Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2012 Aug 15.
Published in final edited form as: J Immunol. 2011 Jul 6;187(4):1591–1600. doi: 10.4049/jimmunol.1100231

Insulinoma-released exosomes or microparticles are immunostimulatory and can activate autoreactive T cells spontaneously developed in non-obese diabetes mice1

Huiming Sheng *, Saleema Hassanali *, Courtney Nugent *, Li Wen †, Emma Hamilton-Williams ‡, Peter Dias *, Yang D Dai *,2
PMCID: PMC3150365  NIHMSID: NIHMS303075  PMID: 21734072

Abstract

Exosomes (EXO) are secreted intracellular microparticles that can trigger inflammation and induce antigen-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 non-obese diabetic (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 MyD88-dependent pathways, and activate purified APC and result in T cell proliferation. To address whether 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 destruction, accumulated more EXO-reactive, IFN-gamma-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 diabetes-resistant NOR mice. Thus, EXO or 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.

Introduction

A tissue-specific autoimmune response is thought to occur following a triggering event, which could be from an exogenous infectious pathogen, an endogenous superantigen, a physiological stress event, or a non-infectious environmental agent (1). For most autoimmune diseases, such events have not been well defined or are too diverse to be confirmed. In the NOD mice, what has been an excellent model for studying genetic susceptibility to human T1D (2), the existence and/or characteristics of such triggering agents or events likewise remains unclear. One interesting feature using the NOD model of disease is that many NOD congenic mouse strains develop insulitis while remaining partially or completely resistant to diabetes (3-5), whereas, other resistant strains such as B10 and C57BL/6 are free from insulitis (6, 7). Also, NOD.scid mice, which lack both T and B cells, have been shown to exhibit abnormal pancreatic physiology including vascular pathology and metabolic alterations (8). This indicates that a triggering event may be associated with initiating the early insulitis, and that progression from insulitis to complete destruction of the pancreatic islets and subsequent diabetes may then be due to a dysfunctional immune regulatory network (9, 10).

It has previously been suggested that a wave of pancreatic islet cell apoptosis during islet development and remodeling in early life might be the triggering event (11). However, such physiological cell death must also occur frequently in resistant mice and whose islets are free from insulitis. Alternatively, increased apoptosis may rather promote immune tolerance (12). Since islet-specific autoimmune attack starts from peri-insulitis, Winer et al proposed that peri-islet Schwann-like glial cells, but not the insulin-producing beta cells, could be the initial targets of the autoimmune response (13). The mechanism that leads to this early peri-insulitis and targeting of the glial cells is unknown. Nevertheless, autoreactive T cells specific for candidate antigens expressed in the glial cells were detectible (14).

EXO are small (50-100nm) microparticles/vesicles that originate from fusion of late endosomal compartments with the plasma membrane (15). Under normal physiological conditions or in responding to stress or tissue damage, various cell types with hematopoietic or non-hematopoietic origins can secret this type of microparticles to transport membrane proteins (15) or genetic materials such as microRNA (16) on purposes of intracellular communications or tissue repair. Recent evidence suggests that secreted membrane microparticles, particularly EXO, can stimulate immune responses (17). Interestingly, many tumor cells also actively release EXO, and some of them may contain strong proinflammatory stimuli capable of inducing tumor-specific immunity (18-20). However, immunization with tumor-derived EXO frequently causes immune-suppression and even promotes tumor growth (21, 22), possibly due to induction of myeloid immune suppressor cells (23) or regulatory T cells (24). This led us to study EXO-induced responses in an autoimmune scenario, at a stage of disease where pathogenic effectors, rather than regulatory T cells may be preferentially activated in genetically susceptible individuals.

We have successfully isolated EXO from mouse insulinoma. We have shown that the EXO preparation has strong adjuvant effect to induce proinflammatory cytokines, including IL-6 and TNF-alpha. We have confirmed that MyD88 is required for this adjuvant effect (25), suggesting an involvement of Toll-like receptor (TLR) -mediated sensing of the innate stimuli enclosed in the insulinoma-derived EXO. We detected candidate islet antigens, particularly glutamic acid decarboxylase 65 KDa (GAD65), expressed in the EXO. Thus, EXO may act as both an endogenous adjuvant and a unique antigen carrier to trigger immune responses. Based on our observations that EXO can cause insulitis in resistant mice and EXO-reactive Th1 cells are enriched in prediabetic NOD mice, we propose that abnormal EXO or a dysregulated EXO-releasing pathway may trigger islet-specific autoimmunity in diabetes-susceptible individuals.

Materials and Methods

Mice

NOD/LtJ (NOD), NOR/LtJ (NOR), NOD.scid and C57BL/6 (B6) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained as inbred strains at the animal facility of the Torrey Pines Institute for Molecular Studies (TPIMS). NOD/MrkTac strain, NOD.B10-H-2b (NOD.H-2b) (7), NOD.B10-Idd5 congenic mice, line 6146 carrying B10-derived Idd5.2 locus (26), were obtained from Taconic (Hudson, NY) and maintained as inbred strain at TPIMS. NOD.Idd3/5 mice, line 1591, which carry both a B6-derived Idd3 and a B10-derived Idd5 T1D-protective loci (27), were housed at the animal facility of the Scripps Research Institute (TSRI), San Diego, CA. Splenocytes from NOD.MyD88 knockout (MyD88.KO) mice were prepared by Dr. Li Wen at the Yale University and shipped to us overnight. Experimental protocols were conducted with approvals from the Ethical Review Committee of TPIMS.

Preparation of EXO

MIN6 insulinoma cell line was used for EXO preparation. This cell line was originally derived from a transgenic C57BL/6 strain that overexpressed SV40 T antigen under an insulin promoter (28). A second insulinoma cell line, NIT-1, was also used for EXO preparation, which was however, established from a SV40 T antigen transgenic NOD strain (29). To prepare EXO, insulinoma cells were cultured in 150 cm2 culture flasks using high glucose DMEM media, supplied with 10% FCS that was pre-centrifuged at 100,000g × 90 min to remove serum EXO. Culture supernatants were harvested every 2 or 3 days depending on the density of the cells in the flasks and stored in 4°C, and when the cells reached confluence, trypsin-EDTA treatment was used to release the cell monolayer and one-third of the cells were inoculated for subculture. Once collected a large volume of culture supernatant (1-2 liters), EXO isolation was performed following a protocol provided by Dr. Clotilde Thery at the Curie Institute in Paris. Briefly, the culture supernatants were centrifuged at 3000rpm × 15 minutes, followed by filtration using 200nm pore-size membrane to remove cell debris and large particles. The filtered supernatants were concentrated in an Amicon Stirred Cells unit (Millipore, Billerica, MA) using an Ultracel membrane disc of 300KDa NMWL (Millipore). EXO in the concentrated supernatant was harvested by spinning in an ultracentrifuge (Sorvall Discovery 90SE, Hitachi) at 100,000g × 90 minutes. The EXO pellets were then washed once with PBS, and resuspended in PBS. Protein concentration was determined by Bradford protein assay (BioRad, Hercules, CA). Normally, 1 liter of MIN6 culture supernatant yields 0.5 – 1.0 mg of EXO.

