In comparing the incidence of virally induced type 1 diabetes in F1 crosses of RIP-LCMV mice to three different mouse strains identical at the major histocompatibility complex H-2Db locus, we surprisingly found that disease development was reduced by 80% in F1 crosses to the SV129 genetic background and by 60% after eight backcrosses to the original C57BL/6 RIP-LCMV mice. In this model, diabetes is strongly dependent on a virally induced H-2Db–restricted cytotoxic T-cell (CTL) response. Importantly, numbers and effector functions of autoaggressive CD4 and CD8 lymphocytes were not decreased in the protected mice, and CTLs were still able to kill syngeneic islet cells in vitro with equal efficacy compared with CTLs from the original RIP-LCMV strain. Furthermore, CTLs were able to extravasate into islets in vivo, and no evidence for induction of regulatory cells was observed. However, regeneration of β-cells in islets under “attack” occurred only in the protected SV129-crossed animals, whereas it was not evident at any time in any mice that developed diabetes. Thus, genetic factors can “override” the diabetogenic potential of high numbers of autoaggressive lymphocytes through, for example, increased islet regeneration. This finding has important implications for interpreting numbers and pathogenicity of autoreactive lymphocytes in prediabetic patients of genetically diverse backgrounds.

Numbers of autoaggressive CD4 and CD8 lymphocytes are thought to directly correlate with the incidence and severity of type 1 diabetes and other autoimmune disorders (1,2,3,4,5,6,7,8,9,10). Indeed, increased numbers are usually present in mice with more severe disease (11,12,13), but the situation in humans is still unclear, and lymphocytes with specificity for islet antigens are not always consistently detectable in peripheral blood of prediabetic or diabetic individuals (unpublished, 4th International Diabetes Workshop, November 1999, Fiuggi, Rome). Thus, it appears possible that genetic, potentially non–major histocompatibility complex (MHC)-linked factors could “override” the aggressive potential of autoreactive lymphocytes. We sought to further investigate this issue in a model of antigen—specifically, (virally) induced diabetes, using varied backcrosses to the SV129 strain (14). This mouse strain is of particular importance because most targeted gene knockout mice are generated on this background, and extensive crossing to different diabetes models is usually required to obtain a particular knockout in a given model (15,16,17,18). The “danger” of carrying over diabetes-protective alleles using this strategy is a concern, especially if the final goal is prevention of disease. The notion that this can be the case emerged from earlier experimentation by our laboratory in which lower diabetes incidence occurred in control groups after crossing transgenic or NOD mice to several SV129 knockout mice (19).

The RIP-LCMV model for virally induced diabetes that we used for experiments reported here was developed in 1990 by the laboratories of Zinkernagel and Oldstone (20,21). These mice have the main advantage over the NOD model of spontaneous diabetes because autoreactive lymphocytes are easily traceable, and the time-point for induction of autoreactivity can be experimentally chosen. In RIP-LCMV mice, the self-antigen is a glycoprotein (GP) derived from lymphocytic choriomeningitis virus (LCMV) and is costimulatively expressed under the control of the rat insulin promoter (RIP) in pancreatic β-cells. Diabetes only develops after autoreactive (LCMV-specific) and CD8 lymphocytes have been activated systemically and reach the islets, where they have to be further driven by local antigen-presenting cells (APCs) that take up β-cell antigens and provide costimulation to cause sufficient β-cell destruction (22,23). Local activation of APCs in the pancreas, which occurs through systemic LCMV infection before autoreactive (anti-LCMV) lymphocytes enter the islets, is required for diabetes development (23). It is important to emphasize that the pancreatic autoimmune process begins after most LCMV has been cleared systemically and only starts when the systemic anti-LCMV response reaches its peak (23). Thus, LCMV infection precipitates and initiates the pancreatic autoimmune process. However, continuation of the autoaggressive response is independent from the presence of LCMV and can be viewed as a true “hit-and-run” event (22,23,24). Thus, insulitis in RIP-LCMV mice is an independent, localized (in islets and pancreatic lymph nodes), and self-perpetuating true autoimmune reaction that does not rely on external influx of LCMV-primed lymphocytes (19).

