protein. A, Images of EXO preparations under an electron microscope after Downloaded from negative staining. Arrow-pointed microparticles are the major structure observed. B, Preparing gel samples for identifying protein content of the EXO by mass spectrometry. MIN6 EXO (∼60 mg) was denatured in sample buffer and separated on a 10% SDS-PAGE gel. The gel was stained by Coomassie dye and cut into four slices, M1, 2, 3, and 4, as indicated, and submitted to the mass spectrometry facility in the Scripps Research Institute (San Diego, CA) for protein identification. C, Expression of GAD65 in insulinoma cell lines as detected in Western blot. Control brain samples were collected from GAD65.KO or wild-type NOD mice, and the blot was reprobed with anti-ERK to indicate similar amount of samples loaded. D, Detecting GAD65 and HSC70 protein in EXO preparations from MIN6 or NIT-1 insulinoma cell lines. The expression of GAD65 protein only in MIN6 EXO was confirmed in three separate experiments. http://www.jimmunol.org/ using the total microparticle preparation identified several signa- Fig. 2A demonstrates that the EXO can stimulate total splenocytes ture proteins of typical EXO (17), including tetraspanins, Alix, to produce inflammatory cytokines, mainly IL-6, IFN-g, and TNF- and TSG101, suggesting that the microparticle preparation con- a, although IL-10 and MCP-1 are also detectable. IL-12p70 was tained EXO. To further analyze the protein content in the EXO, not detected; however, when a blocking anti–IL-12 Ab was added, we separated the EXO on SDS-PAGE; after Coomassie stain- IFN-g release from the EXO-stimulated splenocytes reduced .3- ing, the gel was cut into four slices based on their m.w. (Fig. 1B), fold (Fig. 2B), indicating that IL-12 is actually produced to and each gel slice was subjected to mass spectrometry analysis. promote IFN-g secretion by EXO-activated cells, but its concen- by guest on September 28, 2021 Approximately 200 protein molecules were identified (see Sup- plemental Table); other than molecules involved in intracellu- lar vesicle sorting and protein degradation, some cytosolic and membrane proteins were also detected. Insulin was detected by mass spectrometry only once when total EXO preparation was analyzed. GAD65, one of the diabetes-associated islet Ags, did not appear in the protein list identified by mass spectrometry, al- though the GAD65 protein was expressed abundantly in MIN6 cells (Fig. 1C). A second insulinoma cell line, NIT-1, does not express GAD65 (Fig. 1C). The anti-GAD65 mAb (clone 144) binds to the N terminus of GAD65 protein. GAD65.KO brain tissue was used as a negative control. The band stained on the Western blot membrane was confirmed to be GAD65, not its glutamic acid decarboxylase 67 kDa isoform, by its m.w. Never- theless, the GAD65 protein was detected in MIN6 EXO by Western blot, which might be more sensitive than mass spec- trometry in analyzing a protein mixture. As shown in Fig. 1D, only EXO released by the MIN6, but not NIT-1 insulinoma, was stained positive by the GAD65-specific mAb, whereas both insulinomas-released EXOs contain HSC70 protein, which is commonly found in such microparticles (17). Thus, GAD65 is released by MIN6 cells, possibly via EXO secretion pathway, but FIGURE 2. EXO collected from MIN6 culture supernatant is proin- at a low level under the given culture condition. flammatory. A, Splenocytes (106/200 ml/well) from 8-wk-old NOD female MIN6-derived EXO contains some innate stimuli that can mice were cultured with 5 mg/ml MIN6 EXO for 48 h in a flat-bottom induce production of inflammatory cytokines 96-well plate. A CBA assay was performed to monitor the levels of six different inflammatory cytokines in the culture supernatants or the non- To examine whether the EXO preparations can stimulate an innate stimulated control. Similar results were observed in three separated ex- immune response, splenocytes from 6- to 8-wk-old NOD mice were periments. B, Experiment was similarly performed as in A, except that incubated with EXO for 6–24 h, and the levels of several in- an IL-12–blocking Ab (clone C17.8) or a rat Ig isotype control was added flammatory cytokines in the culture supernatants were measured. at a final concentration of 20 mg/ml during the culture with EXO. 1594 EXOSOMES IN TISSUE-SPECIFIC AUTOIMMUNITY tration might be too low to be detected using this CBA assay. The class II MHC, CD80, CD86, or ICAM-1 on the splenic B220+, cytokine levels produced by the EXO-stimulated splenocytes were CD11c+, and CD11b+ APC subpopulations after EXO stimulation. dose and time dependent (Table I): even after just 6 h of in- Clearly, professional APC were activated effectively by EXO as cubation with a low dose of EXO (1 mg/ml), TNF-a was elevated detected by their upregulated expression of MHC II or costimu- 8-fold above background, indicating that a rapid innate signal was latory molecules. It is noticed that nonstimulated splenic CD11c+ triggered by EXO. This instant TNF-a release was due to EXO- and CD11b+ APC expressed higher level of class II MHC than induced activation of splenocytes because the cytokine was not B220+ APC and EXO stimulation only upregulated CD86 or detected by culturing EXO or splenocytes separately. At 20 mg/ml CD80, but not class II MHC on these two populations (Fig. 4B). of 24-h incubation with EXO, IL-6 increased 32-fold from 15.6 to Because EXO are small particles under 100 nm diameter, it is 497.3 pg/ml, IFN-g increased 22-fold from 2.7 to 59.9 pg/ml, and possible that certain APC subpopulation(s), such as dendritic cells, TNF-a increased 54-fold from 9.0 to 482.7 pg/ml (Table I). In may be more efficient than others, such as macrophages or B cells, contrast, an EXO preparation from cultured NOD splenocytes was in picking up this size range of microparticles. We isolated nonstimulatory, whereas the same volume of supernatant from CD11c+, CD11b+, or B220+ APC populations from NOD spleno- MIN6 culture yielded sufficient EXO to induce the innate re- cytes by a positive selection protocol using MACS microbeads sponse. Therefore, the EXO secreted by the MIN6 cells contain coated with the respective Abs, and examined their responses to some endogenous, adjuvant-like components, capable of inducing EXO. At 5 mg/ml EXO concentration and 12-h incubation time, the inflammatory cytokines. both purified CD11c+ and CD11b+ APC populations responded rapidly (also observed at an 8-h time point) to EXO stimulation The innate response induced by EXO requires by releasing mainly IL-6 and TNF-a (Fig. 4C); the response by Downloaded from MyD88-mediated TLR-signaling pathway the purified B220+ APC (∼90% purity) was much lower, with To examine whether TLR-mediated innate signaling pathway is a marginal (∼2-fold) increase of TNF-a, and IL-6 increased in- required for the EXO-induced cytokine response, we tested total significantly. splenocytes from a MyD88.KO mouse. Fig. 3A shows that the cytokine response to EXO stimulation (12 h) was almost com- T cells contribute to EXO-induced lymphocyte proliferation