Cytokine detection assay

A cytometric bead array (CBA) -based flow cytometry method (BD Biosciences) was used to analyze for six different inflammatory cytokines: IL-6, IL-10, MCP-1, IFN-gamma, TNF-alpha and IL-12p70 according to the manufacturer's protocol. Briefly, 50 μl of the culture supernatant of EXO-stimulated splenocytes that were collected from young (8-12 wk old) NOD or congenic mouse strains was incubated with 50 μl of a mixture of six types of cytokine-capturing beads and 50 μl of the PE-Detection reagent in a round-bottom 96-well plate at room temperature for 2 hours, with protection from light. After two washes, the incubated beads were resuspended in 200 μl of the washing buffer and then subjected to FACS analysis. Since each type of the beads is coated with a different capture antibody specific for one of the six cytokines and has a distinct fluorescent intensity in the red channel when analyzed in a flow cytometer, the six individual cytokines can be simultaneously measured. The concentration of each cytokines was extrapolated from the standard curves by testing with the respective recombinant proteins of the cytokines.

Proliferation assay

To assess T cell activation by EXO, total splenocytes (5×105 cells/200μl/well) were cultured with EXO, Concanavalin A (Con A), or medium only in 96-well flat-bottomed plates for 72 hours followed by pulsing with 0.5 μCi/well of 3H-thymidine (Amersham Biosciences, Pittsburgh, PA) for 18 hours. Cells were harvested with a Micro Cell Harvester (Skatron Instruments, Sterling, VA), and incorporation of 3H-thymidine was measured on a Wallac MicroBeta® Trilux counter (Perkin Elmer, Boston, MA). Stimulation indexes (SI) were calculated as cpm of EXO or Con A / cpm of medium control.

ELISPOT

The number of EXO-reactive, IFN-gamma–secreting cells was evaluated using an enzyme-linked immunosorbent spot (ELISPOT) assay. Briefly, ELISPOT plates (MAHA S4510, Millipore) were pre-coated overnight at 4°C with 5μg/ml of anti–IFN-gamma antibody (BD Biosciences) and blocked with complete 10% FCS RPMI1640 culture medium at room temperature for 2 hours. Splenocytes were added at 106/well in the presence of EXO or Con A, or medium. After culture for 48 hours, the plates were washed, and IFN-gamma-positive spots were detected using 2μg/ml of a biotinylated anti–IFN-gamma antibody (BD Biosciences), followed by addition of horseradish peroxidase-conjugated Streptavidin (Sigma). The spots were developed using 3-amino-9-ethyl-carbazole (AEC) substrate (BD Biosciences).

IFN-gamma secretion assay

IFN-gamma-secreting cells were identified and quantified by flow cytometry using a mouse cytokine secretion-capture assay kit (Miltenyi Biotec, Auburn, CA), which could capture the secreting cytokines and retain on cell surface. Briefly, splenocytes or lymph node cells (106/sample/well) were first activated by EXO for 48 hours, and washed twice with ice-cold buffer. To capture the secreted IFN-gamma, the pre-activated cells were resuspended in 40 μl the ice-cold buffer and 10 μl of the IFN-gamma capture reagent (provided in the kit), followed by incubation on ice for 5 minutes. After adding 1 ml/sample of 37°C warm medium, the cells were incubated again at 37°C for 45 minutes with slow rotation. The cells were then washed once and resuspended in 40 μl of ice-cold buffer with 10 μl of the IFN-gamma detection antibody (provided in the kit), together with additional fluorescent-labeled antibodies to identify subsets of lymphocytes. After staining for another 15 minutes at room temperature, the cells were washed and resuspended in the ice-cold buffer for flow cytometry analysis.

SDS-PAGE and Western blotting analysis

MIN6 or NIT-1 cell lysates were prepared by resuspending cell pelletes (5×106 cells) in 200 μl of a cell-lysing buffer (containing 20 mM Tris.HCl, 5mM EDTA, 1% NP-40, 1mM NaVO4, 1% SDS, and 10 μg/ml Aprotinin/Leupeptin), and then incubated on ice for 15 minutes, followed by centrifugation at 15,000rpm × 20 minutes at 4°C. To perform SDS-PAGE, cell lysates or EXO samples were denatured by incubation with a sample buffer at 95°C for 10 minutes. SDS-PAGE-separated protein samples were electro-transferred to nitrocellulose membrane (Amersham, GE Healthcare Life Sciences, Piscataway, NJ) using a semi-dry trans-blot (BioRad Laboratories, Hercules, CA). The membrane was immuno-blotted with 1 μg/ml of primary antibodies, followed by a respective secondary HRP-labeled anti-IgG (Amersham). The protein bands were visualized with an enhanced chemiluminescence (ECL) detection system (Amersham). Clones 4-17 or 144 (provided by Dr. C. Hampe at the University of Washington, Seattle) were used for detecting GAD65. Anti-HSC70 were purchased from Stressgen (Enzo Life Sciences, Plymouth Meeting, PA). Anti-Chromagranin A was purchased from AbCam (Cambridge, MA).

Results

Mouse MIN6 insulinoma can release EXO that expresses candidate islet antigens

Exosomes were isolated from the culture supernatant of MIN6 insulinoma cells. As demonstrated by electron microscopy, the majority of the microparticles retain a round, vesicular shape, with a diameter less than 100 nm (Fig. 1A). Initial mass spectrometry analysis using the total microparticle preparation identified several signature proteins of typical EXO (17), including tetraspanins, Alix and TSG101, suggesting that the microparticle preparation contained EXO. To further analyze the protein content in the EXO, we separated the EXO on SDS-PAGE, after Coomassie staining, the gel was cut into 4 slices based on their molecular weights (Fig. 1B), and each gel slices were subjected to Mass spectrometry analysis. Around 200 protein molecules were identified (see Supplementary Table); other than molecules involved in intracellular 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 major diabetes-associated islet antigens, did not appear in the protein list identified by mass spectrometry, although 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 monoclonal antibody (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 GAD67 isoform, by its molecular weight. Nevertheless, the GAD65 protein was detected in MIN6 EXO by Western blot, which might be more sensitive than mass spectrometry 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 monoclonal antibody, whereas, both insulinomas released EXO contain heat shock protein cognate 70 (HSC70) protein, which is commonly found in such microparticles (17). Thus, GAD65 is released by MIN6 cells together or via EXO secretion pathway, but at a low level under the given culture condition.

FIGURE 1.

FIGURE 1

Open in a new tab

MIN6 insulinoma cells release EXO that expresses GAD65 protein. (A) Images of EXO preparations under an electron microscope after 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 (about 60 μg) was denatured in sample buffer and separated on a 10% SDS-PAGE gel. The gel was stained by Coomassie dye and cut into 4 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.

MIN6-derived EXO contains some innate stimuli that can induce production of inflammatory cytokines

To examine whether the EXO preparations can stimulate an innate immune response, splenocytes from 6-8 week-old NOD mice were incubated with EXO for 6-24 hours, and the levels of several inflammatory cytokines in the culture supernatants were measured. Fig. 2A demonstrates that the EXO can stimulate total splenocytes to produce inflammatory cytokines, mainly IL-6, IFN-gamma and TNF-alpha, although IL-10 and MCP-1 are also detectable. IL-12p70 was not detected, however, when a blocking anti-IL-12 antibody was added, IFN-gamma release from the EXO-stimulated splenocytes reduced over 3 folds (Fig. 2B), indicating that IL-12 is actually produced to promote IFN-gamma secretion by EXO-activated cells, but its concentration might be too low to be detected using this CBA assay. The cytokine levels produced by the EXO-stimulated splenocytes were dose- and time-dependent (Tab. I): even after just 6 hours of incubation with a low dose of EXO (1 μg/ml), TNF-alpha was elevated 8 folds above background, indicating that a rapid innate signal was triggered by EXO. This instant TNF-alpha release was due to EXO-induced activation of splenocytes since the cytokine was not detected by culturing EXO or splenocytes separately. At 20 μg/ml of 24h incubation with EXO, IL-6 increased 32 folds from 15.6 pg/ml to 497.3 pg/ml, IFN-gamma increased 22 folds from 2.7 pg/ml to 59.9 pg/ml and TNF-alpha increased 54 folds from 9.0 pg/ml to 482.7 pg/ml (Tab. I). In contrast, EXO preparations from 48h-cultured NOD splenocytes were non-stimulatory, whereas, the same volume of supernatant from MIN6 culture yielded sufficient EXO to induce the innate response. Therefore, the EXO secreted by the MIN6 cells contain some endogenous adjuvant-like components, capable of inducing the inflammatory cytokines.