Previous studies have shown that CD8 T-cells, perforin, as well as interferon-γ (IFN-γ) contribute in a multifactorial fashion to islet destruction (22,24,25). Whereas IFN-γ and CD8 cells are absolutely essential, diabetes can occur in the absence of perforin (22). Most dominant and subdominant CD8 and CD4 epitopes have been mapped for the recognition of LCMV antigens on the H-2Db (DbKbIAb) MHC background (26,27). Under these premises, one would assume that the incidence of RIP-LCMV diabetes would be similar on any H-2Db background because autoreactive cytotoxic T-cell (CTL) are likely generated at sufficient quantities. Our earlier studies have demonstrated that >1 in 3,000 CTL precursors are required for autoimmune diabetes in RIP-LCMV mice (6) and that H-2Db mice generate on average 1 in 100 LCMV-specific CTL precursors 7 days after infection (28). Therefore, the reduction of disease in crosses to the SV129 background reported in this article came as a surprise to us, because we did not expect non–MHC-linked factors to overcome the “power” of autoaggressive CTL in this model. As a possible explanation, we provide evidence that attack of islets from SV129 mice induces increased β-cell regeneration, an observation that is not true for other H-2Db RIP-LCMV mouse strains in the prediabetic stage. This finding has important implications when using autoreactive lymphocytes as an indicator for ongoing disease severity, because correlation between their frequencies and disease might possibly be low in an outbred population, where the effect of other genes might be able to overcome their autoaggressive potential.

Islet isolation.

Islets of Langerhans were isolated from mice as previously described (22,29,30). In brief, the common bile duct was cannulated, and the pancreas was distended with 3 ml of Dulbecco’s modified Eagle’s medium containing 1.3 units/ml collagenase P (Boehringer Mannheim, Indianapolis, IN). Pooled pancreases were digested at 37°C for 20 min and were then disrupted by shaking. Islets were purified on Histopaque-1077 density gradients (Sigma, St. Louis, MO). The gradient was centrifuged with gradually increasing speed from 25 to 800g for 4 min then at 800g for 10 min. Islets were aspirated from the media/gradient interface, washed, and hand-picked if necessary. The islets were dispersed into single cells with 0.2% trypsin (Calbiochem, La Jolla, CA) and 10 mmol/l EDTA in Hank’s balanced salt solution (HBSS), and then they were allowed to recover in complete medium (CMRL, 7% fetal calf serum [FCS], 1% penicillin/streptomycin, and 1% glutamine) for 1 h before staining with monoclonal antibodies (mAbs). To ensure the purity of the β-cell preparation, β-cells sorted with a fluorescence-activated cell sorter (FACS) were fixed on immunohistochemistry slides and stained for intracellular insulin as previously described (31). The antibodies used were a polyclonal guinea pig anti-insulin antibody (Dako, Carpinteria, CA) as primary antibody and a biotinylated goat anti–guinea pig IgG antibody (Vector Laboratories, Burlingame, CA) as secondary antibody. Of the isolated islet cells, 56% were β-cells on average, as evidenced by positive insulin stain, and >98% of the cells in the final-gated autofluorescent population were β-cells.

Detection of MHC class I on β-cells.

Whole islets were trypsinized, and islet cells were counted. To stain class I MHCs, we used 28-14-8 (Pharmingen, La Jolla, CA), a biotinylated mouse IgG2a mAb directed against murine H-2Db that only cross-reacts with H-2Ld and H-2Dq. Secondary antibodies were as follows: phycoerythrin-conjugated goat F(ab′)2 anti-rat IgG(H+L) (Caltag Labs, Burlingame, CA) and phycoerythrin-conjugated streptavidin (Caltag Labs). Islet cell or spleen cell suspensions were incubated for 20 min on ice with mAb diluted in Dulbecco’s phosphate-buffered saline (DPBS) containing 1% FCS. They were then washed and incubated for 15 min with a secondary antibody as necessary. Cells were finally washed and resuspended in DPBS with 1% FCS and 2.5 μg/ml propidium iodide to stain dead cells. Analysis was performed on a FACSort or FACScalibur flow cytometer (Becton Dickinson, San Jose, CA) using Cell Quest software (Becton Dickinson). β-Cells were sorted on a FACStarplus (Becton Dickinson) based on flavin adenine dinucleotide auto-fluorescence according to the method previously described (32). Background levels were determined by staining MHC class I–deficient islets from β2m−/− mice.

Cytotoxicity assays.

