http://immunol.nature.com • april 2002 • volume 3 no 4 • nature immunology

Malin Flodström1,Amy Maday1, Deepika Balakrishna1, Mary Malo Cleary1, Akihiko Yoshimura2

and Nora Sarvetnick1

Published online: 4 March 2002, DOI: 10.1038/ni771

The mechanisms that regulate susceptibility to virus-induced autoimmunity remain undefined. Weestablish here a fundamental link between the responsiveness of target pancreatic β cells to inter-ferons (IFNs) and prevention of coxsackievirus B4 (CVB4)-induced diabetes.We found that an intactβ cell response to IFNs was critical in preventing disease in infected hosts. The antiviral defense,raised by β cells in response to IFNs, resulted in a reduced permissiveness to infection and subse-quent natural killer (NK) cell–dependent death.These results show that β cell defenses are critical forβ cell survival during CVB4 infection and suggest an important role for IFNs in preserving NK celltolerance to β cells during viral infection. Thus, alterations in target cell defenses can criticallyinfluence susceptibility to disease.

1Department of Immunology, IMM-23,The Scripps Research Institute, 10550 N.Torrey Pines Road, La Jolla, CA 92037, USA. 2Molecular and Cellular Immunology, MedicalInstitute of Bioregulation, Kyushu University 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan. Correspondence should be addressed to N. S. (noras@scripps.edu).

Target cell defense prevents the development of diabetes

after viral infection

Both genetic and environmental factors are involved in the etiologyof autoimmune disease and viral infections have been implicated asnongenetic triggers of autoimmune reactions to self. Type 1 diabetesis an immune-mediated disease that results from selective loss of theinsulin-producing pancreatic β cell. Numerous epidemiological andclinical studies have linked enteroviral infections, particularly infec-tions with coxsackievirus B4 (CVB4), with a progression to type 1diabetes1–3. Based on animal studies, different models for virus-induced reactions to self, including CVB4-induced diabetes, havebeen proposed. They include molecular mimicry, bystander activa-tion of self-reactive T cells and a direct viral cytolysis of infected tar-get cells4–6. Although coxsackie viral antigens have been found in thepancreatic β cells of newly affected type 1 diabetic patients7–9, littleis known about the antiviral defenses generated by target β cells orhow these defenses can regulate susceptibility to diabetes induced byviral infection.

In vitro, CVB4 and other members of the CVB family infect humanand rodent β cells and many such infections result in widespread β celldeath10–15. In contrast, studies with mice have revealed that althoughsystemic CVB4 infection can cause almost complete destruction of theexocrine pancreas, the pancreatic islet cells, including β cells, areselectively spared from CVB4-induced pathology16–19. These observa-tions show that although there is strong viral tropism for the exocrinepancreas during systemic infection, the net infectivity of β cellsappears to be very low. Accordingly, the majority of systemic infec-tions with CVBs are cleared without β cell destruction and develop-ment of diabetes17,19–21. Nonetheless, several reports of diabetes thatoccurred in close association with a CVB infection suggest that infec-tion in susceptible individuals may still lead to β cell destruction3,6. In

addition, CVB antigens have been found in residual β cells fromhumans who succumbed to a lethal virus infection7,8,10, andenteroviruses, including CVBs such as CVB4, have been isolated fromnewly diagnosed type 1 diabetes patients10,15,22,23. These reports togeth-er with the in vitro findings described above, raise the possibility thatβ cell permissiveness to CVB4 infection may, in part, regulate suscep-tibility to CVB4-induced diabetes. Until now, the host factors regulat-ing the permissiveness of pancreatic β cells to CVB4 infection havenot been fully explored.

Interferon-α (IFN-α), IFN-β and IFN-γ are produced early duringviral infections, including infection with picornaviruses (for example,encephalomyocarditis virus24 and CVB4, M. Flodström and N.Sarvetnick, unpublished data). By inducing an antiviral state in IFN-responsive target cells, IFNs minimize the permissiveness of targetcells to viral infection and/or replication25–27. We sought to understandthe role played by IFNs in regulating the cellular permissiveness ofpancreatic β cells to CVB4 infection. We assessed the relevance ofIFN-induced β cell–specific antiviral defenses in regulating CVB4-induced diabetes. We show that the β cells critically depended on IFNsto lower their permissiveness to CVB4 infection. We also show that inmice with pancreatic β cells that had defective IFN responses, CVB4caused an acute form of diabetes, which resembled the type 1 diabetesthat develops in humans after severe enteroviral infection. Finally, weshow that permissiveness to CVB4 infection resulted in significant β cell damage and an increased susceptibility to natural killer (NK)cell–dependent death in vivo. Thus, our findings show that the β cellitself contains critical circuits that control its survival, which suggeststhat target cell defenses can regulate autoimmune reactions triggered byviral infections.

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ResultsActivation of the Jak-STAT signaling pathwayUpon binding to their respective receptors, type I (IFN-α and IFN-β)and type II (IFN-γ) IFNs transmit their intracellular signals through thephosphorylation of Janus kinases (Jaks) and the signal transducers andactivators of transcription (STAT) family of transcription factors28.Because IFN-γ activates the Jak-STAT pathway in insulin-producingcells29, we tested whether type I IFNs could also initiate a response in β cells by measuring the phosphorylation of STAT1 in NIT-1 cells, apancreatic β cell line established from nonobese diabetic (NOD)mice30. We found that STAT1 phosphorylation increased in cellsexposed to IFN-α or IFN-γ compared to control cells, which were notexposed to IFNs (Fig. 1). Therefore, insulin-producing β cells respondto both type I and type II IFNs.

Mice with IFN-resistant β cellsTo determine whether IFN responsiveness is critical for pancreatic β cell survival during a systemic infection with CVB4, we generatedtransgenic (Tg) NOD mice that expressed the suppressor of cytokinesignaling 1 (SOCS-1)31,32 under the control of the human insulin pro-moter33. SOCS-1 is a negative regulator of IFN signaling that acts byinhibiting Jak1 and Jak231,32; expression of SOCS-1 blocks IFN-γ–induced STAT1 activation in an insulin-secreting cell line34.Transgene expression in both the two SOCS-1–Tg NOD lines (A andB) that we had established was confirmed by immunohistochemistry(Fig. 2 and data not shown). Although SOCS-1 was absent from isletcells and exocrine tissue from non-Tg NOD mice (Fig. 2a), as well asfrom the exocrine tissue of their SOCS-1–Tg littermates (Fig. 2d), weobserved SOCS-1 expression in the islets of Tg mice (Fig. 2d). Thestructure of the pancreatic islets in SOCS-1–Tg NOD mice was nor-mal and insulin+, glucagon+ (Fig. 2e,f) and somatostatin+ cell distribu-tion (data not shown) was similar to that in non-Tg mice (Fig. 2b,c).

