Autoimmune diabetes mellitus in humans is characterized by immunological destruction of pancreatic beta islet cells. We investigated the circumstances under which CD8+ T cells specific for pancreatic beta-islet antigens induce disease in mice expressing lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) as a transgene under the control of the rat insulin promoter. In contrast to infection with LCMV, immunization with LCMV-GP derived peptide did not induce autoimmune diabetes despite large numbers of autoreactive cytotoxic T cells. Only subsequent treatment with Toll-like receptor ligands elicited overt autoimmune disease. This difference was critically regulated by the peripheral target organ itself, which upregulated class I major histocompatibility complex (MHC) in response to systemic Toll-like receptor–triggered interferon-α production. These data identify the 'inflammatory status' of the target organ as a separate and limiting factor determining the development of autoimmune disease.
Autoimmune diabetes in humans is characterized by immunological destruction of beta islet cells in the pancreas and subsequent hyperglycemia. T cells specific for beta islet cell antigens are likely to have a major role in the disease process1 and have been suggested to arise following exposure to viruses2,3,4,5 or infectious agents containing cross-reactive epitopes (molecular mimicry)6. Indeed, Coxsackie virus and human cytomegalovirus contain proteins that share amino acid sequences with human GAD65, which is strongly expressed in the pancreas7,8, and antigenic mimicry may explain the known association of human leukocyte antigen (HLA) haplotype and disease development5. However, several viruses suspected to be associated with diabetes do not contain any known cross-reactive epitopes5, and approximately 50% of the European population is infected with human cytomegalovirus9 without being diabetic, although human cytomegalovirus has a cross-reactive epitope with the beta islet antigen GAD65 (ref. 5). Autoreactive T cells have been detected in individuals who do not show overt disease10, suggesting that the onset of autoimmune disease is not determined solely by the presence of autoreactive T cells.
To find an explanation for these discrepancies, we examined RIP-GP mice, in which LCMV-GP is expressed as a transgene under the control of the rat insulin promoter (RIP)11,12,13,14. These mice have been previously used to show that diabetes can be induced by autoreactive cytotoxic T cells (CTL) that are primed by LCMV infection11 or by injection of peptide-pulsed mature dendritic cells (DCs)13. Disease could not be induced by infection with LCMV-GP-recombinant vaccinia virus (VV-G2), despite the presence of activated autoreactive T cells14. These different outcomes may have resulted from variation in the magnitude of the T-cell response induced. Alternatively, LCMV, or mature DCs, may provide nonspecific inflammatory signals in addition to activating GP-specific T-cell responses, which could influence the efficiency of CD8+ T cell–mediated killing12, for instance by increasing expression of MHC I on the target organ14 or by enhancing migration of CD8+ T cells. Indeed, virus-induced production of chemokines is known to be important for T-cell migration into peripheral tissues and efficient antigen clearance15, and chemokine production in pancreatic islets has been shown to have a role in T-cell infiltration into this organ16. Toll-like receptor (TLR) signaling following recognition of distinct pathogen patterns can also influence the production of proinflammatory cytokines and chemokines by DCs and macrophages17,18,19,20,21. Eleven different TLRs have been identified so far22, and these recognize molecular patterns such as RNA (receptors TLR3, TLR7 and TLR8)23,24, DNA (TLR9)25 or lipopolysaccharide (TLR4)26. The pattern of cytokine production following TLR ligation depends on the type of TLR, which results in a specifically optimized pathogen-host balance and may occasionally result in autoimmune diseases27.
To analyze the role of such nonspecific immune stimuli in autoimmunity, we analyzed the induction of diabetes in RIP-GP mice following immunization with LCMV or with the immunodominant LCMV-GP–derived MHC class I peptide gp33 with or without additional TLR ligation.