FIGURE 2.

FIGURE 2

Open in a new tab

EXO collected from MIN6 culture supernatant is proinflammatory. (A) Splenocytes (106/200μl/well) from 8wk old NOD female mice were cultured with 5 μg/ml of MIN6 EXO for 48 hours in a flat-bottom 96-well plate. A CBA assay was performed to monitor the levels of six different inflammatory cytokines in the culture supernatants or the non-stimulated control. Similar results were observed in three separated experiments. (B) Experiment was similarly performed as in (A), except for that an IL-12-blocking antibody (clone C17.8) or a rat Ig isotype control was added at a final concentration of 20 μg/ml during the culture with EXO.

Table I.

Exosomes stimulate splenocytes to release inflammatory cytokines.

Stimulation times Cytokines (pg/ml) EXO doses (μg/ml)
0.0 1.0 5.0 20.0
6 hours IL-6 15.6 20.3 28.5 35.5
IFNγ 2.7 2.4 5.5 3.5
TNFα 9.0 77.6 121.8 193.5
12 hours IL-6 ND 81.3 116.7 161.3
IFNγ ND 3.4 4.8 10.0
TNFα ND 124.9 172.5 266.9
24 hours IL-6 17.4 334.3 333.5 497.3
IFNγ 8.9 36.2 31.1 59.9
TNFα 11.8 319.0 366.3 482.7
Open in a new tab

Splencoytes (106/200μl/well) from one male NOD were stimulated with different doses of EXO for various periods. Cytokine levels in the culture supernatants were monitored by the CBA assay. ND: not done.

The innate response induced by EXO requires MyD88-mediate TLR signaling pathway

To examine whether TLR-mediated innate signaling pathway is required for the EXO-induced cytokine response, we tested total splenocytes from a MyD88.KO mouse. Fig. 3A shows that the cytokine response to EXO stimulation (12h) was almost completely impaired in the MyD88.KO splenocytes as compared with wild type NOD cells; a similar lack of response in the MyD88.KO cells was observed after a longer (24h) incubation time although IFN-gamma was slightly increased, about 2 folds above the non-stimulated background. In contrast, in the wild type NOD female splencotyes, IFN-gamma increased at least 20 folds at 12h, and reached over 100 fold at 24h after stimulation with 10 μg/ml of MIN6 EXO (data not shown). In a 72-hour lymphocyte proliferation assay, the wild type NOD splenocytes responded to the EXO (10 μg/ml) with a stimulation index over 50. In contrast, the MyD88.KO splenocytes were completely unable to proliferate at the same dose ranges (SI<3), but responded strongly to Con A (Fig. 3B). These results suggest that the possible innate stimuli enclosed in the EXO may activate TLR signals via MyD88-dependent pathways.

FIGURE 3.

FIGURE 3

Open in a new tab

MyD88-dependent innate signaling pathway is required for EXO-induced inflammatory response. (A) Comparing EXO-induced cytokine secretion between two age-matched (within 8-12wk old) NOD and NOD.MyD88.KO female mice. Total splenocytes (106/200μl/well) were stimulated with 5 μg/ml of MIN6 EXO or Con A, or medium (Neg.) for 12 hours. Data are representative of three independent experiments. (B) EXO-induced proliferation of the splenocytes (5×105 cells/200μl/well) collected from the NOD or NOD.MyD88.KO mice. 3H-thymidine (0.5 μCi/well) was added for the last 18h of a 96h culture in a flat-bottom 96-well plate. (Background: NOD = 198 cpm, MyD88 = 672 cpm)

APC and purified subsets are activated by the EXO to upregulate class II MHC and costimulatory molecules

To examine whether EXO could activate APC, we monitored the expression of CD86 (B7.2) on class II MHC-positive splenocytes. Fig. 4A demonstrates that EXO stimulation increased the percentage of CD86 and I-Ag7 MHC double positive population from 2.6% to 11.5% in total splenocytes. Fig. 4B shows the expression of class II MHC, CD80, CD86 or ICAM-1 on the splenic B220+ (top panel), CD11c+ (middle panel) and CD11b+ (bottom panel) APC subpopulations after EXO stimulation. Clearly, professional APC were activated effectively by EXO as detected by their upregulated expression of MHC II or costimulatory molecules. It is noticed that non-stimulated splenic CD11c+ and CD11b+ APC expressed higher level of class II MHC than B220+ APC and EXO stimulation only upregulated CD86 or CD80, but not class II MHC on these two populations (Fig. 4B). Since EXO are small particles under 100 nm diameter, it is possible that certain APC subpopulation(s), such as dendritic cells, may be more efficient than others, such as macrophages or B cells, in picking up this size range of microparticles. We isolated CD11c+, CD11b+ or B220+ APC populations from NOD splenocytes by a positive selection protocol using MACS microbeads coated with the respective antibodies, and examined their responses to EXO. At 5 μg/ml of EXO concentration and 12 hours incubation time, both purified CD11c+ and CD11b+ APC populations responded rapidly (also observed at a 8h time-point) to EXO stimulation by releasing mainly IL-6 and TNF-alpha (Fig. 4C); the response by the purified B220+ APC (about 90% purity) was much lower, with a marginal (about 2 folds) increase of TNF-alpha, and IL-6 increased insignificantly.

FIGURE 4.

FIGURE 4

Open in a new tab

Activation of APC upon EXO stimulation. (A) Upon EXO stimulation (5 μg/ml for 12h), 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 (5μg/ml for 12h). (C) Activation of purified APC subsets by EXO. B220+, CD11b+ or CD11c+ cell populations were isolated using MACS microbeads coated with respective antibodies in a positive selection method, and then stimulated with 1 μg/ml of EXO for 8h, IL-6 and TNF-alpha concentrations in the culture supernatants were measured in the CBA assay.

T cells contribute to EXO-induced lymphocyte proliferation

Since NOD total splenocytes proliferated strongly after EXO stimulation (Fig. 5A), we asked whether T cells are the proliferating population by testing the proliferation response after depleting CD4+ and CD8+ T cells from the splenocytes. As a result, the proliferation capability was severely impaired in the absence of T cells. In one experiment, total NOD splenocytes reached a SI as high as 65 at the dose of 10 μg/ml of the EXO; whereas, the T-cell-depleted splenocytes responded 3-fold less to the same dose of EXO (Fig. 5A) despite that only about 50-60% depletion of T cells was achieved in this experiment. T cell proliferation was also demonstrated by a two-fold increase of the percentage of total CD4+ T cells in the EXO-stimulated splenocytes (Fig. 5B). Strikingly, no reactivity was observed when NOD.scid splencoytes were used in the proliferation assay (Fig. 5A), suggesting that T and/or B lymphocytes were the only populations that proliferated in response to EXO. The lack of proliferation of NOD.scid splenocytes to the EXO was not due to defects in their innate responses since the NOD.scid splenocytes also produced high levels of inflammatory cytokines when stimulated by EXO, as noted by a change of cytokine pattern (Fig. 5C).