Similar to their non-Tg littermates, some of the islets in SOCS-1–TgNOD mice showed a mild peri-insulitis or insulitis at 8 weeks of age(Table 1). In addition, the first incidence of spontaneous diabetes inSOCS-1–Tg NOD mice paralleled that in non-Tg littermates at the ageof 15 weeks (data not shown).

CVB4 replicates in SOCS-1–expressing islet cellsCVBs can infect and replicate in isolated human and rodent β cells10,11,13–15,35. We tested the ability of IFNs to inhibit CVB4 replica-tion in primary islet cells (Fig. 3). In initial experiments, pancreaticislets were isolated from the two murine strains C57BL/6 and NOD(Fig. 3a,b). The islets were treated with IFN-α (1000 U/ml) or PBS for24 h and then infected with CVB4. When no IFN-α was added, theislets from both strains continuously produced infectious CVB4 (Fig.3a,b). On day 6 after infection, light microscopy showed that most ofthe islets had lost their integrity (data not shown). In contrast, viraltiters from IFN-α–treated islets remained two to three logs lower dur-ing the 6-day study period (Fig. 3a,b) and the islets retained theirround dense structure (data not shown). IFN-γ (1000 U/ml) also inhib-ited the generation of infectious CVB4 in pancreatic islets (data notshown). To verify the biological relevance of the IFN action on isletcells, islets from mice that lacked intact IFN receptors (IFN-αβγR–/–

mice)36 and their wild-type counterparts were treated with IFN-α orPBS for 24 h, then infected with CVB4. In wild-type islets, IFN-αtreatment significantly reduced the production of infectious CVB4(Fig. 3c, P<0.05) and loss of integrity (data not shown). IFN-α treat-ment did not inhibit CVB4 replication and islet degeneration in IFN-αβγR–/– islets (Fig. 3d and data not shown). Taken together, these datashow that both type I and II IFNs induced the rapid transition of pan-creatic islet cells to an antiviral state, which was critical for a block inCVB4 replication in vitro.

To determine whether IFNs have a biological effect on the SOCS-1–expressing cells, pancreatic islets were isolated from SOCS-1–TgNOD mice and their non-Tg littermates. In culture media frominfected non-Tg islet cells, IFN-α and IFN-γ inhibited, in a dose-dependent manner, the generation of infectious CVB4 (Fig. 3e anddata not shown). In contrast, CVB4 replication proceeded with littleor no inhibition in IFN-α–treated (Fig. 3f) or IFN-γ–treated (data notshown) SOCS-1–Tg islet cells. In addition, the infection-inducedloss of islet integrity in SOCS-1–Tg islets treated with IFN-α or

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Figure 1. STAT1 is phosphorylated ininsulin-producing cells after stimula-tion with IFNs. NIT-1 cells were exposedto IFN-α (α) or IFN-γ (γ). STAT1 phospho-rylation (PY-STAT) 10 or 20 min after expo-sure was assessed by immunoblotting. Dataare representative of two independentexperiments.

Figure 2. SOCS-1 is expressed in pancreatic islets fromSOCS-1–Tg NOD mice, but is absent in islets from non-Tg littermates. Paraffin sections of formalin-fixed pancreatafrom 8-week-old (a–c) non-Tg and (d–f) Tg mice were stainedwith antibodies to (a,d) SOCS-1 (b,e) insulin or (c,f) glucagon.Original magnifications were (a,d) ×20 and (b,c,e,f) ×40.

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IFN-γ was as severe as the degeneration in islets infected with CVB4alone. Indeed, replication (Fig. 3f) and islet degeneration (data notshown) resembled that in IFN-α–treated islet cells from IFN-αβγR–deficient mice (Fig. 3d and data not shown). Uninfected isletcells treated with IFN-α or IFN-γ remained intact for the 6-day studyperiod, as determined by light microscopy and/or electron micro-scopy (data not shown).

Because the light microscopy analysis showed degenerative changesin the infected islets, an ultrastructural analysis was done. This analy-sis revealed a considerable number of dead cells in CVB4-infectedislets from both SOCS-1–Tg NOD mice and their non-Tg littermateson day 4 post-infection (p.i.) (Fig. 3i,j). Some cells showed the featuresof virus-mediated cytopathic effects, such as cytoplasmic degeneration

and ruptured plasma membranes (Fig. 3i,j,l). Others showed the hall-marks of apoptosis37, including deformed nucleoli, partial chromatincondensation and enlarged peri-nuclear spaces (Fig. 2j,l). In addition,many β cells had undergone some degree of degranulation. In contrast,no such cellular destruction affected non-Tg islets subjected to IFN-αtreatment and CVB4 infection (Fig. 3k). In fact, the ultrastructure ofthese islets was nearly indistinguishable from that of uninfected cellstreated with IFN-α alone (Fig. 3g) or PBS (data not shown). IFN-α didnot rescue SOCS-1–Tg islet cells from severe degenerative changes(Fig. 3l). Indeed, at day 4 p.i. the injury to those islets resembled thatof Tg islets infected with CVB4 alone (Fig. 3j,l). This islet cell destruc-tion appeared to correlate with the replication of CVB4 (Fig. 3f). Theseexperiments showed that ectopic expression of SOCS-1 rendered β cells insensitive to IFN stimulation.

CVB4 induces diabetes in SOCS-1–Tg NOD miceTo determine the consequences of defective β cell responses to IFNsduring systemic CVB4 infection, SOCS-1–Tg NOD mice and theirnon-Tg littermates were infected with CVB4 at 8–9 weeks of age (atleast 6 weeks before the spontaneous onset of diabetes in both groups)and were examined. At this age, the nonfasting blood glucose concen-trations in uninfected SOCS-1–Tg NOD mice were 127±4 mg/dl(n=20) and 114±6 mg/dl (n=20) for lines A and B, respectively, where-as in non-Tg littermates it was 117±4 mg/dl (n=20). In agreement withpublished data17, non-Tg mice developed severe hypoglycemia 3 or 4days after virus inoculation and 4–6 days p.i. 7/40 (18%) mice died.By 12 days p.i., only 1/33 (3%) surviving mice became hyperglycemic(Fig. 4a and data not shown). The rest of the mice remained normo- orhypoglycemic (data not shown). In contrast, after infection withCVB4, most of the SOCS-1–Tg NOD mice developed early and severehyperglycemia; after initial hypoglycemia during the first 3–4 days