pletely impaired in the MyD88.KO splenocytes as compared with Because NOD total splenocytes proliferated strongly after EXO http://www.jimmunol.org/ wild-type NOD cells; a similar lack of response in the MyD88.KO stimulation (Fig. 5A), we asked whether T cells are the pro- cells was observed after a longer (24-h) incubation time, although liferating population by testing the proliferation response after IFN-g was slightly increased, ∼2-fold above the nonstimulated depleting CD4+ and CD8+ T cells from the splenocytes. As a re- background. In contrast, in the wild-type NOD female splenco- sult, the proliferation capability was impaired in the absence of tyes, IFN-g increased at least 20-fold at 12 h, and reached .100- T cells. In one experiment, total NOD splenocytes reached a SI as fold at 24 h after stimulation with 10 mg/ml MIN6 EXO (data not high as 65 at the dose of 10 mg/ml EXO, whereas the T cell- shown). In a 72-h lymphocyte proliferation assay, the wild-type depleted splenocytes responded 3-fold less to the same dose of NOD splenocytes responded to the EXO (10 mg/ml) with a EXO (Fig. 5A) despite that only ∼50–60% depletion of T cells SI .50. In contrast, the MyD88.KO splenocytes were completely was achieved in this experiment. T cell proliferation was also by guest on September 28, 2021 unable to proliferate at the same dose ranges (SI , 3), but re- demonstrated by a 2-fold increase of the percentage of total CD4+ sponded strongly to Con A (Fig. 3B). These results suggest that T cells in the EXO-stimulated splenocytes (Fig. 5B). Strikingly, no the possible innate stimuli enclosed in the EXO may activate TLR reactivity was observed when NOD.scid splencoytes were used signals via MyD88-dependent pathways. in the proliferation assay (Fig. 5A), suggesting that T and/or APC and purified subsets are activated by the EXO to B lymphocytes were the only populations that proliferated in re- upregulate class II MHC and costimulatory molecules sponse to EXO. The lack of proliferation of NOD.scid splenocytes to the EXO was not due to defects in their innate responses be- To examine whether EXO could activate APC, we monitored the cause the NOD.scid splenocytes also produced high levels of in- expression of CD86 (B7.2) on class II MHC-positive splenocytes. flammatory cytokines when stimulated by EXO, although the Fig. 4A demonstrates that EXO stimulation increased the per- cytokine release pattern was different (Fig. 5C). centage of CD86 and I-Ag7 MHC double-positive population from 2.6 to 11.5% in total splenocytes. Fig. 4B shows the expression of Prediabetic animals accumulate EXO-reactive, IFN-g–secreting T cells To examine whether EXO stimulation could activate diabetes- Table I. Exosomes stimulate splenocytes to release inflammatory cytokines associated T cell responses in NOD mice, three different meth- ods were used to monitor the IFN-g response to the EXO: 1) total EXO Doses (mg/ml) EXO-responding, IFN-g–secreting cells in splenocytes were de- Stimulation Cytokines tected by ELISPOT assay; 2) IFN-g and other inflammatory cyto- Times (pg/ml) 0.0 1.0 5.0 20.0 kines in culture supernatants were examined by a flow cytometry- 6 h IL-6 15.6 20.3 28.5 35.5 based CBA assay; and 3) single IFN-g–secreting Th1 cells were IFN-g 2.7 2.4 5.5 3.5 identified by an IFN-g capture assay (see Materials and Methods). TNF-a 9.0 77.6 121.8 193.5 Fig. 6A shows that the levels of EXO-induced IFN-g response 12 h IL-6 ND 81.3 116.7 161.3 IFN-g ND 3.4 4.8 10.0 correlated well with the developmental stages of diabetes in the TNF-a ND 124.9 172.5 266.9 ELISPOT assay, with the oldest NOD females exhibiting the 24 h IL-6 17.4 334.3 333.5 497.3 highest number of IFN-g+ spots, and the male NODs the lowest. In IFN-g 8.9 36.2 31.1 59.9 addition, CBA assay was performed to compare NOD female and TNF-a 11.8 319.0 366.3 482.7 male mice for their responses to EXO stimulation in vitro. Among Splenocytes (106/200 ml/well) from one male NOD were stimulated with differ- the six inflammatory cytokines, IFN-g was found different in that ent doses of EXO for various periods. Cytokine levels in the culture supernatants were monitored by the CBA assay. female splenocytes produced 10-fold higher IFN-g on average ND, not done. than male splenocytes after stimulated with EXO (Fig. 6B). Three The Journal of Immunology 1595