CTL activity was measured in a 5- to 6-h in vitro 51Cr release assay (33,34). Briefly, to determine the amount of MHC-restricted CTL lysis, syngeneic or allogeneic target cells were either infected with LCMV-strain ARMSTRONG (ARM) (multiplicity of infection = 1), or uninfected cells were coated with LCMV peptides GP 33-41 (KAVYNFATC), GP amino acid 276-286 (SGVENPGGYCL), and nucleoprotein (NP) 396-404 (FQPQNGQFI), all of which were H-2Db–restricted (27). Assays used splenic lymphocytes harvested 7 days after primary LCMV infection (105 pfu i.p.) at effector-to-target ratios of 50:1, 25:1, and 12:1 or CTL clones and secondary CTL lines at ratios of 10:1 and 5:1. To determine CTL activity after secondary stimulation, spleen cells harvested from mice 30–180 days after primary inoculation with 1 × 105 pfu LCMV i.p. were incubated with MHC-matched, irradiated, LCMV-infected, or peptide-coated (10−5 mol/l) macrophages in the presence of T-cell growth factor (supernatant from concanavalin A–stimulated rat splenocytes) containing interleukin-2 (IL-2) and irradiated syngeneic spleen feeder cells for 5–12 days (34). The MC57 (H-2KbDb) and BALB/Cl7 (H-2d) cells used as CTL targets were grown as previously reported (34). When islet cells were used as targets, whole islets were isolated from collagenase-digested pancreases of at least three mice and labeled for 1 h at 37°C in the presence of 1 mCi of 50Cr. After washing three times in glucose-free HBSS (Gibco, Gaithersburg, MD), islets were dispersed by trypsinization, washed again three times, and then plated at 1–2 × 104 cells/well in 96-well flat-bottom plates for the cytotoxicity assay. In some assays, cytokines (IFN-γ, tumor necrosis factor-α [TNF-α], and IL-1β) were added directly to the target cells at 1 μg/ml (22). Where indicated, cytotoxicity assays were carried out over an extended time period of 20 h. Precursor frequencies of LCMV-specific CTL were determined as described (28).

Transgenic mice.

Generation and characterization of RIP-LCMV transgenic mice that develop type 1 diabetes after LCMV infection has been previously described (21,34). For this report, we used only RIP-GP 34-20 (H-2Db) transgenic mice, which express viral GP from the LCMV strain ARMSTRONG only in the β-cells of their islets and not in any other organs (34). SV129 and BALB/b mice were obtained from Jackson Laboratories (Bar Harbor, ME) (14). NOD mice were obtained from the rodent breeding colony at the Scripps Research Institute. F1 crosses were obtained in a single mating of RIP-GP mice with SV129, BALB/b, or NOD mice. Backcrosses to the C57BL/6 up to N8 were generated from a single RIP-GPX SV129 progenitor pair. Note that RIP-LCMV-NP mice develop diabetes on both B6 (H-2b) and BALB/c (H-2d) backgrounds, because the CTL response to LCMV NP has both a dominant H-2d–restricted (RPQASGVYM) and an H-2b–restricted (FQPQNGQFI) epitope.

Virus.

Virus stocks consisted of the LCMV strain ARMSTRONG (clone 53b). LCMV was plaque-purified three times on Vero cells, and stocks were prepared by a single passage on BHK-21 cells (35).

Blood glucose measurements.

Blood samples were obtained from the retro-orbital plexus of mice, and plasma glucose concentration was determined using Accucheck III (Boehringer Mannheim). Mice with blood glucose values >300 mg/dl were considered diabetic (36).

Histological and immunochemical analysis of tissues.

Tissues taken for histological analysis were fixed in 10% zinc formalin and stained with hematoxylin and eosin. Immunochemical studies were carried out on 6- to 10-μm freshly frozen cryomicrotome sections using immunostaining of islets to detect expression of MHC class I and II, Db, insulin, CD4, CD8, B220, and F4/80. Primary antibodies were applied for 1 h. These consisted of rat anti-mouse CD4 (clone RM 4-5), anti-CD8 (clone 53-6.7), anti-B220 (clone RA3 6B2), anti-F4/80 (clone A3-1), anti–MAC-1 (clone M 1/70), anti–MHC class I (clone M 1/42), and anti–class II (clone M5/114) (Pharmingen, San Diego, CA, and Boehringer Mannheim). After washing in phosphate-buffered saline, the secondary antibody (biotinylated goat anti-rat or anti-mouse IgG [Vector Laboratories]) was applied for 1 h. Color reaction was developed with sequential treatment using avidin–horseradish peroxidase conjugate (Boehringer-Mannheim, La Jolla, CA) and diaminobenzidine–hydrogen peroxide.