Activated autoreactive T cells without disease
Mice expressing LCMV-GP as a transgene under the control of RIP were infected with 200 plaque-forming units (p.f.u.) of LCMV-WE (intravenous). Consistent with previous observations11,14, infection resulted in expansion of LCMV-GP33–41-specific CD8+ T cells identified by tetramer binding (tet-gp33+ cells, Fig. 1a), and all infected mice developed hyperglycemia by day 8–10 (Fig. 1a,e). Infection with the vaccinia virus expressing the LCMV-GP recombinant VV-G2 led to reduced expansion of tet-gp33+ cells (Fig. 1b), and no diabetes occurred (Fig. 1b,e). To directly investigate the role of CTL frequency in the induction of autoimmune disease, we immunized RIP-GP mice with the LCMV-GP-derived peptide gp33 together with CpG (ODN-1826) to deliver optimal costimulation. This immunization induced massive expansion of gp33-specific CD8+ T cells such that the frequency of these cells reached 40% of total CD8+ T cells (Fig. 1c). Nevertheless, all mice showed normal blood glucose concentrations, indicating the absence of disease (Fig. 1c,e). To further increase autoreactive T-cell frequencies, we adoptively transferred gp33-specific TCR-transgenic splenocytes (318 CD8+ T cells) into RIP-GP mice and immunized recipient mice with gp33 peptide alone. This protocol resulted in an expansion of the gp33-specific T cells up to a frequency of 80% of total CD8+ T cells (corresponding to 30% of total lymphocytes) even in the absence of costimulatory CpG (Fig. 1d). Still, only 20% of these mice developed hyperglycemia (Fig. 1d,e).
We reasoned that the absence of diabetes in RIP-GP mice showing high frequencies of gp33-specific CD8+ T cells could be explained by inadequate CTL effector function following peptide priming. We therefore examined the functional status of the gp33-specific 318 CD8+ T cells following peptide immunization. Analysis of surface marker expression showed a CD44high CD62Llow phenotype suggestive of activation (Fig. 2a). These cells also expressed high levels of the markers Ly6c+g and IL-2 receptor β-chain (CD122, Fig. 2a), generally expressed by activated T cells. Peptide-stimulated TCR transgenic CD8+ T cells produced interferon-γ (IFN-γ) following in vitro restimulation (Fig. 2b) and lysed peptide-pulsed target cells in ex vivo primary and secondary 51Cr release cytotoxicity assays (Fig. 2c). Furthermore, peptide-immunized mice were able to eliminate gp33-labeled target splenocytes within 4 h in vivo, suggesting full lytic function in vivo of the gp33-specific 318 CTLs present in these mice (Fig. 2d).
Mice immunized with gp33 plus CpG (ODN-1826) without previous transfer of transgenic 318 CD8+ T cells mounted a similar functional CTL response as shown by the ability of these mice to clear gp33-pulsed splenocytes in vivo within 4 h and an LCMV infection within 4 d (Fig. 2e,f).
Pancreatic T-cell infiltrations without disease
We next examined whether the functional beta islet cell–specific CTLs present in gp33 immunized mice did not induce diabetes because of the lack of migration into the pancreas. To address this issue, we crossed the RIP-GP mice with mice expressing the chemokine CXCL13/BLC under control of the rat insulin promoter (RIP-BLC), which have been previously described to display lymphoid infiltrations within the endocrine pancreas28. Mice doubly transgenic for RIP-GP and RIP-BLC were transfused with transgenic gp33-specific splenocytes (318), and recipient mice were immunized with gp33. This resulted in a rapid expansion of gp33-specific CD8+ T cells, such that their frequency approached 50% of total lymphocytes in blood (Fig. 3a). Infiltration of these peptide-activated CTLs into the pancreatic islets was shown by immunohistochemical analysis of the Vα2 TCR expressed by 318 CD8+ T cells (Fig. 3c), yet none of the mice developed diabetes (Fig. 3a). This indicated that the presence of functional CTLs within the pancreas was not sufficient to induce diabetes. In contrast, infection of these mice with LCMV resulted in hyperglycemia (Fig. 3b), suggesting that some factor (other than CTL activation and migration into the pancreas) associated with viral infection had mediated induction of disease.