FIGURE 3. MyD88-dependent innate signaling pathway is required for EXO-induced inflammatory response. A, Comparing EXO-induced cytokine secretion between two age-matched (within 8- to 12-wk-old) NOD and NOD.MyD88.KO female mice. Total splenocytes (106/200 ml/well) were stimulated with 5 mg/ml MIN6 EXO or Con A, or medium (Neg.) for 12 h. Data are representative of three independent experiments. B, EXO-induced proliferation of the splenocytes (5 3 105 cells/200 ml/well) collected from the NOD or NOD.MyD88.KO mice. [3H]thymidine (0.5 mCi/well) was added for the last 18 h of a 96-h culture in a flat-bottom 96-well plate. (Background: NOD = 198 cpm; MyD88 = 672 cpm.) of four 9- to 12-wk-old females produced large amount of IFN-g crease of the Th1 cells observed in the Pan-LN of NOD female Downloaded from (632, 688, and 1044 pg/ml) after 48-h stimulation, whereas all mice after EXO stimulation, from 0.17% increased to 1.8% of three age-matched males responded below 100 pg/ml (35, 72, and total Pan-LN, whereas no increase was observed for a resistant 87 pg/ml). Thus, the EXO-responding, IFN-g–producing cells NOD congenic strain, NOD.Idd3/5. It is noticed that many Pan- are accumulated in prediabetic NOD female mice. This correla- LN of NOD increased their IFN-g secretion after EXO stimula- tion was further confirmed by testing NOD congenic strains with tion, but at a low level, as indicated in a histogram plot in Fig. 7B different susceptibility to T1D. As shown in Fig. 6C, NOD, the (top panel), rather than a polarized, fully activated, Th1 pheno- most susceptible strain, produced the highest number of IFN-g type. Control Ing-LN of the NOD mice showed less of an increase http://www.jimmunol.org/ spots (∼100 spots/106 splenocytes), whereas only ,10 spots were (from 0.25 to 0.48%) after EXO stimulation (Fig. 7B, bottom identified when NOR splenocytes were stimulated by the EXO panel), further confirming that EXO-reactive Th1 cells preferen- and a partially resistant strain, NOD.B10 Idd5.2, exhibited an tially accumulate in the Pan-LN of prediabetic animals. intermediate response. Similarly, IFN-g, but not the other five inflammatory cytokines, was also found different between NOD EXO immunization accelerates insulitis in diabetes-resistant and the resistant strain. Fig. 6D shows one example that spleno- NOR mice cytes from a NOD female produced 3- to 5-fold higher amount of To examine whether EXO can trigger or accelerate islet in-