Double-staining for insulin and 5′-bromo-2′-deoxyuridine (BrDU) (Fig. 5) was performed by first staining freshly thawed pancreatic sections for insulin using a primary guinea pig anti insulin antibody (DACO) and biotinylated anti guinea pig secondary antibody combined with an avidin-alkaline-phosphatase conjugate and Vector-red staining kit (both from Vector Laboratories). Thereafter, staining for BrDU was performed as described using the Pharmingen BrDU staining kit, using rat anti-BrDU as primary antibody to avoid cross-reactivity with the insulin staining and a peroxidase-coupled secondary antibody with 3,3′- diaminobenzidine tetrahydrochloride as a color substrate (brown).

Flow cytometry.

Staining of cell surface antigen and intracellular antigens was performed as described. Virus-specific stimulation was provided by the addition of 1 μg/μl of class I and 2 μg/μl of class II LCMV-CD4 or -CD8 peptides (27,37,38,39) in the presence of 50 units/ml recombinant human IL-2. All stimulation cultures contained 1 μg/ml brefeldin A (BFA B7651; Sigma) to block protein transport into post-Golgi compartments and to allow cytokines to accumulate within cells. Negative controls were stained with cytokine-specific phycoethrin-conjugated antibodies preincubated for 30 min at 4°C with an excess of recombinant cytokine. Cells were acquired and analyzed on a FACSort or FACScalibur flow cytometer (Beckton Dickinson, San Jose, CA) using Cell Quest software (Beckton Dickinson).

Low-dose streptozodozin treatment.

For the studies displayed in Fig. 5, groups of 10 C57BL/6 or SV129 H-2b males were injected with 60 mg/kg body wt i.p. streptozodozin three times, which was dissolved immediately before injection in 100 mmol/l citrate buffer at pH 4.2. BrDU was provided in the drinking water at 0.8 mg/ml continuously.

Incidence of virally induced autoimmune diabetes is only reduced in H-2Db RIP-LCMV transgenic mice when crossed to the SV129 genetic background.

Because SV129 embryonic stem cells are preferably used to generate targeted gene knockout mice, our intention was to evaluate whether the genetic background of this strain could influence the development of autoimmune disease per se. For the first experiment, RIP-LCMV-GP mice that develop diabetes dependent on H-2Db–restricted cytotoxic CD8 lymphocytes but independent from CD4 cells (34) were crossed to various H-2Db mouse strains, and the incidence of hyperglycemia was tracked after LCMV infection. As shown in Fig. 1, only the SV129 genetic constellation appeared to prevent disease development over up to 10 backcrosses to the original RIP-LCMV-GP C57BL/6 background (data shown for N8 but not N10). In contrast, a high degree of diabetes was observed in crosses to BALB/b and NOD H-2Db mice. This finding was surprising because all of these mice express the H-2Db MHC class I allele, to which the vast majority of the H-2Db LCMV (anti-self) CTL response is restricted (27,40). In addition, because all F1 offspring will still express 50% of the original C57BL/6 H-2Db MHC molecules (and in N8 backcrosses to the original C57BL/6 will express even more), the lower diabetes incidence in SV129 F1 crosses is likely not caused by MHC-linked factors. We further investigated this issue by precisely determining autoreactive (LCMV-specific) CD8 and CD4 effector functions.

Autoreactive CTL activities are not reduced in RIP-LCMV-GP × SV129 N8 offspring.

The anti-LCMV CD8 (and CD4) responses can be considered as true autoreactive lymphocytes in RIP-LCMV mice because the transgene is expressed as a pancreatic self-antigen, and mice are tolerant unless systemic LCMV infection is initiated (34). Therefore, the enumeration of these populations according to their effector functions is a very close and accurate measurement of the initial autoreactive islet-specific response in such mice, if lymphocytes can actually enter the islets, which is equivalent between RIP-GP and RIP-GPX SV129 N8 mice (Table 1). As displayed in Fig. 2, perforin-mediated cytotoxic responses to the LCMV GP (islet antigen expressed transgenically) and NP were reduced 7 days after infection during the primary ex vivo response in SV129 mice compared with C57BL/6 RIP-LCMV mice. Interestingly, this defect was seen neither in the memory response (Fig. 2) nor in the N8 RIP-LCMV/SV129 mice that had been backcrossed to the original RIP-LCMV-GP C57BL/6 background for eight generations but were still largely protected from diabetes (Figs. 1 and 2). Similarly, the inflammatory autoreactive cytokine response reflected in LCMV-specific IFN-γ production (Fig. 3) was also reduced in SV129 mice but not in the N8 backcrosses (Fig. 3). Thus, because diabetes was still reduced after eight backcrosses to the original C57BL/6 genetic background (Fig. 1), but the autoreactive CTL response was not impaired compared with the original RIP-LCMV-GP mice (that developed a high degree of diabetes) (Figs. 2 and 3), other genetic, likely non–MHC-linked, factors have to account for the protection from diabetes. Along with the similarities in anti-LCMV CTL in B6- and SV129-crossed mice, viral (LCMV) clearance occurred with similar kinetics and was accomplished by days 8–10 from major organs (data not shown).