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Figure 3.An intact β cell response to IFNsis critical for the inhibition of CVB4 repli-cation in vitro. Pancreatic islets were isolatedfrom (a) C57BL/6 (b) NOD (c) 129S6/SvEv(IFN-αβγR+/+) (d) IFN-αβγR–/– (e) non-Tg NODand (f) SOCS-1–Tg NOD mice that were infect-ed with CVB4 in vitro. Replication of CVB4 wasimpaired after treatment with IFN-α. (d) Thiseffect was dependent on intact IFN receptorsand (f) was prevented by SOCS-1 expression.Data are mean±s.e.m. from two to eight inde-pendent experiments. *P<0.05 and **P<0.01 ver-sus PBS-treated islets from the same mousestrain. (g–l) CVB4 infection caused ultrastruc-tural changes in pancreatic islet cells. Pancreaticislet cells were taken from (g,i,k) non-Tg or(h,j,l) SOCS-1–Tg NOD mice after 5 days ofexposure (g–h) to IFN-α alone (i–j) CVB4infection for 4 days or (h–l) exposure to IFN-αfor 24 h followed by infection with CVB4 for 4 days. (k) Islet cells from non-Tg NOD micewere protected from CVB4-induced damagewhen treated with IFN-α. (l) In contrast, IFN-αtreatment did not prevent islet cell destructionin SOCS-1–Tg NOD mice. Images are repre-sentative of two independent experiments.Original magnification: ×5200.

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Figure 4. Acute onset of diabetes in SOCS-1–Tg NOD and NOD-SCIDmice infected with CVB4. (a) Non-Tg (n=5) and (b) SOCS-1–Tg (n=5) NODmice were infected with CVB4, although only SOCS-1–Tg mice developed diabetesafter infection. Data are representative of 40 and 21 CVB4-infected non-Tg and Tgmice, respectively.

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p.i., their blood glucose concentrations increased greatly, usuallyexceeding 500 mg/dl (27.5 mM) by day 6–8 p.i. (Fig. 4b). Only 1/21(5%) Tg mice succumbed to the viral infection before day 6 p.i., butthe accumulated incidence of diabetes among the survivors was 95%(19/20) at 12 days p.i. (P<0.01, non-Tg versus SOCS-1–Tg NOD miceby χ2-test).

CVB4 infection may precipitate autoimmune-mediated destructionof pancreatic β cells in NOD mice38, so we crossed the SOCS-1–TgNOD mouse onto a C57BL/6 background for one generation.(C57BL/6×NOD)F1 mice do not develop diabetes or insulitis39, and wefound that no diabetes occurred in CVB4-infected non-Tg(NOD×C57BL/6)F1 mice after CVB4 infection (n=4). In contrast, theSOCS-1–Tg (NOD×C57BL/6)F1 mice rapidly developed hyper-glycemia, and 4/5 mice were diabetic by day 12 p.i. (data not shown).Hence, the rapid onset of diabetes was not restricted to SOCS-1–Tgmice on a pure NOD background.

Histological evaluation of the pancreata showed that, although theexocrine tissue had undergone massive degeneration in the infectedSOCS-1–Tg NOD mice and their non-Tg littermates, marked differ-ences were apparent. In non-Tg mice, most pancreatic islets wereintact, with a normal distribution of insulin+, glucagon+ (Fig. 5a,b) andsomatostatin+ cells (data not shown). The degree of insulitis and isletdestruction (Table 1) was similar to that in uninfected non-Tg NODmice (Table 1). However, the islets of SOCS-1–Tg NOD mice were

disrupted, and the number of insulin-staining β cells was greatlyreduced (Fig. 5c). These islets showed severe insulitis (Table 1) orwere atrophied, containing mainly glucagon+ (Fig. 5d) and somato-statin+ (data not shown) cells. Hence, the infected mice whose pancre-atic β cells were incapable of responding to IFNs lost their insulin+, butnot glucagon+ and somatostatin+ islet cells after CVB4 infection.Therefore, as a consequence of the β cell loss, the SOCS-1–Tg NODmice developed diabetes.

CVB4 infects SOCS-1–expressing β cells in vivoWe next determined whether β cell destruction in SOCS-1–Tg NODmice correlated with a general increase in CVB4 replication in the hostand/or with a specific increase in the pancreatic islets. Inoculation ofmice with CVB4 typically results in rapid dissemination of the virus tovital organs such as the liver, kidneys, spleen, heart and pancreas19,40.We measured high titers of replicating virus in several organs on day 3p.i., a time when viral titers generally peak in such organs19,40. We foundthat the titers in pancreata, spleens, kidneys and livers, respectively,were similar in both groups; data are mean±s.e.m. log10 plaque-formingunits (PFU) per gram of tissue. SOCS-1–Tg NOD mice (n=3):12.1±0.3, 9.1±0.3, 7.3±0.2 and 7.4±0.3; non-Tg littermates (n=3):11.6±0.3, 8.7±0.3, 7.1±0.4 and 7.7±0.7. Also on day 4 p.i. (when hyper-glycemia was first observed in infected SOCS-1–Tg NOD mice, seeFig. 4b), the viral loads in pancreata from both groups were similar.

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Figure 5. Insulin-staining β cells are lost in SOCS-I–Tg NOD mice infected with CVB4. Representative immunostaining of pancreatic sections from (a,b) non-Tgand (c,d) SOCS-1– Tg NOD mice infected with CVB4 and killed 8–10 days later. Consecutive serial sections were stained with antibodies to (a,c) insulin or (b,d) glucagon.(a,b) The pancreas from a non-Tg mouse had intact islets with both (a) insulin+ and (b) glucagon+ cells. (c,d) The pancreas of a SOCS-1–Tg mouse had a severely distortedislet with (c) only a few insulin+ cells but (d) near to normal numbers of glucagon+ cells. Sections are representative of pancreata from at least ten infected animals. Originalmagnification: ×40.

Table 1. Histological analysis of pancreatic sections from non-Tg and SOCS-1–Tg NOD mice that were untreated or treatedwith anti–AGM-1 and infected with CVB4

Insulitis rankNumber of mice Number of islets CBV4 infection Anti–AGM-1 A+Ba C D

Line ANon-Tg NOD 5 128 – – 77% (99) 19% (24) 4% (5)Non-Tg NOD 3 30 + – 73% (22) 27% (8) 0% (0)SOCS-1–Tg NOD 5 116 – – 81% (94) 11% (13) 8% (9)SOCS-1–Tg NOD 5 90 + – 0% (0) 11% (10) 89% (80)Line BNon-Tg NOD 3 50 + – 90% (45) 8% (4) 2% (1)Non-Tg NOD 2 33 + + 36% (12) 58% (19) 6% (2)SOCS-1–Tg NOD 5 64 + – 0% (0) 3% (2) 97% (62)SOCS-1–Tg NOD 8 98 + + 30% (29) 43% (42) 28% (27)

Pancreatic sections (from two or three levels per organ) were stained with H&E and insulitis was ranked A–D (see Methods). aDue to difficulties in separating peri-insulitisthat normally occurs in pancreata of NOD mice from mononuclear cells that were infiltrating the exocrine area of the pancreata of infected mice, the two first classeswere grouped together.