IFN-g than an age- and gender-matched NOD.B10 Idd5.2 mouse. flammation or insulitis in diabetes-resistant mice, we directly by guest on September 28, 2021 To quantify EXO-responding, IFN-g–secreting Th cells, we per- injected EXO i.v., without addition of other adjuvant, into three formed IFN-g–secreting/capturing assay after stimulating pan- diabetes-resistant mouse strains, NOR, NOD.H-2b, and C57BL/6 creatic lymph node cells (Pan-LN) or inguinal lymph node cells mice. After 3 or 7 d, their pancreatic glands were collected for (Ing-LN) with EXO in culture. Fig. 7A shows a .10-fold in- histological examination. It has been reported that both NOD.H-2b

FIGURE 4. Activation of APC upon EXO stimulation. A, Upon EXO stimulation (5 mg/ml for 12 h), the percentage of activated APC (MHC-II+CD86+) in NOD splenocytes increased from 2.6 to 11.5%. B, Upregulation of class II MHC and costimulatory molecules, ICAM-1, CD80, and CD86 on splenic B220+ (top panel), CD11c+ (middle panel), or CD11b+ (bottom panel) APC subpopulations after EXO stimulation (5 mg/ml for 12 h). C, Activation of purified APC subsets by EXO. B220+, CD11b+, or CD11c+ cell populations were isolated using MACS microbeads coated with respective Abs in a positive selection method, and then stimulated with 1 mg/ml EXO for 8 h; IL-6 and TNF-a concentrations in the culture supernatants were measured in the CBA assay. 1596 EXOSOMES IN TISSUE-SPECIFIC AUTOIMMUNITY

than the untreated group. Fig. 8 shows the insulitis levels for the individual NOR mouse with or without EXO injection. The se- verity of insulitis or lymphocyte infiltration is significantly in- creased in the group treated with EXO for 7 d (p = 0.01), but the difference between the untreated and 3-d–treated groups was in- significant.

Discussion Exosomes derived from tumor cells are naturally occurring small nanoparticles that not only contain potent adjuvant-like substances, but also carry tumor-specific Ags (19, 20). It has also been reported that EXO can activate myeloid-derived suppressor cells (30) and regulatory T cells (24, 31), which may explain the difficulties in designing EXO vaccine to treat cancer patients (32). Because these physiologically important microparticles contain mostly self Ags, to which immune tolerance is well established, except for those rare individuals who are highly susceptible to a particular autoimmune disease, the chance of successfully inducing effective

antitumor immunity or breaking self tolerance using EXO as an Downloaded from immunogen is low. Thus, the consequence of EXO-induced im- mune responses might be heavily dependent on the types/contents of EXO as well as the genetic background of the hosts. To our knowledge, this is the first study characterizing immune responses to EXO in an autoimmune scenario, the NOD mouse model of