SV129 islets can be killed by autoreactive CTL in vitro.

We next tested whether islets from SV129 mice might be resistant to destruction by autoreactive perforin-positive CTLs or by inflammatory cytokines in general. Previous studies have implicated both of these pathways in islet destruction (22). As shown in Fig. 4, this is not the case, and islets of SV129 mice are as susceptible as islets from C57BL/6 mice or RIP-LCMVxC57BL/6 N8 mice (not shown) to CTL killing and cytokine-mediated death. As expected, MHC class I levels on islets from both strains were upregulated to very similar levels after LCMV infection, an event that is essential in sensitizing β-cells to CTL recognition (data not shown) (22). This observation strengthens the notion that genetic factors other than MHC class I could be responsible for protecting SV129-crossed RIP-LCMV mice from autoaggressive CTL and diabetes. We further dissected the underlying mechanism of protection.

Islet cells from SV129 × RIP-LCMV-GP mice regenerate “under attack” during the prediabetic phase.

Islets from the different inter- and backcrossed RIP-LCMV mice shown in Fig. 1 were evaluated histologically. Double-staining for insulin (red) and BrDu incorporation (brown) was performed during the prediabetic, diabetic, and nondiseased stages and is shown in Fig. 5. Interestingly, signs of β-cell regeneration, as evidenced by double-staining for BrDU as well as insulin, were only observed in the majority of SV129 crosses that had not developed autoimmune diabetes. In contrast, uninfected islets as well as islets from diabetic (Fig. 5) or nondiabetic C57BL/6 controls do not exhibit this phenotype, and β-cell regeneration is not seen. On average, 5 of 9 β-cells double-stained for insulin and BrDU were typically found per islet from the nondiabetic RIP-LCMV × SV129 (C57BL/6 N8 [Fig. 5B] or F1 [Fig. 5E]) mice, whereas no BrDU incorporation was observed in any of the diabetic or uninfected normoglycemic controls. Interestingly, low-dose streptozodozin treatment appeared to have a similar effect on islets of SV129 but not C57BL/6 mice, as shown in Fig. 5D. Thus, β-cells appear to generate only under attack in SV129 but not B6 mice, and increasing the islet cell mass by regeneration is likely one of the pathways preventing diabetes in mice crossed to the SV129 background. Indeed, nondiabetic but LCMV-infected RIP-LCMV-GP × SV129 C57BL/6-N8 mice had close to normal insulin content in the pancreas (data not shown).

Although autoreactive lymphocytes can enter islets of C57BL/6 and SV129 mice to a similar degree in vivo, fewer lymphocytes and activated APCs are found in islets of RIP-LCMV × SV129 mice protected from diabetes.

As shown in Fig. 6, islet infiltration in nondiabetic RIP-LCMV-GP mice crossed to the SV129 strain and back-crossed for eight generations to the C57BL/6 original genetic background is reduced, and fewer CD4 and CD8 lymphocytes are found at day 14 after the triggering LCMV infection (Table 1). It is difficult to distinguish precisely between cause and effect from such data, but the histological findings at earlier stages post–LCMV infection (Table 1) indicate that lymphocytes can still enter into the islet area of SV129-crossed transgenic mice but are present to a lesser extent inside islets in protected animals compared with diabetic non-SV129 controls (Table 1). A primary defect in lymphocytes trafficking therefore appears unlikely because similar numbers of CD4 and CD8 lymphocytes are able to reach the islets initially after LCMV infection in both mouse strains (Table 1). Furthermore, no change in the production of protective cytokines such as IL-4 or IL-10 was evident by Elispot analysis (data not shown) when comparing RIP-LCMV-GP C57BL/6 and RIP-LCMV-GP × SV129(C57BL/6-F8) mice at day 14 post–LCMV infection, and immune deviation is consequently not the likely cause for the observed lack of diabetes in the SV129-crossed animals.