MHC class I upregulation by LCMV versus pepitide
We reasoned that the ability of LCMV infection to induce diabetes could be explained by virus-induced cytokine production rendering pancreatic beta islet cells susceptible to CTL-mediated destruction. Four days after LCMV infection, we detected strongly elevated serum IFN-α (Fig. 4a), a cytokine known to induce the upregulation of MHC class I29. Indeed, immunohistochemical analysis of beta islet cells infected with LCMV showed enhanced expression of MHC class I in LCMV-infected mice (Fig. 4b), consistent with previous reports12,14. In contrast, immunization with gp33 enhanced neither systemic IFN-α production nor MHC class I expression in pancreatic beta islet cells (Fig. 4a,b). Rag1−/− mice (lacking T and B cells) showed a smaller enhancement of MHC I expression after LCMV infection when compared with naive and LCMV-infected C57BL/6 mice (Fig. 4c). Additional functional depletion of the interferon (IFN) system in mice lacking type I (Ifnar−/−), type II (Ifngr−/−) or both (Ifnagr−/−Rag1−/−) IFN receptors abrogated MHC class I expression after LCMV infection. Notably, virus replication within beta islet cells was detected only in mice lacking IFN type I receptors (Fig. 4c).
TLRs regulate MHC class I expression on beta cells
IFN-α is produced after ligation of TLRs with virus-related products (e.g., RNA) specifically engaging TLR3 and TLR7 (refs. 24,30,31). We therefore analyzed the involvement of TLR stimulation in the production of systemic IFN-α and upregulation of MHC class I on beta islet cells. Administration of the virus-related double-stranded RNA repeat poly(I:C)23 to naive C57BL/6 mice resulted in substantial production of IFN-α and upregulation of MHC class I expression on beta islet cells (Fig. 5a,c and Supplementary Fig. 1 online). This effect could be mimicked by comparable amounts of recombinant IFN-α (Fig. 5b). As observed for LCMV infection, poly(I:C)-induced upregulation of MHC I expression on beta islet cells required functional IFN type I receptor signaling, as shown using Ifnar−/− mice (Fig. 5d). Poly(I:C)-triggered MHC I upregulation was mediated by TLR3 stimulation, being absent in Tlr3−/− mice (Fig. 5d). Treatment of C57BL/6 mice with the low molecular weight immune modifier R-848 (a ligand for TLR7)32 similarly enhanced serum IFN-α production and pancreatic MHC class I expression (Fig. 5a,c and Supplementary Fig. 1 online). We found that IFN-α was essential for R-848–mediated upregulation of MHC class I expression, as this response was blocked by a monoclonal antibody specific for IFN-α (RMMA-1, Fig. 5e). In addition, mice deficient in type I IFN receptor (Ifnar−/−) were not able to upregulate MHC I following R-848 treatment (Fig. 5e). Upregulation of pancreatic MHC class I expression following R-848 treatment was mediated by specific TLR signaling as shown in Tlr7−/− mice (Fig. 5e). R-848 induced upregulation of MHC class I expression required the presence of the common TLR adaptor protein MyD88 expressed on bone marrow–derived cells (Fig. 5e). This strongly suggested that bone marrow derived cells are responsible for the observed MHC I upregulation, whereas pancreatic islet cells themselves do not respond to R-848. Triggering of TLR4 by lipopolysaccharide, or TLR9 by CpG (ODN-1826) did not result in upregulated MHC class I expression on beta islet cells (Fig. 5c and Supplementary Fig. 1 online), consistent with the inability of the employed dose of CpG to enhance systemic IFN-α levels (Fig. 5a)33. Thus, in our experiments, the quantities of IFN-α required for pancreatic islet cell MHC class I upregulation could only be provided through LCMV infection or by stimulation of TLR-3 and TLR-7.
To determine the source of IFN-α following poly(I:C) treatment, we analyzed sequential sections of spleens taken from mice that had received poly(I:C) 3 h earlier. We detected the majority of IFN-α in cell clusters in the marginal zone (Supplementary Fig. 2 online), consistent with earlier reports20,34.