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SOCS-1–Tg NOD mice: 11.8±0.3 log10 PFU per gram of tissue (n=4);non-Tg mice: 11.5±0.3 log10 PFU per gram of tissue (n=4). Because nogeneralized increase in the replication of CVB4 occurred in infectedSOCS-1–Tg NOD mice, pancreatic viral load apparently did not con-tribute to their marked β cell loss after infection.

The pancreatic islets typically represent only 1–2% of the total pan-creas mass; therefore, the amount of virus in homogenates of thewhole pancreas may not accurately reflect an enhanced amount ofCVB4 replication specific to the islet cells. Consequently, the abilityof SOCS-1 expression to enhance CVB4 replication specifically inislet cells was examined. Because islets are difficult to isolate frominfected mice, immunohistochemistry was chosen for this analysis. Tothis end, SOCS-1–Tg NOD mice and their non-Tg littermates were

infected and killed at day 3 or 4 p.i. and examined (Fig. 6). Pancreatawere removed and CVB4 was detected by immunohistochemistry withan antibody specific for VP-1 (a capsid protein conserved within themembers of the enterovirus family41). Although CVB4 was present inthe exocrine part of the pancreata of non-Tg mice on days 3 and 4 p.i.(Fig. 6b and data not shown), the virus was virtually absent from theirislets (day 3 p.i., n=2, Fig. 6b; day 4 p.i., n=5, data not shown). Viruswas also detected in the exocrine tissue of infected SOCS-1–Tg NODmice; however, in marked contrast to non-Tg littermates, CVB4 wasdetectable in the islets of infected SOCS-1–Tg NOD mice (Fig. 6c,d).Indeed, at 3 days p.i., virus was detected to varying degrees in theislets of all the SOCS-1–Tg NOD mice studied (n=3, Fig. 6c). On day4 p.i., islet structures and/or endocrine cells were visible in only 4/6

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Figure 6.The tropism of CVB4 for islet cells is altered when IFN signaling is perturbed. Pancreatic sections were analyzed with VP-1 (an antibody that detectsa CVB4 coat protein). No VP-1 staining was evident (a) in the pancreas of an uninfected SOCS-1–Tg NOD mouse or in (b) the islets of a CVB4-infected non-Tg NODmouse killed on day 3 p.i. Note the presence of CVB4 in exocrine pancreata from both (b) non-Tg and (c,d) Tg mice killed on day 3 and 4 p.i., respectively; VP-1 stainingappeared exclusively in the islets of Tg mice. Original magnifications were (a,c) ×40 and (b,d) ×20.

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Figure 7. CVB4 and insulin colocalize in the pancreatic islet cells of infected SOCS-1–Tg NOD mice. (a–d) Little if any colocalization of CVB4 and insulinstaining was seen within islet cells from an infected non-Tg NOD mouse killed on day 3 p.i. (e–h) In infected SOCS-1–Tg NOD, however, pancreatic sections showed thelocalization of CVB4 within insulin+ β cells. Note the presence of CVB4 in pancreatic exocrine tissue from both mice. (a,e) The FITC channel (green) shows cells stainedfor CVB4 with an antibody to VP-1. (b,f): Rhodamine staining (red) shows insulin+ cells. (c,g) A composite image of both channels. (d,h) Phase contrast images of the respec-tive tissue sections.

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infected SOCS-1–Tg NOD mice (Fig. 6d and data not shown), andCVB4 was present in the endocrine cells of all four mice with remain-ing islet structures. In the two other pancreata, the islet cell structureswere so distorted that individual endocrine cells could not be dis-cerned, which precluded analysis. Double immunofluorescence analy-sis (Fig. 7) showed that CVNB and insulin colocalized in several cellsfrom the islets of SOCS-1–Tg NOD mice (Fig. 7g, and data notshown). Little, if any, colocalization of CVB4 and insulin was foundin the islets of infected non-Tg mice (Fig. 7c). Taken together, thesefindings showed that the apparent tropism of CVB4 was altered in theTg mice, with virus being present in the pancreatic islets.

In additional studies, IFN-αβγR–/– mice and their wild-type controlswere infected with CVB4. All IFN-αβγR–/– mice (n=11) died within 4 days of infection, whereas most of the receptor-sufficient controlmice (10/12) survived the whole 28-day study period. Immunohisto-chemical analysis of pancreata from infected mice killed at days 2, 3or 5 p.i. did not reveal CVB4-staining cells in the pancreatic islets ofwild-type mice (n=2 mice for each timepoint, data not shown). In IFN-αβγR–/– mice that survived 2 or 3 days p.i., 5–10% of the total numberof endocrine cells within each islet stained positively for CVB4 (n=3or 4 mice per timepoint, data not shown), which showed that islet cellCVB4 tropism was altered in IFN-αβγR–/– mice. Together, theseresults suggested that intact β cell responses to IFNs were critical inpreventing CVB4 from infecting β cells in vivo.

Diabetes evolves without adaptive immunityBecause virus-infected cells are often attacked by activated immunecells (in particular CD8+ T cells42), we next determined whether theunresponsiveness to IFN caused infected SOCS-1–Tg β cells tobecome the targets of immune-mediated destruction during CVB4infection. In addition, the NOD mouse has a pool of autoreactive T lymphocytes that are responsive to pancreatic β cell antigens43.Hence, antigen leakage from CVB4-infected SOCS-1–Tg β cells couldpotentially have activated quiescent autoreactive T cells in SOCS-1–TgNOD mice. To assess the participation of CD8+ T cells, SOCS-1–TgNOD mice and their non-Tg littermates were depleted of CD8+ T cellsbefore infection with CVB4. We found that depletion did not inhibit

diabetes development in SOCS-1–Tg mice; these mice all becameseverely hyperglycemic within 6–7 days p.i. (n=3, data not shown).However, in antibody-treated non-Tg littermates, blood glucose con-centrations remained low (below 125 mg/dl, n=2). Histological analy-sis of pancreata from mice killed on day 8 p.i. showed that in theSOCS-1–Tg NOD mice the pancreatic islets were severely distortedand almost devoid of insulin+ cells (data not shown). These experimentsruled out a prominent role for CD8+ T cells in the destruction of SOCS-1–Tg β cells during systemic CVB4 infection.

To expand our studies on the role of adaptive immune responses indisease development, the SOCS-1–Tg mice were bred with nonobese-diabetic–severe-combined immunodeficient (NOD-SCID) mice, whichlack mature T and B lymphocytes44. SOCS-1–Tg NOD-SCID and theirnon-Tg NOD-SCID littermates both lost a substantial number of pan-creatic acinar cells after infection (data not shown). No diabetes (Fig.8a) or histological signs of β cell loss were observed in CVB4-infect-ed non-Tg littermates (n=5). However, most SOCS-1–Tg NOD-SCIDmice (8/11, 73%) infected with CVB4 underwent rapid and extensiveislet damage and developed severe hyperglycemia (Fig. 8b). Thus, theadaptive immune response played little, if any, role in CVB4-induceddiabetes in SOCS-1–Tg NOD mice.