FIGURE 5.

FIGURE 5

Open in a new tab

EXO-induced T cell proliferative response. (A) Reduced proliferative response to EXO by T-cell-depleted NOD splenocytes. T cells were depleted using two types of MACS microbeads coated with anti-CD4 and anti-CD8, respectively. NOD splenocytes or T-depleted (no T), or NOD.scid splenocytes were stimulated with 1 μg/ml (EXO-1) or 10 μg/ml (EXO-10) of EXO or Con A (3 μg/ml) in a total 96h proliferation assay. (B) NOD splencoytes were stimulated with 5 μg/ml of EXO for 72 hours, and then stained with anti-CD4. Fresh NOD splenocytes were used as control (Neg). (C) Comparing EXO-induced cytokine secretion between two NOD and NOD.scid 10-12wk old female mice. Total splenocytes (106/200μl/well) were stimulated with 5 μg/ml of EXO for 24 hours, and the CBA cytokine analysis was performed.

Prediabetic animals accumulate EXO-reactive, IFN-gamma-secreting T cells

To examine whether EXO stimulation could activate diabetes-associated T cell responses in NOD mice, three different methods were used to monitor the IFN-gamma response to the EXO: (1) total EXO-responding, IFN-gamma secreting cells in splenocytes were detected by ELISPOT assay, (2) IFN-gamma and other inflammatory cytokines in culture supernatants were examined by a flow cytometry-based CBA assay (see Materials and Methods), and (3) single IFN-gamma-secreting Th1 cells were identified by an IFN-gamma-capture assay. Fig. 6A shows that the levels of EXO-induced IFN-gamma response correlated well with the developmental stages of diabetes in the ELISPOT assay, with the oldest NOD females exhibiting the highest number of IFN-gamma+ spots, and the male NODs the lowest. In addition, CBA assay was performed to compare NOD female and male mice for their responses to EXO stimulation in vitro. Among the six inflammatory cytokines, IFN-gamma was found different in that female splenocytes produced 10 fold higher of IFN-gamma on average than male splenocytes after stimulated with EXO (Fig. 6B). Three out of four 9-12 wk old females produced large amount of IFN-gamma (632, 688 and 1044 pg/ml) after 48h stimulation, whereas all three age-matched males responded below 100 pg/ml (35, 72 and 87 pg/ml). Thus, the EXO-responding, IFN-gamma-producing cells are accumulated in prediabetic NOD female mice. This correlation was further confirmed by testing NOD congenic strains with different susceptibility to T1D. As shown in Fig. 6C, NOD, the most susceptible strain, produced the highest number of IFN-gamma spots (about 100 spots/106 splenocytes); whereas, only less than 10 spots were identified when NOR splenocytes were stimulated by the EXO and the partially resistant strain, NOD.B10 Idd5.2, exhibited an intermediate response. Similarly, IFN-gamma but not the other five inflammatory cytokines was also found different between NOD and the resistant strain. Fig. 6D shows one example that splenocytes from a NOD female produced 3-5 fold higher amount of IFN-gamma than an age- and gender-matched NOD.B10 Idd5.2 mouse.

FIGURE 6.

FIGURE 6

Open in a new tab

EXO can activate diabetes-associated, IFN-gamma-secreting cells. (A) Total splenocytes (106/200μl/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 μg/ml) in an IFN-gamma 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 4 female and 3 male NOD mice (9-12 wk old) were stimulated with EXO for 48h, 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-gamma responses among different NOD congenic strains (8-10 wk old female mice) were compared by ELISPOT method. Data represent one of two experiments with similar observation. (D) IFN-gamma 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.

To quantify EXO-responding, IFN-gamma-secreting T helper cells, we performed IFN-gamma secreting/capturing assay after stimulating pancreatic lymph node cells (Pan-LN) or inguinal lymph node cells (Ing-LN) with EXO in culture. Fig. 7A shows an over 10 fold increase of the Th1 cells observed in the Pan-LN of NOD female mice after EXO stimulation, from 0.17% increased to 1.8% of total Pan-LN; whereas, no increase was observed for a resistant NOD congenic strain, NOD.Idd3/5. It is noticed that many Pan-LN of NOD increased their IFN-gamma secretion after EXO stimulation, but at a low level, as indicated in a histogram plot in Fig. 7B (top panel), rather than a polarized, fully activated, Th1 phenotype. Control Ing-LN of the NOD mice showed less of an increase (from 0.25% to 0.48%) after EXO stimulation (Fig. 7B, bottom panel), further confirming that EXO-reactive Th1 cells preferentially accumulate in the Pan-LN of prediabetic animals.

FIGURE 7.

FIGURE 7

Open in a new tab

Accumulation of EXO-responding, IFN-gamma-secreting T helper cells in pancreatic lymph nodes of NOD mice. (A) Pancreatic lymph node (Pan-LN) cells (5×105/well) from age-matched NOD and NOD.B10 Idd3/5 (Idd3/5) female mice (3 mice/group) were mixed with irradiated syngenic splenic APC (1:1) and then cultured with medium or 5μg/ml of EXO for 48h. IFN-gamma-secreting CD4+ T cells were identified in an IFN-gamma 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 48h). Inguinal lymph nodes (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).

EXO immunization accelerates insulitis in diabetes-resistant NOR mice

To examine whether EXO can trigger or accelerate islet inflammation or insulitis in diabetes-resistant mice, we directly injected EXO intravenously, without addition of other adjuvant, into three diabetes-resistant mouse strains, NOR, NOD.H-2b and C57BL/6 mice. After 3 or 7 days, their pancreatic glands were collected for histological examination. It has been reported that both NOD.H-2b and C57BL/6 mice do not develop insulitis (6, 7), and after 7 days of EXO injection, all islets from these two strains (four mice per strain, and 40-50 islets per mouse) remained completely free from lymphocyte infiltration (data not shown). Interestingly, we noticed that not all adult NOR mice are free from insulitis, confirming a previous report that almost half examined islets had some level of lymphocyte infiltration (5). Notably, EXO injection accelerated insulitis in this strain. In the untreated group of nine 4-5 month old NOR female mice, out of total 457 islets examined, only 53 islets or 11.6% showed severe insulitis (over 50% area of one islet is infiltrated by lymphocytes). The mean of lymphocyte infiltration per islet for this untreated group is 12.9%. In contrast, in the experiment group 7 days after treated with EXO, there were 94 islets or 42% out of total 226 examined showed severe insulitis, and the mean of lymphocyte infiltration is 37.7%, which is three fold higher than the untreated group. Fig. 8 shows the insulitis levels for the individual NOR mouse with or without EXO injection. The severity of insulitis or lymphocyte infiltration is significantly increased in the group treated with EXO for 7 days (p = 0.01), but the difference between the untreated and 3 day-treated groups was insignificant.

FIGURE 8.

FIGURE 8

Open in a new tab

Induction of insulitis in NOR mice by EXO immunization. 4-5 month-old NOR females mice were injected (i.v.) once with 15 μg of the EXO in PBS. After 3 or 7 days, pancreases were collected and submitted for hematoxylin and eosin stain. Levels of islet infiltration were scored in blind, and the percentages of islets within the each level of lymphocyte infiltration are shown for individual mice. Nine age/gender-matched NOR mice purchased from the Jackson Laboratory were used as untreated controls. The mean of infiltration for each mouse was calculated as: the sum of percentages of infiltration for each islets / # of total islets examined for each mouse.