In this study, we demonstrate that genes other than the MHC class I genes can overcome the autoaggressive potential of high numbers of self-reactive CD8 (and CD4) lymphocytes. Autoimmune diabetes development is drastically reduced in the RIP-LCMV transgenic mouse model when crossed once to SV129 mice and is even reduced after eight back-crosses to the original C57BL/6 background, whereas equally high levels of autoreactive CD8 and CD4 lymphocytes are generated systemically. Islets can be killed by such CTLs and their cytokines in vitro. However, β-cells regenerate in vivo under attack in protected mice, a finding that we think accounts for at least some of the protection from diabetes in SV129 crosses that maintain sufficient insulin production. Of course, additional, still unknown factors might contribute to this protection as well. Our observation is important in illustrating that genetic factors can easily overcome the autoaggressive potential of islet antigen–specific CD8 and CD4 lymphocytes. As a consequence, one might expect that the correlation between autoreactive lymphocytes and diabetes development could possibly be low in human populations with diverse genetic backgrounds.

Previous studies using tetramers and other functional in vitro assays have shown that up to one in three lymphocytes becomes an activated CTL during acute LCMV infection in C57/BL/6 mice (39,41). This translates into a very high degree of autoreactivity in RIP-LCMV-GP mice that have a fully functional anti-LCMV T-cell repertoire and that do not delete or anergize LCMV CD8 or CD4 cells as a consequence of expressing the LCMV antigen on β-cells (34). In humans, such high frequencies of autoreactive cells are never present systemically, and it is unlikely and has never been shown that they might reach such numbers locally in the islets (4). Thus, the pathogenic potential of autoreactive lymphocytes, even if they were restricted exclusively to diabetes susceptibility MHC alleles, might be overestimated, and other factors can be at least equally as important for the pathogenesis of autoimmune diabetes. One of these factors, which is illustrated in this report, is the potential of β-cells to regenerate under attack, but others are likely also playing a role. For example, local inflammation involving the activation of APCs in the pancreas and islets might be important in providing a suitable environment for autoaggressive lymphocytes (23). We find fewer activated APCs in islets of protected RIP-GP/SV129 mice (Table 1); however, this is likely the consequence of the prevention of diabetes (Table 1). Further studies will be required to clarify this issue. Second, the susceptibility of islet cells to cytokines or CTL killing contribute to diabetes pathogenesis, but in the present study, no differences were noted between the SV129 and C57BL/6 mouse strains, ruling out this possibility as a contributing factor out (Fig. 4). Third, the degree of extravasation of lymphocytes into the pancreas and islets might differ between SV129 and C57BL/6 mouse strains. Again, in this study, it is experimentally difficult to distinguish between cause and effect, as seen with the APCs. Fewer lymphocytes are present in the islets of nondiabetic RIP-LCMV-GP × SV129 mice; however, after LCMV infection, initially the same numbers of CD4 and CD8 lymphocytes enter the islets of RIP-LCMV SV129 and C57BL/6 transgenics (Table 1). Therefore, the lower degree of infiltration in SV129 mice is more likely a consequence of maintaining sufficient islet cell mass as well as a slowly subsiding inflammatory process. Finally, because differences in cytokine profiles were not observed (Table 1), differences in effector function of autoreactive CD4 and CD8 lymphocytes are less likely. However, it cannot be excluded that hence unknown effector functions other than cell number, CTL killing, and cytokine production determine the diabetogenicity of CD4 or CD8 lymphocytes, and that these functions differ between SV129 and C57BL/6 H-2Db mouse strains.

In summary, the degree of systemically activated autoreactive CD4 and CD8 lymphocytes does not predict the degree of autoimmune diabetes in RIP-LCMV mice, which diverges drastically between C57BL/6 and SV129-N8 genetic backgrounds. Islet cell regeneration is likely one of the factors accounting for this difference, but other non–MHC-linked genes have possible additional effects that still need to be discovered. It is important to consider that our findings might not be generally applicable to every diabetes model crossed to the SV129, as evidenced by good induction of type 1 diabetes in SV129 mice by low-dose streptozodozin (M.G.V.H, unpublished findings) and good diabetes incidence found in other models after back-crossing to SV129 (42). However, our findings suggest being careful with the interpretation of protection from diabetes in SV129-crossed models and, most importantly, exercising caution toward using autoreactive T-cell activity as a sole correlate for disease activity in type 1 diabetes.