NK cells contribute to CVB4-induced diabetesWe next assessed the role played by NK cells in causing β celldestruction and diabetes in CVB4-infected SOCS-1–Tg NOD mice.NK cells contribute to the host’s first line of defense against infect-ing viruses by producing cytokines and directly destroying virus-infected cells45,46. Because our previous experiments showed thatSOCS-1 expression rendered β cells permissive to early CVB4infection, we examined NK cells as mediators of β cell destruction.To determine whether uninfected SOCS-1–Tg β cell were destroyednonspecifically by activated NK cells, SOCS-1–Tg NOD mice andtheir non-Tg littermates were exposed to double-stranded RNA inthe form of poly(I)·poly(C)47. Despite mimicking a viral infectionand activating NK cells, SOCS-1–Tg NOD mice (n=4, data notshown) and non-Tg mice (n=3, data not shown) treated withpoly(I)·poly(C) remained normoglycemic for the 28-days study

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Figure 8. Adaptive immunity is not necessary for the swift induction of diabetes in CVB4-infectedSOCS-1–Tg NOD mice, but depletion of NK cells prevents diabetes development. (a) NOD-SCID mice didnot develop diabetes after CVB4 infection (n=4). (b) However, an acute form of diabetes did occur in infected SOCS-1–Tg NOD-SCID mice (n=5). Data are representative of 11 Tg and five non-Tg mice. (c) Diabetes was not apparent innon-Tg NOD mice that were depleted of NK cells using anti–asialo-GM1. (d) NK cell depletion with anti–asialo-GM1prevented diabetes development in CVB4-infected SOCS-1–Tg NOD mice (n=6). Data are representative of three non-Tg and 11 Tg SOCS-1–Tg NOD mice; mice were treated with anti–asialo-GM1 both before and after CVB4 infection.(e) Most of the SOCS-1–Tg (NOD×C57BL6)F1 mice (5/9) developed diabetes after infection with CVB4.Treatment withNK cell–depleting anti-NK1.1 blocked diabetes development.

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period. This suggested that activated NK cells per se had no pro-nounced effect on β cell survival.

Although generalized activation of NK cells did not result in isletcell destruction, the targeting of CVB4-infected β cells by NK cellswas not ruled out. To address this, SOCS-1–Tg NOD mice and theirnon-Tg littermates were treated with anti–asialo-GM1 or PBS beforeand during infection with CVB4. Anti–asialo-GM1 treatment effec-tively depletes NK cells48. Although one non-Tg mouse treated withanti–asialo-GM1 succumbed to the viral infection on day 4 p.i., noneof the surviving mice (n=2) developed diabetes (Fig. 8c) and no dia-betes occurred in non-Tg mice treated with PBS (n=5) (data notshown). Of the antibody-treated SOCS-1–Tg NOD mice, 2/11 diedwithin 5 days of infection, and only one of the surviving mice devel-oped hyperglycemia when examined to 10 days p.i. (Fig. 8d). In addi-tion, 5/5 mock-treated SOCS-1–Tg NOD mice developed a severehyperglycemia within 5–7 days p.i. (data not shown). Histologicalevaluation of pancreata from infected mice showed milder insulitis(Table 1) and less islet destruction in SOCS-1–Tg NOD mice treatedwith anti-asialo-GM1 (Fig. 9a,b) than in mice treated with vehiclealone (Table 1 and data not shown).

Because prototypical NK cell markers, such as asialo-GM1, areexpressed on NK cells as well as on some cells of the T cell lin-eage49–52, it was possible that anti–asialo-GM1 treatment depleted notonly NK cells but also virus-specific T cells52. The cell surface mark-er NK1.1 is not expressed on NK cells or on NK T cells in the NODmouse53, but it is present on these cell types in C57BL/6 mice and in(C57BL/6×DBA/2)F1 mice54. The percentage of NK1.1+ TCRαβ–

splenocytes in non-Tg and SOCS-1–Tg (NOD×C57BL/6)F1 mice aresimilar to those in C57BL/6 mice, and (NOD×C57BL/6)F1 hybrids canbe successfully depleted of NK cells with anti-NK1.1 (M. Flodströmand N. Sarvetnick, unpublished data). We found that although infec-tion with CVB4 resulted in a rapid development of diabetes in themajority of SOCS-1–Tg (NOD×C57BL/6 mice)F1 (5/9), none of theanti-NK1.1–treated SOCS-1–Tg (NOD×C57BL/6)F1 mice (0/6) devel-oped diabetes at day 28 p.i. (Fig. 8e). Together, these data show thatNK cells contribute to β cell destruction in SOCS-1–Tg NOD miceafter CVB4 infection.

DiscussionIt has been proposed that viral infections trigger or precipitate autoim-mune reactions to self1,2,4–6. We have defined here an additional way in

which infection-induced reactions to autologous cells can be regulated.Our data show that target cell responses will critically determine the out-come of a viral infection: antiviral defenses expressed by the pancreaticβ cell are necessary in preventing diabetes after infection with CVB4.

Type I IFNs secreted during early viral infection act in a paracrinemanner to lower the permissiveness of neighboring and distant cells toviral infection. During certain viral infections, early innate productionof IFN-γ may also contribute to the transition to an antiviral state25–27.Our data suggest that IFNs provide the stimuli necessary to activateantiviral defenses in pancreatic β cells. Treatment of pancreatic isletswith IFNs before and during infection in vitro blocked CVB4 replica-tion and cell death, which shows that IFNs prevented islet cells fromundergoing viral cytolysis and/or infection-induced apoptosis. CVB4was detected in the islet cells of mice with β cells that were eitherexpressing SOCS-1 or lacking IFN receptors. This shows that the tro-pism of CVB4 for islet cells was altered when the engagement of IFNreceptors or IFN signaling was perturbed in β cells. In SOCS-1–Tgmice, infection resulted in β cell loss and diabetes, which showed thatan intact β cell response to IFN is required to prevent diabetes after asystemic CVB4 infection.