Discussion

Exosomes derived from tumor cells are naturally occurring small nanoparticles that not only contain potent adjuvant-like substances, but also carry tumor-specific antigens (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). Since these physiologically important microparticles contain mostly self antigens, to which immune tolerance or regulation is well established, except for those rare individuals who are highly susceptible to a particular autoimmune disease, the chance of successly inducing effective anti-tumor immunity or breaking self tolerance using EXO as an immunogen is low. Thus, the consequence of EXO-induced immune responses might be heavily dependent on the types/contents of EXO as well as the genetic background of the hosts. To our knowledge, we believe 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 immunosuppression 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 unknown whether the accumulated EXO-reactive Th1 cells in the prediabetic NOD mice were primed directly by the endogenous EXO secreted by the pancreatic islet cells, or via cross-reaction to other antigens. Nevertheless, EXO or microparticles secreted by islet cells should be useful tools for studying tissue-specific autoantigens and their cognate autoreactive T cells.

It has been previously shown that tumor cell-derived EXO requires MyD88-dependent TLR-signaling pathway to induce IL-6 and TNF-alpha secretion from myeloid cells (25). EXO derived from different tissues or cell lines may vary in their proinflammatory activity, and the characteristics of the possible innate stimuli within MIN6 insulinoma remain unknown, but we confirmed that the innate response also required MyD88. Several HSPs were found in our EXO preparations. Chalmin et al demonstrated that Hsp72 expressed on tumor cell-derived EXO could activate myeloid-derived suppressor cells in a TLR2/MyD88-dependent manner (30); whereas, Zhang et al demonstrated that neither TLR2 nor TLR4 were involved in their system testing EXO-induced activation of the myeloid-derived suppressor cells (25). EXO released from Leishmania could modulate monocytes’ cytokine response by promoting IL-10 but inhibiting TNF-alpha (33). Interestingly, human primary astrocytes can release exosomes carrying mitochondrial DNA (34), which were shown to have innate stimulatory activity, possibly via the TLR9 signaling pathway (35). It was noteworthy that our EXO preparation contains endogenous gag and env proteins (Supplementary Table); whether endogenous retroviruses, which have been implicated as etiological agents of T1D (36, 37), contribute to the immune responses to EXO is an interesting possibility. Whether EXO produced by different types of tumor cells or primary tissues or organisms vary in their usages of different innate pathways or TLRs remains to be studied.

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

Among the 6 inflammatory cytokines we examined, IL-6 and TNF-alpha were the major cytokines produced by purified splenic APC populations (CD11c+ DC, CD11b+ macrophages and B220+ cells) although IL-10 was also detected after EXO stimulation. MCP-1 was produced only by CD11b+ macrophages, and IFN-gamma was undetectable for all three purified APC populations. When total splenocytes were cultured with EXO, IFN-gamma level increased after 24h culture, indicating an activation of T cells at this time point. However, some CD4-CD8- splenocytes were also found to produce IFN-gamma when cultured with EXO. This is particular interesting since it has been shown that NK cells may be one of the early islet infiltrators triggering the inflammatory response (42), and an abnormal EXO release from the islets may attribute to this early NK infiltration.

T1D is a Th1-mediated autoimmune disease and activation of tissue-specific, IFN-gamma secreting T helper cells is a crucial checkpoint controlling disease progression (43). However, the endogenous pathway inducing these pathogenic effector cells is not fully understood. Since I-Ag7 class II MHC is required for the insulitis and diabetes in NOD mice and T cells are among the earliest islet infiltrating lymphocytes (44), we proposed that altered presentation of islet antigens may contribute to specific activation of high affinity Th cells (45, 46). We found that EXO were effective in activating IFN-gamma-secreting Th1 cells in NOD mice. Presumably, these Th1 cells represent the islet-antigen-specific memory cells that are primed endogenously in the pancreatic islets or the draining lymph nodes. We noticed that despite a significant increase of EXO-reactive Th1 cells in old NOD female mice, the number of highly polarized Th1 cells was very low as indicated in the ELISPOT assay. Also, although many EXO-activated T helper cells in the Pan-LN of NOD mice can produce IFN-gamma, the amount of IFN-gamma they produced was small as measured in the IFN-gamma secreting/capturing assay. One possibility is due to a low expression of candidate autoantigens within the EXO. It might be interesting to test whether increased expression of the autoantigens could enhance IFN-gamma production. It is also possible that other islet antigen sources such as apoptotic bodies released following beta cell death (47) that may express large amount of candidate antigens can stimulate T cells to produce more IFN-gamma. Alternatively, polyclonal activation of many T helper cell clones that are not highly polarized but biased to produce some IFN-gamma might be sufficient to cause damage of the islets. In fact, it has been shown that even at very early stage of prediabetic NOD mice, TCR repertoire in pancreatic lymph nodes, although restricted, is highly diverse and dynamic (48), and we had similar observation in studying the TCR repertoire of islet-infiltrating T cells (Marrero et al. manuscript submitted).

It will be helpful to test whether insulinoma-derived EXO can activate some highly diabetogenic T cell clones (49, 50), providing that EXO and its antigens may be processed and presented via some unique pathway(s). Our study is inconclusive about whether the EXO-induced IFN-gamma-secreting cells could be due to partial cross-reactivity (51) of the disease-related memory precursors to some unknown exosomal antigens or even due to some bystander mechanism (52). Study of a particular antigen and its cognate T cells using EXO as an antigen carrier may help address further whether EXO could be responsible for inducing a tissue-specific autoimmunity. It is noteworthy that of the five different inflammatory cytokines we examined, only IFN-gamma was found to differ between diabetes susceptible and resistant strains, or between NOD male and female mice when total splenocytes were cultured with EXO. Such difference cannot be identified after stimulation with LPS. This indicates that EXO may be a possible endogenous target that can preferentially activate a small percent of memory autoreactive T cells in vitro.

We have found that candidate diabetes-associated autoantigens, particularly GAD65, are expressed in the EXO produced by MIN6 insulinoma cells. GAD65-specific autoantibodies and T cell responses are detected very early in prediabetic patients (53). The GAD65 protein in the pancreatic islets of NOD mice (54) and the NIT-1 insulinoma cells is barely detectible, but its expression in the MIN6 cells and their secreted EXO is effective (Fig. 1C). We have also detected another candidate diabetic autoantigens, Chromogranin A (55), in MIN6 cells as well as in their secreted EXO (data not shown). We have learned that most of candidate diabetes antigens such as GAD65 and IA-2, except for insulin, are not a necessity causing diabetes in NOD mice (56-58); it might be an interesting question whether a disease-triggering event, maybe abnormal EXO secretion, could cause autoimmune targeting at several islet antigens simultaneously. The hypothesis that insulin acts as the primary islet antigen (58) is intriguing since islet beta cells apparently are not the only cell populations attacked in the islets and might be secondary to the peri-insulitis targeting at the islet glial cells (13), which we found also express GAD65 protein and contain innate stimuli (Unpublished data by Sheng et al.). This leads us to wondering whether the GAD65-specific T cell responses developed early in NOD mice (59, 60) may be triggered by the glial cells. Interestingly, not all types of EXO are proinflammatory; for example, oligodendrocytes-released EXO can carry large amount of myelin autoantigens, but are incapable of inducing a concomitant inflammatory response (61). Whether NOD mouse strain and/or the islet glial cells are producing abnormal EXO that are more inflammatory than diabetes-resistant strains remains to be studied.