FIG. 1.

Protection from autoimmune diabetes in RIP-LCMV × SV129 F1 crosses. Groups of RIP-LCMV-GP or -NP mice were back-crossed to different genetic H-2Db backgrounds as indicated. Blood glucose levels were assessed by Accucheck, as described in research design and methods, at weekly intervals. Some nondiabetic mice of each group were observed beyond 3 months, but blood glucose levels were not increasing after that time. Please note that the LCMV NP expressed as a transgene in RIP-LCMV-NP mice has both an H-2b–restricted and an H-2d–restricted CTL epitope (see research design and methods) to which significant numbers of CTL are generated in vivo. Therefore, RIP-NP mice crossed to the BALB/c background develop diabetes, as we have described previously (34).

FIG. 1.

Protection from autoimmune diabetes in RIP-LCMV × SV129 F1 crosses. Groups of RIP-LCMV-GP or -NP mice were back-crossed to different genetic H-2Db backgrounds as indicated. Blood glucose levels were assessed by Accucheck, as described in research design and methods, at weekly intervals. Some nondiabetic mice of each group were observed beyond 3 months, but blood glucose levels were not increasing after that time. Please note that the LCMV NP expressed as a transgene in RIP-LCMV-NP mice has both an H-2b–restricted and an H-2d–restricted CTL epitope (see research design and methods) to which significant numbers of CTL are generated in vivo. Therefore, RIP-NP mice crossed to the BALB/c background develop diabetes, as we have described previously (34).

FIG. 2.

Generation of autoaggressive perforin-positive CD8 CTL is reduced in SV129 mice but not in RIP-LCMV-GP × SV129 N8 back-crosses. CTL assays were performed as described in research design and methods. Target cells were H-2Db MC57 fibroblasts infected with LCMV or coated with LCMV MHC class I peptides. Primary CTL levels were measured in mice 7 days after infection with 1 × 105 pfu LCMV i.p. directly ex vivo, whereas secondary (“memory”) CTL levels were determined after a 7–10 day in vitro stimulation of H-2Db APCs, as described in detail in research design and methods. Values and means for primary CTL assays were done at 50:1 effector-to-target ratios, whereas secondary CTLs were assessed at 10:1 ratios. All comparisons were carried out in parallel using three mice per group. The means ± 1 SE are displayed.

FIG. 2.

Generation of autoaggressive perforin-positive CD8 CTL is reduced in SV129 mice but not in RIP-LCMV-GP × SV129 N8 back-crosses. CTL assays were performed as described in research design and methods. Target cells were H-2Db MC57 fibroblasts infected with LCMV or coated with LCMV MHC class I peptides. Primary CTL levels were measured in mice 7 days after infection with 1 × 105 pfu LCMV i.p. directly ex vivo, whereas secondary (“memory”) CTL levels were determined after a 7–10 day in vitro stimulation of H-2Db APCs, as described in detail in research design and methods. Values and means for primary CTL assays were done at 50:1 effector-to-target ratios, whereas secondary CTLs were assessed at 10:1 ratios. All comparisons were carried out in parallel using three mice per group. The means ± 1 SE are displayed.

FIG. 3.

Numbers of IFN-γ–producing autoaggressive CD8 and CD4 lymphocytes are equal in RIP-LCMV (C57BL/6) and RIP-LCMV × SV129 mice. A: Lymphocytes were harvested from spleens of LCMV (1 × 105 pfu)–infected mice (day 7 postinfection) and stimulated in vitro for 6 h in the presence of LCMV H-2Db–restricted CD4 and CD8 peptides as described in research design and methods. The overnight addition of brefeldin A resulted in an accumulation of intracellular cytokines that were determined by intracellular FACS analysis (see research design and methods). The values and means displayed were generated in a simultaneous experiment using three mice per group and represent LCMV antigen–specific cytokine generation. General background levels of cytokine generation were subtracted for each peptide and did not exceed 15% of the experimental values shown. Note that the reduction of CD8 IFN-γ in SV129 but not B6×129 N8 is statistically significant from C57BL/6. The means ± 1 SE are displayed. B: A representative intracellular IFN-γ and TNF-α staining for CD4 and CD8 lymphocytes is shown (see research design and methods). Non–peptide-stimulated lymphocytes harvested from spleens at day 7 (not shown) or splenocyte populations harvested at day 0 before LCMV infection (left panels) did not show intracellular cytokine staining ever exceeding >15% of the measured peptide-specific amount of IFN-γ–producing lymphocytes and were both used as negative controls and subtracted to obtain the antigen-specific IFN-γ production values shown in A. The upper two panels show cells stimulated in the presence of LCMV CD8 peptide NP396 (see research design and methods), whereas the lower two panels show cells stimulated in the presence of LCMV CD4 peptide GP61 (see research design and methods).