TLRS convert autoreactivity into overt disease
We showed that LCMV infection leads to the upregulation of MHC class I on beta islet cells and that this effect could be mimicked by injection of recombinant IFN-α, the TLR3 ligand poly(I:C), or the TLR7 ligand R-848. This prompted us to investigate whether autoimmune diabetes could be elicited by administration of recombinant IFN-α or by ligation of TLR3 or TLR7 in RIP-GP mice that were preimmunized with gp33 in the absence of virus infection. Indeed, administration of IFN-α, poly(I:C) or R-848 to these peptide-immunized mice resulted in a rapid and sometimes transient development of hyperglycemia (Fig. 5f). The ability of the TLR ligands to induce disease thus correlated strictly with their capacity to induce systemic IFN-α and to upregulate MHC class I on pancreatic islet cells (Fig. 5a–c and Supplementary Fig. 1 online). In contrast to naive mice, gp33-immunized mice did not become diabetic after LCMV infection (Fig. 5g). This was possibly due to rapid viral clearance by primed CD8+ T cells and thus reduced IFN-α production in immunized mice (Fig. 5g). To further strengthen our hypothesis that TLR signals are required in addition to autoreactive T cells to induce overt autoimmune disease, we investigated RIP-GP mice deficient in the common TLR adaptor molecule MyD88. Bone marrow chimeras were generated by injection of Myd88−/− bone marrow into lethally irradiated RIP-GP or C57BL/6 mice. After LCMV immunization, C57BL/6 bone marrow chimeras (Myd88−/− bone marrow into C57BL/6) had reduced pancreatic MHC I expression on day 8 after infection (Fig. 6a), and no autoimmune disease was observed in RIP-GP chimeras (Myd88−/− bone marrow into RIP-GP; Fig. 6b). It is noteworthy that in these mice, autoreactive CD8+ T cells showed a delayed expansion after LCMV infection (Fig. 6c), indicating a role for TLR signals in both innate and adaptive immune responses. But tet-gp33+ CD8+ T cells that were present were functional, as assessed by intracellular IFN-γ staining and FACS analysis (Fig. 6e) as well as in a direct ex vivo cytotoxicity assay (Fig. 6d). Chimeras lacking MyD88 (Myd88−/− bone marrow into C57BL/6) were able to initially suppress virus replication in a similar manner that observed in control chimeras (C57BL/6-negative bone marrow into C57BL/6).
IFN type I is essential for virus-induced diabetes
To directly examine the requirement for type I IFNs in mediating MHC class I upregulation on pancreatic islet cells and the subsequent induction of autoimmune disease, we crossed RIP-GP mice to Ifnar−/− mice and then infected these mice with LCMV. IFN type I receptor–competent RIP-GP mice developed diabetes with normal kinetics, whereas type I IFN receptor–deficient littermates remained healthy (Fig. 6g). Consistent with published evidence35, we observed normal CD8+ T-cell priming in the Ifnar−/− mice, as assessed in a direct ex vivo cytotoxicity assay (Fig. 6h). MHC class I expression within pancreatic islet cells was reduced in mice lacking type I IFN signals (Fig. 6f).
We have shown that the presence of activated and functional gp33-specific CD8+ T cells was not sufficient for induction of autoimmune disease in mice expressing LCMV-GP under control of the rat insulin promoter. It has previously been shown that autoreactive CD8+ T cells without the chemokine CXCR3 cannot induce diabetes because of inefficient homing16. In our experiments using double transgenic RIP-GP × RIP-BLC mice, we found that GP-specific CTLs can be located within pancreatic endocrine tissue yet still not cause disease as long as the beta islet cells express only low levels of MHC I. Thus, although an initial clonal burst of autoreactive CD8+ T cells and their recruitment into the autoantigen-presenting peripheral organ are prerequisites for disease induction, other factors exert additional control over the onset of autoimmune disease.
In our experiments, disease onset and progression was further determined by the 'inflammatory status' of the target organ, as indicated by MHC class I expression by beta islet cells. This upregulation of MHC class I expression on beta islet cells was dependent on functional IFN type I receptors and seemed to be critically regulated by production of systemic IFN-α. It is likely that activation of IFN type I receptors by IFN-β can also upregulate MHC I expression. IFN-β is also produced during LCMV infection36, and it is possible that it contributes to diabetes in the LCMV system. This would be consistent with the finding that neutralizing IFN-α antibody treatment substantially delayed the onset of disease but did not prevent diabetes completely (K.L. and M.R., unpublished data), whereas a blockade of IFN type I receptor completely prevented diabetes.
Notably, cells within the pancreatic islets were more susceptible to upregulation of MHC class I expression than exocrine pancreatic tissue, consistent with previous observations12,14. We are currently evaluating whether this mechanism holds true for other endocrine cells; it may partially explain why autoimmune diseases often target endocrine organs. There has been no previous characterization of the inflammatory status of beta islet cells during health or disease; however, treating hepatitis C virus–infected human individuals with IFN-α occasionally led to diabetes if beta cell–specific antibodies were present in the serum37. Mechanisms underlying this antibody-mediated destruction of beta islets remain to be established.