Supplementary Material

1
2

ACKNOWLEDGMENTS

We thank Drs. Linda Wicker at the University of Cambridge, UK, and Linda Sherman at The Scripps Research Institute, San Diego, USA, for their support and guidance, as well as editing the manuscript. We also thank Dr. Christiane Hampe at the University of Washington for providing monoclonal antibodies specific for several regions of GAD65, Dr. Sally Sarawar (TPIMS) for providing the IL-12-blocking antibody (clone C17.8) and rat Ig isotype control, and Dr. Todd Braciak (TPIMS) for critical reading of the manuscript.

Footnotes

1

This work was supported by a career grant to Y.D.D. from the National Institutes of Health (DK076741) and a research grant to Y.D.D. from the Diabetes National Research Group (DNRG).

REFERENCES

  • 1.Mackay IR, Leskovsek NV, Rose NR. Cell damage and autoimmunity: a critical appraisal. J Autoimmun. 2008;30:5–11. doi: 10.1016/j.jaut.2007.11.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Wicker LS, Clark J, Fraser HI, Garner VE, Gonzalez-Munoz A, Healy B, Howlett S, Hunter K, Rainbow D, Rosa RL, Smink LJ, Todd JA, Peterson LB. Type 1 diabetes genes and pathways shared by humans and NOD mice. J Autoimmun. 2005;25(Suppl):29–33. doi: 10.1016/j.jaut.2005.09.009. [DOI] [PubMed] [Google Scholar]
  • 3.Hanson MS, Cetkovic-Cvrlje M, Ramiya VK, Atkinson MA, Maclaren NK, Singh B, Elliott JF, Serreze DV, Leiter EH. Quantitative thresholds of MHC class II I-E expressed on hemopoietically derived antigen-presenting cells in transgenic NOD/Lt mice determine level of diabetes resistance and indicate mechanism of protection. J Immunol. 1996;157:1279–1287. [PubMed] [Google Scholar]
  • 4.Serreze DV, Prochazka M, Reifsnyder PC, Bridgett MM, Leiter EH. Use of recombinant congenic and congenic strains of NOD mice to identify a new insulindependent diabetes resistance gene. J Exp Med. 1994;180:1553–1558. doi: 10.1084/jem.180.4.1553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Rothe H, Ito Y, Kolb H. Disease resistant, NOD-related strains reveal checkpoints of immunoregulation in the pancreas. J Mol Med. 2001;79:190–197. doi: 10.1007/s001090000182. [DOI] [PubMed] [Google Scholar]
  • 6.Yui MA, Muralidharan K, Moreno-Altamirano B, Perrin G, Chestnut K, Wakeland EK. Production of congenic mouse strains carrying NOD-derived diabetogenic genetic intervals: an approach for the genetic dissection of complex traits. Mamm Genome. 1996;7:331–334. doi: 10.1007/s003359900097. [DOI] [PubMed] [Google Scholar]
  • 7.Wicker LS, Appel MC, Dotta F, Pressey A, Miller BJ, DeLarato NH, Fischer PA, Boltz RC, Jr., Peterson LB. Autoimmune syndromes in major histocompatibility complex (MHC) congenic strains of nonobese diabetic (NOD) mice. The NOD MHC is dominant for insulitis and cyclophosphamide-induced diabetes. J Exp Med. 1992;176:67–77. doi: 10.1084/jem.176.1.67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Chaparro RJ, Konigshofer Y, Beilhack GF, Shizuru JA, McDevitt HO, Chien YH. Nonobese diabetic mice express aspects of both type 1 and type 2 diabetes. Proc Natl Acad Sci U S A. 2006;103:12475–12480. doi: 10.1073/pnas.0604317103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Yamanouchi J, Rainbow D, Serra P, Howlett S, Hunter K, Garner VE, Gonzalez-Munoz A, Clark J, Veijola R, Cubbon R, Chen SL, Rosa R, Cumiskey AM, Serreze DV, Gregory S, Rogers J, Lyons PA, Healy B, Smink LJ, Todd JA, Peterson LB, Wicker LS, Santamaria P. Interleukin-2 gene variation impairs regulatory T cell function and causes autoimmunity. Nat Genet. 2007;39:329–337. doi: 10.1038/ng1958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Tang Q, Adams JY, Tooley AJ, Bi M, Fife BT, Serra P, Santamaria P, Locksley RM, Krummel MF, Bluestone JA. Visualizing regulatory T cell control of autoimmune responses in nonobese diabetic mice. Nat Immunol. 2006;7:83–92. doi: 10.1038/ni1289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Trudeau JD, Dutz JP, Arany E, Hill DJ, Fieldus WE, Finegood DT. Neonatal beta-cell apoptosis: a trigger for autoimmune diabetes? Diabetes. 2000;49:1–7. doi: 10.2337/diabetes.49.1.1. [DOI] [PubMed] [Google Scholar]
  • 12.Xia CQ, Peng R, Qiu Y, Annamalai M, Gordon D, Clare-Salzler MJ. Transfusion of apoptotic beta-cells induces immune tolerance to beta-cell antigens and prevents type 1 diabetes in NOD mice. Diabetes. 2007;56:2116–2123. doi: 10.2337/db06-0825. [DOI] [PubMed] [Google Scholar]
  • 13.Winer S, Tsui H, Lau A, Song A, Li X, Cheung RK, Sampson A, Afifiyan F, Elford A, Jackowski G, Becker DJ, Santamaria P, Ohashi P, Dosch HM. Autoimmune islet destruction in spontaneous type 1 diabetes is not beta-cell exclusive. Nat Med. 2003;9:198–205. doi: 10.1038/nm818. [DOI] [PubMed] [Google Scholar]
  • 14.Tsui H, Chan Y, Tang L, Winer S, Cheung RK, Paltser G, Selvanantham T, Elford AR, Ellis JR, Becker DJ, Ohashi PS, Dosch HM. Targeting of pancreatic glia in type 1 diabetes. Diabetes. 2008;57:918–928. doi: 10.2337/db07-0226. [DOI] [PubMed] [Google Scholar]
  • 15.Johnstone RM. Exosomes biological significance: A concise review. Blood Cells Mol Dis. 2006;36:315–321. doi: 10.1016/j.bcmd.2005.12.001. [DOI] [PubMed] [Google Scholar]
  • 16.Pegtel DM, Cosmopoulos K, Thorley-Lawson DA, van Eijndhoven MA, Hopmans ES, Lindenberg JL, de Gruijl TD, Wurdinger T, Middeldorp JM. Functional delivery of viral miRNAs via exosomes. Proc Natl Acad Sci U S A. 107:6328–6333. doi: 10.1073/pnas.0914843107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Thery C, Ostrowski M, Segura E. Membrane vesicles as conveyors of immune responses. Nat Rev Immunol. 2009;9:581–593. doi: 10.1038/nri2567. [DOI] [PubMed] [Google Scholar]
  • 18.Zitvogel L, Regnault A, Lozier A, Wolfers J, Flament C, Tenza D, Ricciardi-Castagnoli P, Raposo G, Amigorena S. Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes. Nat Med. 1998;4:594–600. doi: 10.1038/nm0598-594. [DOI] [PubMed] [Google Scholar]