T1D, in which immunopathology rather than immunosuppres- http://www.jimmunol.org/ sion is the outcome of an organ-specific inflammatory response. We found that EXO can induce innate immunity via a MyD88- mediated TLR-signaling pathway, and that EXO-reactive Th1 cells accumulated in prediabetic NOD female mice, but not in NOD males or resistant congenic strains. These findings emphasize the importance of genetic background and memory precursors in controlling adaptive immune responses to EXO. It remains un- known whether the accumulated EXO-reactive Th1 cells in the prediabetic NOD mice were primed directly by the endogenous by guest on September 28, 2021 EXO secreted by the pancreatic islet cells, or via cross-reaction to other Ags. Nevertheless, EXO or microparticles secreted by islet FIGURE 5. EXO-induced T cell proliferative response. A, Reduced cells should be useful tools for studying tissue-specific auto- proliferative response to EXO by T cell-depleted NOD splenocytes. T cells antigens and their cognate autoreactive T cells. were depleted using two types of MACS microbeads coated with anti-CD4 It has been shown previously that tumor cell-derived EXO and anti-CD8, respectively. NOD splenocytes or T-depleted (no T), or requires MyD88-dependent TLR-signaling pathway to induce IL-6 NOD.scid splenocytes were stimulated with 1 mg/ml (EXO-1) or 10 mg/ml and TNF-a secretion from myeloid cells (25). EXO derived from (EXO-10) EXO or Con A (3 mg/ml) in a total 96-h proliferation assay. B, different tissues or cell lines may vary in their proinflammatory NOD splencoytes were stimulated with 5 mg/ml EXO for 72 h, and then activity, and the characteristics of the possible innate stimuli stained with anti-CD4. Fresh NOD splenocytes were used as control (Neg.). C, Comparing EXO-induced cytokine secretion between two NOD within MIN6 insulinoma remain unknown, but we confirmed that and NOD.scid 10- to 12-wk-old female mice. Total splenocytes (106/200 the innate response also required MyD88. Several heat shock ml/well) were stimulated with 5 mg/ml EXO for 24 h, and the CBA cy- proteins were found in our EXO preparations. Chalmin et al. (30) tokine analysis was performed. demonstrated that heat shock protein 72 expressed on tumor cell-derived EXO could activate myeloid-derived suppressor cells in a TLR2/MyD88-dependent manner, whereas Zhang and col- and C57BL/6 mice do not develop insulitis (6, 7), and after 7 d of leagues (25) demonstrated that neither TLR2 nor TLR4 was in- EXO injection, all islets from these two strains (four mice per volved in their system testing EXO-induced activation of the strain, and 40–50 islets per mouse) remained completely free myeloid-derived suppressor cells. EXO released from Leishma- from lymphocyte infiltration (data not shown). Interestingly, we nia could modulate monocytes’ cytokine response by promoting noticed that not all adult NOR mice are free from insulitis, con- IL-10, but inhibiting TNF-a (33). Interestingly, human primary firming a previous report that almost half examined islets had astrocytes can release exosomes carrying mitochondrial DNA some level of lymphocyte infiltration (5). Notably, EXO injection (34), which were shown to have innate stimulatory activity, accelerated insulitis in this strain. In the untreated group of nine 4- possibly via the TLR9-signaling pathway (35). It was noteworthy to 5-mo-old NOR female mice, of total 457 islets examined, only that our EXO preparation contains endogenous gag and env pro- 53 islets or 11.6% showed severe insulitis (.50% area of one islet teins (Supplemental Table); whether endogenous retroviruses, is infiltrated by lymphocytes). The mean of lymphocyte infiltration which have been implicated as etiological agents of T1D (36, 37), per islet for this untreated group is 12.9%. In contrast, in the contribute to the immune responses to EXO is an interesting experiment group 7 d after treated with EXO, there were 94 islets possibility. Whether EXO produced by different types of tumor or 42% of total 226 examined showed severe insulitis, and the cells or primary tissues or organisms vary in their usages of dif- mean of lymphocyte infiltration is 37.7%, which is 3-fold higher ferent innate pathways or TLRs remains to be studied. The Journal of Immunology 1597 Downloaded from