Poly(I:C) was able to prevent diabetes in NOD (non-obese diabetic) mice38, but it was shown in diabetes-prone bio-breeding (DP-BB) rats that poly(I:C) can accelerate39,40 or prevent diabetes, depending on the dosage used41. These contradictory observations may be partially explained by differences in doses and different kinetics of poly(I:C). Chronic application of TLR ligands is known to tolerize subsequent TLR triggering42,43. In addition, different doses of TLR ligands might influence the activation status of responding or anergized autoreactive CD8+ T cells differently.
Our manuscript shows how innate immune activation influences the adaptive immune response. Blocking TLR signaling (e.g., Myd88−/− mice) not only abrogates innate cytokine secretion44 but also delays the priming of specific T cells. Specific T cells may themselves augment 'inflammatory' signals by production of cytokines (e.g., IFN-γ), which in our model may also contribute to upregulation of pancreatic MHC class I expression at late time points. In addition to TLR signaling, MyD88 is involved in signaling through the IL-1 and IL-18 receptors45,46. Notably, IFN responses induced by virus infection are enhanced in the presence of IL-18 (ref. 47). We therefore cannot exclude a role for these cytokines in the delayed diabetes induction found in our Myd88−/− experiments.
In conclusion, our experiments using the LCMV RIP-GP model have shown that highly activated CD8+ T cells can 'peacefully coexist' with pancreatic beta islet cells expressing the relevant autoantigen. Engagement of TLRs was required to break this coexistence and converted potential autoreactivity into overt autoimmune disease. This may indicate an important role for systemic virus infections in the disease-triggering phase of autoimmune diabetes in humans. In particular, a role for TLR signaling is consistent with the observation that overt diabetes often coincides with viral infections in human patients5. Alternatively, local inflammation within the pancreas may cause upregulation of MHC class I and promote autoimmunity. Taken together, our results suggest that pathogenesis of clinically relevant autoimmune diseases can be viewed as a two step process, requiring both the generation of autoreactive T cells and TLR-mediated inflammation that renders the target organ susceptible to immune attack.
Mice and viruses.
LCMV strain WE was originally obtained from F. Lehmann-Grube (Heinrich Pette Institute) and was propagated in L929 cells. Virus titers were measured using a focus-forming assay as described48. Mice were infected with 200 p.f.u. LCMV-WE or with 2 × 106 p.f.u. recombinant vaccinia virus expressing the LCMV-GP (VV-G2). Mice transgenic for a T-cell receptor recognizing LCMV-GP33–41 (LCMV-gp33/H-2Db-specific TCR, 318)13, RIP-GP mice11, RIP-GP × RIP-BLC mice28, Rag1−/− mice (Jackson Laboratory), Myd88−/− mice32, Tlr7−/− mice32 (N4, backcrossed on C57BL/6) and DTR mice (for depletion of dendritic cells, DTR Jackson Laboratory) were maintained on the C57BL/6 genetic background, whereas Ifnagr−/− mice35 were of the 129Sv genetic background. Tlr3−/− mice49 mice were on a BALB/c background. Ifnar−/− and Ifnar+/− mice crossed on RIP-GP mice were of mixed background (129Sv × C57BL/6). For generation of bone marrow chimeras, recipient mice were irradiated with 950 rad on day −1. On day 0, 107 bone marrow cells were transferred and mice were used for experiments 7 weeks later. All mice were purchased from the Institute for Labortierkunde and housed under specific pathogen-free conditions. RIP-GP × RIP-BLC (and the corresponding RIP-GP mice) were used at an age of more than 10 weeks (to ensure the development of lymphoid structures within the pancreas of RIP-GP × RIP-BLC mice). Animal experiments including infection with recombinant viruses were carried out with authorization of the Veterinäramt of the Kanton Zurich and in accordance with the Swiss law for animal protection.