  • 19.Chen W, Wang J, Shao C, Liu S, Yu Y, Wang Q, Cao X. Efficient induction of antitumor T cell immunity by exosomes derived from heat-shocked lymphoma cells. Eur J Immunol. 2006;36:1598–1607. doi: 10.1002/eji.200535501. [DOI] [PubMed] [Google Scholar]
  • 20.Zeelenberg IS, Ostrowski M, Krumeich S, Bobrie A, Jancic C, Boissonnas A, Delcayre A, Le Pecq JB, Combadiere B, Amigorena S, Thery C. Targeting tumor antigens to secreted membrane vesicles in vivo induces efficient antitumor immune responses. Cancer Res. 2008;68:1228–1235. doi: 10.1158/0008-5472.CAN-07-3163. [DOI] [PubMed] [Google Scholar]
  • 21.Escudier B, Dorval T, Chaput N, Andre F, Caby MP, Novault S, Flament C, Leboulaire C, Borg C, Amigorena S, Boccaccio C, Bonnerot C, Dhellin O, Movassagh M, Piperno S, Robert C, Serra V, Valente N, Le Pecq JB, Spatz A, Lantz O, Tursz T, Angevin E, Zitvogel L. Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of thefirst phase I clinical trial. J Transl Med. 2005;3:10. doi: 10.1186/1479-5876-3-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Morse MA, Garst J, Osada T, Khan S, Hobeika A, Clay TM, Valente N, Shreeniwas R, Sutton MA, Delcayre A, Hsu DH, Le Pecq JB, Lyerly HK. A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer. J Transl Med. 2005;3:9. doi: 10.1186/1479-5876-3-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Xiang X, Poliakov A, Liu C, Liu Y, Deng ZB, Wang J, Cheng Z, Shah SV, Wang GJ, Zhang L, Grizzle WE, Mobley J, Zhang HG. Induction of myeloid-derived suppressor cells by tumor exosomes. Int J Cancer. 2009;124:2621–2633. doi: 10.1002/ijc.24249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Wieckowski EU, Visus C, Szajnik M, Szczepanski MJ, Storkus WJ, Whiteside TL. Tumor-derived microvesicles promote regulatory T cell expansion and induce apoptosis in tumor-reactive activated CD8+ T lymphocytes. J Immunol. 2009;183:3720–3730. doi: 10.4049/jimmunol.0900970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Liu Y, Xiang X, Zhuang X, Zhang S, Liu C, Cheng Z, Michalek S, Grizzle W, Zhang HG. Contribution of MyD88 to the tumor exosome-mediated induction of myeloid derived suppressor cells. Am J Pathol. 176:2490–2499. doi: 10.2353/ajpath.2010.090777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Hunter K, Rainbow D, Plagnol V, Todd JA, Peterson LB, Wicker LS. Interactions between Idd5.1/Ctla4 and other type 1 diabetes genes. J Immunol. 2007;179:8341–8349. doi: 10.4049/jimmunol.179.12.8341. [DOI] [PubMed] [Google Scholar]
  • 27.Wicker LS, Chamberlain G, Hunter K, Rainbow D, Howlett S, Tiffen P, Clark J, Gonzalez-Munoz A, Cumiskey AM, Rosa RL, Howson JM, Smink LJ, Kingsnorth A, Lyons PA, Gregory S, Rogers J, Todd JA, Peterson LB. Fine mapping, gene content, comparative sequencing, and expression analyses support Ctla4 and Nramp1 as candidates for Idd5.1 and Idd5.2 in the nonobese diabetic mouse. J Immunol. 2004;173:164–173. doi: 10.4049/jimmunol.173.1.164. [DOI] [PubMed] [Google Scholar]
  • 28.Miyazaki J, Araki K, Yamato E, Ikegami H, Asano T, Shibasaki Y, Oka Y, Yamamura K. Establishment of a pancreatic beta cell line that retains glucose-inducible insulin secretion: special reference to expression of glucose transporter isoforms. Endocrinology. 1990;127:126–132. doi: 10.1210/endo-127-1-126. [DOI] [PubMed] [Google Scholar]
  • 29.Hamaguchi K, Gaskins HR, Leiter EH. NIT-1, a pancreatic beta-cell line established from a transgenic NOD/Lt mouse. Diabetes. 1991;40:842–849. doi: 10.2337/diab.40.7.842. [DOI] [PubMed] [Google Scholar]
  • 30.Chalmin F, Ladoire S, Mignot G, Vincent J, Bruchard M, Remy-Martin JP, Boireau W, Rouleau A, Simon B, Lanneau D, De Thonel A, Multhoff G, Hamman A, Martin F, Chauffert B, Solary E, Zitvogel L, Garrido C, Ryffel B, Borg C, Apetoh L, Rebe C, Ghiringhelli F. Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells. J Clin Invest. 120:457–471. doi: 10.1172/JCI40483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Wang GJ, Liu Y, Qin A, Shah SV, Deng ZB, Xiang X, Cheng Z, Liu C, Wang J, Zhang L, Grizzle WE, Zhang HG. Thymus exosomes-like particles induce regulatory T cells. J Immunol. 2008;181:5242–5248. doi: 10.4049/jimmunol.181.8.5242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Viaud S, Thery C, Ploix S, Tursz T, Lapierre V, Lantz O, Zitvogel L, Chaput N. Dendritic cell-derived exosomes for cancer immunotherapy: what's next? Cancer Res. 70:1281–1285. doi: 10.1158/0008-5472.CAN-09-3276. [DOI] [PubMed] [Google Scholar]
  • 33.Silverman JM, Clos J, Horakova E, Wang AY, Wiesgigl M, Kelly I, Lynn MA, McMaster WR, Foster LJ, Levings MK, Reiner NE. Leishmania exosomes modulate innate and adaptive immune responses through effects on monocytes and dendritic cells. J Immunol. 185:5011–5022. doi: 10.4049/jimmunol.1000541. [DOI] [PubMed] [Google Scholar]
  • 34.Guescini M, Genedani S, Stocchi V, Agnati LF. Astrocytes and Glioblastoma cells release exosomes carrying mtDNA. J Neural Transm. 117:1–4. doi: 10.1007/s00702-009-0288-8. [DOI] [PubMed] [Google Scholar]
  • 35.Zhang Q, Raoof M, Chen Y, Sumi Y, Sursal T, Junger W, Brohi K, Itagaki K, Hauser CJ. Circulating mitochondrial DAMPs cause inflammatory responses to injury. Nature. 464:104–107. doi: 10.1038/nature08780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Suenaga K, Yoon JW. Association of beta-cell-specific expression of endogenous retrovirus with development of insulitis and diabetes in NOD mouse. Diabetes. 1988;37:1722–1726. doi: 10.2337/diab.37.12.1722. [DOI] [PubMed] [Google Scholar]
  • 37.Gaskins HR, Prochazka M, Hamaguchi K, Serreze DV, Leiter EH. Beta cell expression of endogenous xenotropic retrovirus distinguishes diabetes-susceptible NOD/Lt from resistant NON/Lt mice. J Clin Invest. 1992;90:2220–2227. doi: 10.1172/JCI116107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Wen L, Ley RE, Volchkov PY, Stranges PB, Avanesyan L, Stonebraker AC, Hu C, Wong FS, Szot GL, Bluestone JA, Gordon JI, Chervonsky AV. Innate immunity and intestinal microbiota in the development of Type 1 diabetes. Nature. 2008;455:1109–1113. doi: 10.1038/nature07336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Wong FS, Hu C, Zhang L, Du W, Alexopoulou L, Flavell RA, Wen L. The role of Toll-like receptors 3 and 9 in the development of autoimmune diabetes in NOD mice. Ann N Y Acad Sci. 2008;1150:146–148. doi: 10.1196/annals.1447.039. [DOI] [PubMed] [Google Scholar]