FIGURE 6. EXO can activate diabetes-associated, IFN-g–secreting cells. A, Total splenocytes (106/200 ml/well) from NOD mice of different ages or genders (wk, weeks old; F, females; M, males) were tested for their reactivity to MIN6.EXO (1 mg/ml) in an IFN-g ELISPOT assay. All mice were not diabetic at the time of testing, as indicated by urine glucose. The results were reproducible in a separate experiment, with similar observation. B, Splenocytes from four female and three male NOD mice (9–12 wk old) were stimulated with EXO for 48 h, and CBA assay was performed to measure cytokines in the culture supernatants. The average cytokine concentrations for each gender are shown. C, EXO-induced IFN-g responses among different

NOD congenic strains (8- to 10-wk-old female mice) were compared by ELISPOT method. Data represent one of two experiments with similar observation. http://www.jimmunol.org/ D, IFN-g levels in the culture supernatants of EXO-stimulated splenocytes were compared between two age-matched NOD and NOD.B10 Idd5.2 (Idd5.2) female mice in a CBA cytokine assay.

Without the MyD88 gene, both female and male NOD mice the TLR2 or TLR9 pathways had some moderate effects on T1D were almost completely resistant to diabetes development at 30 development in NOD mice. Interestingly, MyD88.KO NOD mice wk of age (38), indicating an essential contribution of the innate gained their diabetes susceptibility when housed under germ-free TLR-signaling pathway to this T cell-mediated autoimmunity. environment (38). Thus, gut flora must affect disease development Wen et al. (38) reported that neither TLR3 or TLR4 deficiencies in NOD mice, possibly by shaping the immune repertoire or in NOD mice could mediate protection from diabetes, whereas promoting immune regulation. The requirements for sensing in- by guest on September 28, 2021 TLR9-deficient NOD mice were partially protected (39); Kim flammatory stimuli in the pancreas may be different from that of et al. (40) showed that their TLR2.KO NOD mice were partially responding to the microflora in the gut. Because MyD88 is re- protected; Dutz and colleagues (41) reported that altering either quired for the EXO-induced innate response, determining which

FIGURE 7. Accumulation of EXO-responding, IFN-g–secreting Th cells in pancreatic lymph nodes of NOD mice. A, Pan-LN (5 3 105/well) from age- matched NOD and NOD Idd3/5 (Idd3/5) female mice (three mice/group) were mixed with irradiated syngenic splenic APC (1:1) and then cultured with medium or 5 mg/ml EXO for 48 h. IFN-g–secreting CD4+ T cells were identified in an IFN-g–secreting/capturing assay (see Materials and Methods). B, Histogram plot of EXO-stimulated Pan-LN is presented by gating on CD4+ T cells (top panel, filled, medium control; empty, cultured with EXO for 48 h). Ing-LN from the NOD and Idd3/5 mice were used as controls to culture with (empty) or without EXO (filled), and the results are shown in histogram plot (bottom panel). 1598 EXOSOMES IN TISSUE-SPECIFIC AUTOIMMUNITY

FIGURE 8. Induction of insulitis in NOR mice by EXO immunization. The 4- to 5-mo-old NOR female mice were injected (i.v.) once with 15 mg EXO in PBS. After 3 or 7 d, pancreases were collected and submitted for H&E stain. Levels of islet infiltration were scored in blind, and the percentages of islets

within each level of lymphocyte infiltration are shown for individual mice. Nine age/gender-matched NOR mice purchased from The Jackson Laboratory Downloaded from were used as untreated controls. The mean of infiltration for each mouse was calculated as follows: the sum of percentages of infiltration for each islet/ number of total islets examined for each mouse.

TLR(s) may be involved in responding to EXO will help further tivation of many Th cell clones that are not highly polarized, clarify the endogenous innate stimulatory materials enclosed in but biased to produce some IFN-g, might be sufficient to cause