Immunization with peptides or Toll-like-receptor ligands, and adoptive transfer of CD8+ T cells.
gp33 (KAVYNFATM) was synthesized by Neosystems. Mice were immunized on day 0, 2 and 5 with 1 mg gp33 and 50 μg CpG (ODN1826, Coley Pharmacia) and then boosted with peptide alone on day 9. For priming of LCMV-gp33/H-2Db-specific TCR-transgenic CD8+ T cells, 107 splenocytes were adoptively transferred in RIP-GP mice on day −1, and mice were immunized with 1 mg gp33 on day 0, day 4 and day 9. As the precursor frequency exceeded 1% of total CD8+ T cells, no further costimulation with CpG was required for priming. To analyze the influence of TLRs, we treated mice with 200 μg poly(I:C) (Sigma-Aldrich), a ligand for TLR3; 20 μg R-848 (InvivoGen), a ligand for TLR7; 50 μg CpG (ODN 1826), a ligand for TLR9; or 50 μg lipopolysaccharide (Sigma-Aldrich) a ligand for TLR4. Recombinant mouse IFN-α was obtained from Hycult biotechnology (104 units/mouse). For in vivo depletion of IFN-α, a rat monoclonal antibody was used (RMMA-1, Biomedical Laboratories, PBL).
FACS analysis was performed as described previously50. Briefly, splenocytes or peripheral blood lymphocytes were stained using phycoerythrin-labeled gp33 MHC class I tetramer (gp33/H-2Db) for 15 min at 37 °C, followed by staining with antibody specific for CD8-APC (BD PharMingen) for 30 min at 4 °C. For determination of their activation status, lymphocytes were stained with antibodies specific for CD62L, CD44, CD122 and Ly6c+g (BD PharMingen) for 30 min at 4 °C.
51Cr release assays were performed as previously described.50 EL4 target cells were loaded with 51Cr and pulsed with or without gp33. Splenocytes of immunized mice were incubated directly ex vivo (Primary) or after restimulation with gp33 for 5 d (Secondary) with the target cells. Supernatant was assessed after 8 h. We performed assays for in vivo cytotoxicity using C57BL/6 splenocytes incubated for 1 h with or without gp33 and labeled for 10 min with 5 μg/ml (peptide-labeled splenocytes) or 0.5μg/ml (peptide nonlabeled splenocytes) carboxyfluorescein diacetate succinimidyl ester (CFSE, Molecular Probes), respectively. We injected 107 cells of each fraction intravenously. The number of CFSE-positive cells remaining in the spleen after 4 h was determined by FACS analysis.
Blood glucose concentrations.
Blood glucose concentrations were analyzed from a drop of blood using a Glucometer Elite (Bayer).
We performed IFN-α ELISA according to the manufacturers' specifications (Research Diagnostics RDI).
Histological analyses were performed on snap-frozen tissue. We stained sections with monoclonal antibodies against murine MHC I (M1/42)14, Vα2 (Pharmingen), CD11b (BMA Biomedicals AG), CD11c (KB90, M0732; DAKO), B220 (RA3-6B2, 01121D; Pharmingen), or with VL4, a monoclonal antibody against the LCMV nucleoprotein48. Marginal zone was stained with MOMA-1 (BMA Biomedicals). For staining of IFN-α, a polyclonal sheep antibody specific for mouse IFN-α antiserum was used (Biomedical Laboratories). Staining was developed using a goat anti-rat antibody (Caltag Laboratories) and an alkaline phosphatase-coupled donkey anti-goat antibody (Jackson ImmunoResearch) with naphthol AS-BI (6-bromo-2-hydroxy-3-naphtholic acid 2-methoxy anilide) phosphate and new fuchsin as a substrate. The presence of alkaline phosphatase activity yielded a red reaction product. The sections were counterstained with hemalum.
Note: Supplementary Information is available on the Nature Medicine website.
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We thank A. Aguzzi, M. Heikenwälder and C. Sigurdson for ideas, discussions and critical comments. Additionally, we thank K. Tschannen for technical support and A. Nowotny and S. Behnke for histological analysis. This study was supported by the Swiss National Science Foundation and Deutsche Forschungsgemeinschaft (DFG) LA1419/1-1.
The authors declare no competing financial interests.
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Lang, K., Recher, M., Junt, T. et al. Toll-like receptor engagement converts T-cell autoreactivity into overt autoimmune disease. Nat Med 11, 138–145 (2005). https://doi.org/10.1038/nm1176
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