  • 40.Kim HS, Han MS, Chung KW, Kim S, Kim E, Kim MJ, Jang E, Lee HA, Youn J, Akira S, Lee MS. Toll-like receptor 2 senses beta-cell death and contributes to the initiation of autoimmune diabetes. Immunity. 2007;27:321–333. doi: 10.1016/j.immuni.2007.06.010. [DOI] [PubMed] [Google Scholar]
  • 41.Zhang Y, Lee AS, Shameli A, Geng X, Finegood D, Santamaria P, Dutz JP. TLR9 blockade inhibits activation of diabetogenic CD8+ T cells and delays autoimmune diabetes. J Immunol. 184:5645–5653. doi: 10.4049/jimmunol.0901814. [DOI] [PubMed] [Google Scholar]
  • 42.Brauner H, Elemans M, Lemos S, Broberger C, Holmberg D, Flodstrom-Tullberg M, Karre K, Hoglund P. Distinct phenotype and function of NK cells in the pancreas of nonobese diabetic mice. J Immunol. 184:2272–2280. doi: 10.4049/jimmunol.0804358. [DOI] [PubMed] [Google Scholar]
  • 43.Liblau RS, Singer SM, McDevitt HO. Th1 and Th2 CD4+ T cells in the pathogenesis of organ-specific autoimmune diseases. Immunol Today. 1995;16:34–38. doi: 10.1016/0167-5699(95)80068-9. [DOI] [PubMed] [Google Scholar]
  • 44.Wicker LS, Todd JA, Peterson LB. Genetic control of autoimmune diabetes in the NOD mouse. Annu Rev Immunol. 1995;13:179–200. doi: 10.1146/annurev.iy.13.040195.001143. [DOI] [PubMed] [Google Scholar]
  • 45.Dai YD, Sercarz EE. Antigen processing patterns determine GAD65-specific regulation vs. pathogenesis. Front Biosci. 2009;14:344–351. doi: 10.2741/3248. [DOI] [PubMed] [Google Scholar]
  • 46.Dai YD, Marrero IG, Gros P, Zaghouani H, Wicker LS, Sercarz EE. Slc11a1 enhances the autoimmune diabetogenic T-cell response by altering processing and presentation of pancreatic islet antigens. Diabetes. 2009;58:156–164. doi: 10.2337/db07-1608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Kurrer MO, Pakala SV, Hanson HL, Katz JD. Beta cell apoptosis in T cell-mediated autoimmune diabetes. Proc Natl Acad Sci U S A. 1997;94:213–218. doi: 10.1073/pnas.94.1.213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Petrovc Berglund J, Mariotti-Ferrandiz E, Rosmaraki E, Hall H, Cazenave PA, Six A, Hoglund P. TCR repertoire dynamics in the pancreatic lymph nodes of non-obese diabetic (NOD) mice at the time of disease initiation. Mol Immunol. 2008;45:3059–3064. doi: 10.1016/j.molimm.2008.03.009. [DOI] [PubMed] [Google Scholar]
  • 49.Bergman B, Haskins K. Islet-specific T-cell clones from the NOD mouse respond to beta-granule antigen. Diabetes. 1994;43:197–203. doi: 10.2337/diab.43.2.197. [DOI] [PubMed] [Google Scholar]
  • 50.Burton AR, Vincent E, Arnold PY, Lennon GP, Smeltzer M, Li CS, Haskins K, Hutton J, Tisch RM, Sercarz EE, Santamaria P, Workman CJ, Vignali DA. On the pathogenicity of autoantigen-specific T-cell receptors. Diabetes. 2008;57:1321–1330. doi: 10.2337/db07-1129. [DOI] [PubMed] [Google Scholar]
  • 51.Sercarz EE, Maverakis E. Recognition and function in a degenerate immune system. Mol Immunol. 2004;40:1003–1008. doi: 10.1016/j.molimm.2003.11.002. [DOI] [PubMed] [Google Scholar]
  • 52.Bitmansour AD, Douek DC, Maino VC, Picker LJ. Direct ex vivo analysis of human CD4(+) memory T cell activation requirements at the single clonotype level. J Immunol. 2002;169:1207–1218. doi: 10.4049/jimmunol.169.3.1207. [DOI] [PubMed] [Google Scholar]
  • 53.Lernmark A. Glutamic acid decarboxylase--gene to antigen to disease. J Intern Med. 1996;240:259–277. doi: 10.1046/j.1365-2796.1996.27859000.x. [DOI] [PubMed] [Google Scholar]
  • 54.Kim J, Richter W, Aanstoot HJ, Shi Y, Fu Q, Rajotte R, Warnock G, Baekkeskov S. Differential expression of GAD65 and GAD67 in human, rat, and mouse pancreatic islets. Diabetes. 1993;42:1799–1808. doi: 10.2337/diab.42.12.1799. [DOI] [PubMed] [Google Scholar]
  • 55.Stadinski BD, Delong T, Reisdorph N, Reisdorph R, Powell RL, Armstrong M, Piganelli JD, Barbour G, Bradley B, Crawford F, Marrack P, Mahata SK, Kappler JW, Haskins K. Chromogranin A is an autoantigen in type 1 diabetes. Nat Immunol. 11:225–231. doi: 10.1038/ni.1844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Kash SF, Condie BG, Baekkeskov S. Glutamate decarboxylase and GABA in pancreatic islets: lessons from knock-out mice. Horm Metab Res. 1999;31:340–344. doi: 10.1055/s-2007-978750. [DOI] [PubMed] [Google Scholar]
  • 57.Kubosaki A, Nakamura S, Notkins AL. Dense core vesicle proteins IA-2 and IA-2beta: metabolic alterations in double knockout mice. Diabetes. 2005;54(Suppl 2):S46–51. doi: 10.2337/diabetes.54.suppl_2.s46. [DOI] [PubMed] [Google Scholar]
  • 58.Nakayama M, Abiru N, Moriyama H, Babaya N, Liu E, Miao D, Yu L, Wegmann DR, Hutton JC, Elliott JF, Eisenbarth GS. Prime role for an insulin epitope in the development of type 1 diabetes in NOD mice. Nature. 2005;435:220–223. doi: 10.1038/nature03523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Kaufman DL, Clare-Salzler M, Tian J, Forsthuber T, Ting GS, Robinson P, Atkinson MA, Sercarz EE, Tobin AJ, Lehmann PV. Spontaneous loss of T-cell tolerance to glutamic acid decarboxylase in murine insulin-dependent diabetes. Nature. 1993;366:69–72. doi: 10.1038/366069a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Tisch R, Yang XD, Singer SM, Liblau RS, Fugger L, McDevitt HO. Immune response to glutamic acid decarboxylase correlates with insulitis in non-obese diabetic mice. Nature. 1993;366:72–75. doi: 10.1038/366072a0. [DOI] [PubMed] [Google Scholar]
  • 61.Fitzner D, Schnaars M, van Rossum D, Krishnamoorthy G, Dibaj P, Bakhti M, Regen T, Hanisch UK, Simons M. Selective transfer of exosomes from oligodendrocytes to microglia by macropinocytosis. J Cell Sci. 124:447–458. doi: 10.1242/jcs.074088. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS303075-supplement-1.doc (23.5KB, doc)
2
NIHMS303075-supplement-2.xls (41.5KB, xls)

RESOURCES