the EXO. damage of the islets. In fact, it has been shown that even at very http://www.jimmunol.org/ Among the six inflammatory cytokines we examined, IL-6 and early stage of prediabetic NOD mice, TCR repertoire in pancreatic TNF-a were the major cytokines produced by purified splenic lymph nodes, although restricted, is highly diverse and dynamic APC populations (CD11c+ DC, CD11b+ macrophages, and B220+ (48), and we had similar observation in studying the TCR reper- cells), although IL-10 was also detected after EXO stimulation. toire of islet-infiltrating T cells (I. Marrero, A. Vong, Y. Dai, and MCP-1 was produced only by CD11b+ macrophages, and IFN-g J. Davies, submitted for publication). was undetectable for all three purified APC populations. When It will be helpful to test whether insulinoma-derived EXO can total splenocytes were cultured with EXO, IFN-g level increased activate some highly diabetogenic T cell clones (49, 50), providing after 24-h culture, indicating an activation of T cells at this time that EXO and its Ags may be processed and presented via some point. However, some CD42CD82 splenocytes were also found to unique pathway(s). Our study is inconclusive about whether the by guest on September 28, 2021 produce IFN-g when cultured with EXO. This is particularly in- EXO-induced IFN-g–secreting cells could be due to partial cross- teresting because it has been shown that NK cells may be one reactivity (51) of the disease-related memory precursors to some of the early islet infiltrators triggering the inflammatory response unknown exosomal Ags or even due to bystander activation (52). (42), and an abnormal EXO release from the islets may attribute to Study of a particular Ag and its cognate T cells using EXO as this early NK infiltration. an Ag carrier may help address further whether EXO could be T1D is a Th1-mediated autoimmune disease, and activation of responsible for inducing a tissue-specific autoimmunity. It is tissue-specific, IFN-g–secreting Th cells is a crucial checkpoint noteworthy that of the five different inflammatory cytokines controlling disease progression (43). However, the endogenous we examined, only IFN-g was found to differ between diabetes- pathway inducing these pathogenic effector cells is not fully un- susceptible and -resistant strains, or between NOD male and fe- derstood. Because I-Ag7 class II MHC is required for the insulitis male mice when total splenocytes were cultured with EXO. Such and diabetes in NOD mice and T cells are among the earliest islet- difference cannot be identified after stimulation with LPS. This infiltrating lymphocytes (44), we proposed that altered presen- indicates that EXO may be a possible endogenous target that can tation of islet Ags may contribute to specific activation of high- preferentially activate a small percentage of memory autoreactive affinity Th cells (45, 46). We found that EXO were effective in T cells in vitro. activating IFN-g–secreting Th1 cells in NOD mice. Presumably, We have found that candidate diabetes-associated autoantigens, these Th1 cells represent the islet-Ag–specific memory cells that particularly GAD65, are expressed in the EXO produced by MIN6 are primed endogenously in the pancreatic islets or the draining insulinoma cells. GAD65-specific autoantibodies and T cell re- lymph nodes. We noticed that despite a significant increase of sponses are detected very early in prediabetic patients (53). The EXO-reactive Th1 cells in old NOD female mice, the number GAD65 protein in the pancreatic islets of NOD mice (54) and the of highly polarized Th1 cells was very low, as indicated in the NIT-1 insulinoma cells is barely detectible, but its expression in ELISPOT assay. Also, although many EXO-activated Th cells in the MIN6 cells and their secreted EXO is effective (Fig. 1C). We the Pan-LN of NOD mice can produce IFN-g, the amount of IFN- have also detected another candidate diabetic autoantigen, chro- g they produced was small, as measured in the IFN-g–secreting/ mogranin A (55), in MIN6 cells, as well as in their secreted EXO capturing assay. One possibility is due to a low expression of (data not shown). We have learned that most of candidate diabetes candidate autoantigens within the EXO. It might be interesting to Ags such as GAD65 and IA-2, except for insulin, are not a ne- test whether increased expression of the autoantigens could en- cessity causing diabetes in NOD mice (56–58); it might be an hance IFN-g production. It is also possible that other islet Ag interesting question whether a disease-triggering event, maybe sources such as apoptotic bodies released following b cell death abnormal EXO secretion, could cause autoimmune targeting at (47) that may express large amount of candidate Ags can stimu- several islet Ags simultaneously. The hypothesis that insulin acts late T cells to produce more IFN-g. Alternatively, polyclonal ac- as the primary islet Ag (58) is intriguing because islet b cells The Journal of Immunology 1599 apparently are not the only cell populations attacked in the islets 14. Tsui, H., Y. Chan, L. Tang, S. Winer, R. K. Cheung, G. Paltser, T. Selvanantham, A. R. Elford, J. R. Ellis, D. J. Becker, et al. 2008. 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