The SOCS-1–Tg model suggests that IFNs are critical in preventingdirect virus-mediated killing of β cells. This deduction is based on acorrelation between no suppression of CVB4 replication in IFN-treat-ed SOCS-1–Tg islet cells in vitro and fast induction of diabetes inCVB4-infected SOCS-1–Tg mice, and the observation that the pres-ence of CVB4 in the pancreatic islet cells preceded the disappearanceof β cells in infected SOCS-1–Tg mice. From additional studies withNOD-SCID and (NOD×C57BL6)F1 mice, we deduced that it wasunlikely that β cell–specific responses to IFNs were required to pre-vent either an attack by the host’s adaptive antiviral immune responseor an attack from autoreactive T cells that could have been activatedthrough bystander mechanisms17,38. Depletion of NK cells affordedprotection from CVB4-induced diabetes and led to a marked decreasein β cell destruction after infection in SOCS-1–Tg mice. NK cells playa role in the host’s defense against coxsackievirus infection55,56; how-ever, a direct attack on pancreatic β cells had not previously beenreported. We showed here that systemic activation of NK cells doesnot result in β cell destruction and diabetes in SOCS-1–Tg mice.Together, these findings suggest that an enhanced permissiveness ofSOCS-1–expressing β cells to CVB4 replication is paralleled by theirincreased sensitivity to NK cell–mediated destruction. Whether adirect killing pathway or other mechanisms contribute to the NKcell–dependent destruction of SOCS-1–expressing β cells, these stud-ies suggest that the IFN response not only lowers β cell permissivenessto CVB4 infection, but also contributes to the escape from NKcell–dependent killing. These data raise the possibility that β cellresponses elicited by IFNs preserve self-tolerance of NK cells to pan-creatic β cells during a CVB4 infection.

The human coxsackievirus adenovirus receptor (CAR) protein is areceptor for several CVB serotypes55–57, although the existence of otherreceptors has also been proposed58,59. CAR plays a central role in theproductive infection of human pancreatic islet cells60. The murinehomolog of CAR (mCAR) can determine CVB tropism in murinecells61,62, but whether mCAR and/or other receptor(s) contribute toCVB4 attachment and entry to murine pancreatic islet cells is present-ly unknown. It has been suggested that differing amount of mCARexpression might account for the high tropism of CVB3 for pancreaticacinar cells and a low tropism for pancreatic β cells18. Indeed, viral tro-pism is initially determined by the tissue-specific expression of viralreceptors. Nonetheless, for many RNA and some DNA viruses, the

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Figure 9. Depletion of NK cells prevents the loss of insulin-staining β cellsafter CVB4 infection of SOCS-1–Tg NOD mice. Representative immuno-staining of pancreatic sections from the pancreas of an infected SOCS-1–Tg NODmouse treated with anti–asialo-GM1 to deplete NK cells: the structure of the pan-creatic islet is close to normal and contains numerous (a) insulin+ and (b) glucagon+

cells. Sections are representative of at least eight infected animals. Original magnifi-cation: ×40.

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ability of the receptor-expressing cell to mount an antiviral defense inresponse to type I IFNs is the major determinant for successful virusreplication63. We found that pancreatic islet cells, which are normallyhighly permissive to an in vitro infection with CVB410,11,13,35, did notsupport abundant viral replication after treatment with IFNs. In addi-tion, by infecting SOCS-1–Tg or IFN-αβγR–/– mice, we showed that theIFN action was likely to be crucial also in regulating β cell permissive-ness to CVB4 infection during infection in vivo. Our data provide apossible explanation for the paradoxical finding that although most sys-temic CVB infections pass without causing diabetes in the infectedhost16–19, in vitro infection with CVBs often results in β celldeath10,11,13–15,35. Indeed, although IFNs released during systemic CVB4infection ensured the efficient transition of β cells into an antiviralstate, a lack of adequate IFN stimulation could reasonably account forthe marked effects on β cell function and survival during infection invitro that we observed here and others have also reported10,11,13–15,35.However, whether IFNs mediate inhibition of viral entry and/or viralreplication remains to be determined.

IFN-α is expressed in the pancreata of diabetic patients64–66.Published data showing that human pancreatic β cells infected withCVB in vitro produce IFN-α60 support the view that these individualsmight have carried a persistent viral infection. Animal studies have pro-vided evidence to suggest that type I IFNs (IFN-α or IFN-β) could beinvolved in the pathogenesis of type 1 diabetes by exerting negativeeffects on the β cell67–72. In addition, IFN-α induces the expression ofhuman retroviral superantigens that activate Vβ7+ T cells, which pro-vides a possible link between viral infections and the activation ofpotentially autoreactive T cells72. In contrast to these studies, our datashow that an early response to IFNs is essential for β cell survival inresponse to CVB4 infection. Additional support for a critical roleplayed by IFN-α in preventing CVB-induced pathologies comes fromdata showing that human islets infected with CVBs in vitro rapidly suc-cumb to the viral infection when IFN-α in the culture media is neutral-ized60. These findings suggest that IFN-α can act as a “double-edgedsword” in the pathogenesis of virus-induced autoimmune disease fortwo reasons. First, IFN-α ensures that pancreatic β cells and other cells,as suggested by the rapid death that occurs in CVB4-infected IFN-αβγR–/– mice, enter an antiviral state that is critical for their survivalduring early infection. Second, a prolonged increase in systemic orlocal amounts of type I IFN contributes to β cell demise by either act-ing directly on the β cell or through the activation of self-reactive T cells. Nonetheless, the theory that IFNs play a nonredundant role inpreventing β cell death after CVB4 infection could certainly extend toinfections with other pancreatrophic viruses. Hence, as research seeksto develop methods for preventing type 1 diabetes, these data—whichshow that a block of IFN actions can be devastating to pancreatic β cells and lead to diabetes in hosts infected with CVB4—will be a keyconsideration.

Although diabetes is an unusual outcome of acute CVB infections,one exception is patients diagnosed with type 1 diabetes when suffer-ing from severe, sometimes fatal, enteroviral infections7,10,15,23. Ourmodel for CVB4-induced type 1 diabetes resembles this clinical pic-ture. Thus, some cases of diabetes, which occur in close associationwith a viral infection, could be the result of a failing β cell defenseagainst the infecting virus. In addition, the rapid onset of diabetes par-alleled by pancreatitis in CVB4-infected SOCS-1–Tg mice shares someof the clinical features of a new subtype of nonautoimmune type 1 dia-betes73. Diabetes in this group of patients had an acute onset and wascharacterized by high serum pancreatic enzyme concentrations, theabsence of glutamic acid decarboxylase 65 (GAD65) autoantibodies,

low C peptide concentrations and normal glycosylated hemoglobinA(1c) (HbA1c) concentrations. Although the etiology remains unknown,it was suggested that a viral infection could have caused the rapiddestruction of the pancreatic β cells in these patients73,74. Hence, anincreased understanding of the molecular mechanisms behind IFN-induced antiviral defenses in pancreatic β cells could facilitate thedevelopment of antiviral therapy that may provide effective prophylax-is for humans with acute-onset of type 1 diabetes. Finally, CVB infec-tions may result in β cell damage, an event that might be necessary inorder for an autoimmune-mediated β cell destruction to progress3,17,38,75.Several clinical studies have emphasized the role of enteroviral infec-tions in accelerating the progression of diabetes in humans76–79.Defective β cell antiviral defenses could therefore augment cellulardamage and the release of otherwise sequestered β cell antigens, pro-viding a rich pool of epitopes to prime self-reactive lymphocytes andinitiating autoimmunity4,80. Thus, enhanced antiviral defenses may alsobenefit individuals with a genetic predisposition to develop autoim-mune type 1 diabetes.

In summary, our data show that by responding to IFNs, the β cell notonly restrains CVB4 infection and replication, but also escapes antivi-ral activities raised by the host’s innate immune system. The pancreat-ic β cell was once called the “innocent bystander” and was believed tohave little involvement in its own demise during type 1 diabetes81.However, our results suggest that during CVB4 infection, the host iscritically dependent on the early antiviral defenses raised by the pan-creatic β cell. Conversely, if β cell antiviral defense fails, the host willimmediately succumb to diabetes irrespective of any other defensemechanisms that are awakened. These observations also imply thatindividual variations in the target β cell–specific antiviral defense caninfluence susceptibility to virus-induced type 1 diabetes. This scenariocan most likely be extended to other organ-specific autoimmune dis-eases where genes expressed within the target tissue itself could influ-ence inflammation and thereby regulate disease susceptibility.

MethodsAnimal husbandry. NOD/Shi, NOD-SCID and C57BL/6 were from the rodent-breedingcolony at The Scripps Research Institute (TSRI). Breeding pairs of IFN-αβγR–/–

(129S6/SvEv)36 mice were a gift of S. Virgin (Washington University School of Medicine,St. Louis, MO) and wild-type 129S6/SvEv mice were from Taconic Laboratories(Germantown, NY). Mice were bred and maintained at TSRI, where they were kept in a spe-cific pathogen–free environment. The overall incidence of diabetes in the NOD/Shi colonywas 60–70% for females and 25–30% for males. All live animal experiments were approvedby the Institutional Animal Care and Use Committee (IACUC) and the Animal ResearchCommittee (ARC) and were conducted in accordance with institutional guidelines for ani-mal care and use.

Generation of SOCS-1–Tg mice. SOCS-1–Tg mice were generated by placing a 673–basepair DNA fragment encompassing SOCS-1 cDNA82–84 under the transcriptional control ofthe human insulin promoter33. The transgene construct (3.3-kb) was microinjected into oneof the pronuclei of fertilized eggs derived from NOD/shi donors at the Transgenic andEmbryonic Stem Cell Facility at TSRI. Transgenic progeny were detected as described33. Ofthe 60 mice born, five founders had integrated copies of the transgene encoding SOCS-1.Of these, three mice transmitted the transgene to their progeny, and two of these lines (linesA and B) were kept for the initial studies. Tg mice from both lines behaved similarly wheninfected with CVB4, that is, they quickly developed diabetes after infection with CVB4.Experiments to evaluate the mechanisms behind CVB4-induced diabetes were done withmice from line A.

Virus strain and propagation of viral stocks. CVB4 Edwards strain 2 (E2) was from C. Gauntt (University of Texas, San Antonio, Texas). A stock of CVB4 was prepared andthe titer was determined as described17.

Pancreatic islet isolation and culture. Pancreatic islets were isolated and cultured asdescribed85. Islets were isolated from 5–6-week-old NOD mice in order to retrieve islets thatdid not have significant numbers of mononuclear infiltrates or significant β cell destruction.All other mice used were aged 8–12 weeks. The islet preparations were cultured for at least6 days before experiments began and, as judged by light microscopy and electron

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microscopy (data not shown), mononuclear cells surrounding the islets were released dur-ing this preculture period.

In vivo and in vitro viral infection and in vivo poly(I)·poly(C) treatment. Mice wereinfected with one i.p. injection of CVB4 (50 or 100 PFU) at age 8–9 weeks. Age-matchedanimals that were mock-infected with vehicle alone served as controls. In some experi-ments, the mice were given one i.p. injection of 100 µg of poly(I)·poly(C) (Sigma, St. Louis,MO). A 14 h poly(I)·poly(C) treatment increased NK cell activities in splenic cell popula-tions by >240% (n=2) compared to controls (determined by lysis of YAC-1 target cells in astandard 51Cr-release assay). For in vitro islet infections, a described method60 was used withsome modifications. Briefly, the pancreatic islets (20 per condition) were washed once inHank’s balanced salt solution (HBSS) followed by infection with CVB4 in 2 ml of HBSScontaining 2×105 PFU CVB4/ml (2×104 PFU/islet). After 1.5 h of incubation at 37 °C, isletswere washed three times in HBSS and placed in Millicell culture plate inserts (MilliporeCorp., Bedford, MA) that contained fresh media (1 ml). The plates were incubated at 37 °Cand media was changed every second day for up to day 6 p.i. In some experiments, the isletswere treated with IFN-α (100 or 1000 U/ml, Calbiochem, La Jolla, CA), IFN-γ (1000 U/ml.Pharmingen, San Diego, CA) or vehicle alone for 24 h before infection. Fresh IFN wasadded at each media change. Culture supernatants were retrieved for the determination ofviral titers (see below). The HBSS from the last wash p.i. and media from mock-infectedislets served as controls. Initial experiments showed that the amount of virus that was trans-ferred together with islets in the media from the last wash was below the detection limit ofour plaque assay (see below).

Virus recovery from infected mice and infected pancreatic islets and determination ofviral titers. Titers of infectious virus in the separate organs of infected mice or in culturemedia from infected pancreatic islets (the latter retrieved every 48 h p.i.) were quantified inHeLa cells with a standard plaque assay technique19. Viral titers were quantified as PFU pergram of tissue or per islet, and the results were presented as relative PFU per gram of tissueor islet. The lower detection limit of this assay was 50 PFU per gram of tissue (1.7log10PFU/g) or 50 PFU per ml of islet culture media (2.5 PFU/islet or 0.4 log10PFU/islet).

Ultrastructural analysis of cell death. Infected and control islets cells were evaluated byelectron microscopy. For this ultrastructural analysis, islets were fixed in glutaraldehyde(2.5% glutaraldehyde, 0.1 M Na cacodylate (pH 7.3) and 1 mM CaCl2) and processed forepon-araldite resin embedding by standard procedures. Ultrathin sections were stained withuranyl acetate followed by staining with Reynold’s lead citrate; they were examined at theCore Electron Microscope Facility, TSRI.

Blood glucose assessment and diabetes monitoring. Diabetes was assessed by measuringvenous blood glucose concentrations in nonfasting mice with Glucometer Elite strips(Bayer, Pittsburgh, Pennsylvania). Animals were considered diabetic after at least two con-secutive blood glucose measurements of >250 mg/dl (13.8 mM). The date of diabetes onsetwas taken as the first date these measurements were made. Moribund or infected mice pre-senting nonfasting blood glucose readings above 250 mg/dl for more than 2–3 consecutivedays were killed and the pancreata removed for histological analysis.

Histology, immunohistochemistry and immunofluorescence. Paraffin sections of forma-lin-fixed organs were prepared, cut into 5-µm thick sections and stained with hematoxylinand eosin (H&E) or with primary antibodies to insulin, glucagon (Dako, Carpinteria, CA),SOCS-1 (J192, Immuno-Biological Laboratories, Tokyo, Japan) or VP-1 (Dako)19. Slideswere counterstained in Mayer’s hematoxylin (for insulin, glucagon and VP-1 staining) ormethyl green (for SOCS-1). For double immunofluorescence, primary antibodies weredetected with fluorescein isothiocyanate (FITC)- or Texas red–conjugated secondary anti-bodies (Vector, Burlingame, CA). The Slowfade Light kit (Molecular Probes, Inc. Eugene,OR) was used to ensure minimal fluorescence fading. Sections were analyzed by immuno-fluorescence on a BioRad MRC 1024 scanning confocal microscope (Richmond, CA),mounted on a Zeiss Axiovert TV-100 with a ×40 objective (Thornwood, NY). A z-stack(with 0.3-µm steps) was then made through the entirety of each sample. Each z-stack wasimaged with the same iris, gain and black level settings. The images were then saved in TIFformat and imported into SoftWoRx 2.5 (Applied Precision, Issaquah, WA). Using a pointspread function measured on the MRC 1024, the image stacks were deconvolved with aninverse matrix algorithm with ten iterations. A maximal projection was then made of eachimage and saved in TIF format.

Insulitis scoring. Pancreatic sections (at two or three points per organ) were stained withH&E and ranked for insulitis with the following classes86. A, normal islet morphology withno peri-insulitis or insulitis; B, peri-insulitis (mononuclear cells in the peri-insular space);C, insulitis (substantial mononuclear cell infiltration); and D, islet remnant. Due to the dif-ficulty of distinguishing peri-insulitic infiltrates normally occurring in NOD mice aged 8–9from mononuclear cells that were infiltrating the exocrine part of pancreata from infectedmice, the two first classes were combined when ranking infected mice.

Immunoblot analysis. Protein extracts from NIT-1 cells (ATCC, Rockville, MD) exposedto IFN-α (250 U/ml, Calbiochem) or IFN-γ (250 U/ml, Pharmingen) or vehicle alone werehalved, separated under denaturing and reducing conditions on SDS-PAGE and transferredto nitrocellulose membranes. Membranes were incubated with primary antibodies tomurine STAT1 (which detects both unphosphorylated and phosphorylated STAT1) or

murine phospho–STAT-1 (Upstate Biotechnology, Lake Placid, NY). Signal detection wasdone as described85.

Flow cytometric analysis and cell depletion studies. Single-cell suspensions of spleen orlymph nodes were prepared as described17. The following antibodies were generated andconjugated to FITC or phycoerythrin (PE): anti-CD16/32 (2.4G2), anti-CD8 (53-6.7) andanti-CD4 (RM4-5). PE-anti–pan NK cell (DX5), allophycocyanin–anti-CD3 (145-2C11)and fluorochrome-labeled isotype-matched controls were from BD PharMingen (SanDiego, CA). Samples acquired on a FACScan or a FACSCalibur yielded data for analysisby CELLQuest software (both from Becton Dickinson, San Jose, CA). To deplete CD8+

T cells we used monoclonal rat immunoglobulin G2b, which was specific for mouse CD8(YTS169). Each of three i.p. injections given to mice every other day for 6 days contained1.0 mg of antibody. Mice were infected with CVB4 8 days after the first antibody injection.Thereafter, the presence of CD8+ T cells in single-cell suspensions from the spleens andperipheral lymph nodes (axillar, inguinal and pancreatic) of treated mice were assessed byflow cytometry and showed consistent 90–95% depletion of CD8+ cells in all compartmentstested. To deplete NK cells, SOCS-1–Tg and non-Tg NOD littermates mice were given oneintravenous (i.v.) 50 µl injection of anti–asialo-GM1 (Wako Chemicals, Richmond, VA)—which was diluted in PBS to a total volume of 200 µl—3 days before CVB4 infection, theni.p. 20 µl injections of anti–asialo-GM1 1 day before and then 1 and 4 days after CVB4infection. Control mice were given vehicle alone, then were infected with CVB4. To pre-vent the animals from developing serum sickness (anti–asialo-GM1 is of rabbit origin) theSOCS-1–Tg NOD mice were given four doses of anti–asialo-GM1 and the experimentswere terminated on days 10–12. Mice on the (NOD×C57BL/6)F1 background received onei.p. 200 µg injection of anti-NK1.1 (clone PK136) or vehicle 3, 6 or 9 days before infectionand 2 and 7 days after infection. The depletion of NK cells was verified with flow cyto-metric analysis of splenocytes and intrahepatic lymphocytes or a standard YAC-1 killingassay. Antibody-treated mice showed <1% and <2% CD3–DX5+ cells in their spleens(anti–asialo-GM1 and anti-NK1.1) and liver (anti-NK1.1), respectively. Compared to vehi-cle-treated control mice, the YAC-1 killing activities of splenocytes from anti–asialo-GM1–treated mice were reduced by 85–95% at an effector:target cell ratio of 50:1. All micewere challenged with 100 µg of poly(I)·poly(C), which was given intraperitoneally 14 hbefore killing activities were measured.

Statistical analysis. Data are expressed as mean±s.e.m. values. When experiments weredone in duplicate, the average of the two values was considered as one independent obser-vation. Statistical analysis was done with Student’s unpaired t-tests (single comparisons) orANOVA tests (multiple comparisons). Accumulated incidence of diabetes was determinedwith χ2-tests.

Acknowledgments

We thank L. Mocknic,A. Ilic and L.Tucker for excellent technical assistance; N. Hill,M. Horwitz, C. King, M. Kritzik, S. Pakala, F. Shi and other members of the Sarvetnick labo-ratory for discussions and suggestions; P. Minick for editing the manuscript; and B. Smithand M.Wood (Core Electron Microscope Facility,The Scripps Research Institute) forassistance with electron and confocal microscopy. Supported by National Institutes ofHealth grants (ROI:AI42231), the National Multiple Sclerosis Society (M. F.) and theJuvenile Diabetes Research Foundation (M. F.).

Competing interests statementThe authors declare that they have no competing financial interests.

Received 19 December 2001; accepted 12 February 2002.

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