FIG. 3.

Numbers of IFN-γ–producing autoaggressive CD8 and CD4 lymphocytes are equal in RIP-LCMV (C57BL/6) and RIP-LCMV × SV129 mice. A: Lymphocytes were harvested from spleens of LCMV (1 × 105 pfu)–infected mice (day 7 postinfection) and stimulated in vitro for 6 h in the presence of LCMV H-2Db–restricted CD4 and CD8 peptides as described in research design and methods. The overnight addition of brefeldin A resulted in an accumulation of intracellular cytokines that were determined by intracellular FACS analysis (see research design and methods). The values and means displayed were generated in a simultaneous experiment using three mice per group and represent LCMV antigen–specific cytokine generation. General background levels of cytokine generation were subtracted for each peptide and did not exceed 15% of the experimental values shown. Note that the reduction of CD8 IFN-γ in SV129 but not B6×129 N8 is statistically significant from C57BL/6. The means ± 1 SE are displayed. B: A representative intracellular IFN-γ and TNF-α staining for CD4 and CD8 lymphocytes is shown (see research design and methods). Non–peptide-stimulated lymphocytes harvested from spleens at day 7 (not shown) or splenocyte populations harvested at day 0 before LCMV infection (left panels) did not show intracellular cytokine staining ever exceeding >15% of the measured peptide-specific amount of IFN-γ–producing lymphocytes and were both used as negative controls and subtracted to obtain the antigen-specific IFN-γ production values shown in A. The upper two panels show cells stimulated in the presence of LCMV CD8 peptide NP396 (see research design and methods), whereas the lower two panels show cells stimulated in the presence of LCMV CD4 peptide GP61 (see research design and methods).

FIG. 4.

Despite the observed reduction in type 1 diabetes, CTLs as well as cytokines kill islets of SV129 mice with equal efficacy in vitro. Islet (β-cell) killing assays were performed as described in research design and methods and our previous publications (22). Cytokines and effector CTL (syngeneic spleens day 7 post–LCMV infection with 1 × 105 pfu i.p.) were added directly to purified single-cell suspensions of islet cells labeled with Cr51 for 1 h. All islets were harvested from LCMV-infected H-2b mouse strains 7 days after LCMV infection to ensure sufficient upregulation of MHC class I to allow for CTL recognition and perforin-mediated killing, as we have described previously (22). CTL (effectors) and islets (targets) were derived from the same mouse for each Cr51 reaction well, respectively. Studies were carried out simultaneously with three mice per group, and the means ± 1 SE is shown.

FIG. 4.

Despite the observed reduction in type 1 diabetes, CTLs as well as cytokines kill islets of SV129 mice with equal efficacy in vitro. Islet (β-cell) killing assays were performed as described in research design and methods and our previous publications (22). Cytokines and effector CTL (syngeneic spleens day 7 post–LCMV infection with 1 × 105 pfu i.p.) were added directly to purified single-cell suspensions of islet cells labeled with Cr51 for 1 h. All islets were harvested from LCMV-infected H-2b mouse strains 7 days after LCMV infection to ensure sufficient upregulation of MHC class I to allow for CTL recognition and perforin-mediated killing, as we have described previously (22). CTL (effectors) and islets (targets) were derived from the same mouse for each Cr51 reaction well, respectively. Studies were carried out simultaneously with three mice per group, and the means ± 1 SE is shown.

FIG. 5.

Islets from SV129 × RIP-GP N8 (back-cross) mice regenerate under attack, whereas islets from RIP-GP C57BL/6 mice do not. To selectively label dividing cells, all mice were injected 24 h before euthanasia with BrDU as previously described (36). LCMV infection was established with 1 × 105 pfu when indicated, and all mice were RIP-LCMV-GP transgenic and crossed for eight generations (N8) to the original C57BL/6 H-2Db background. Six mice were analyzed for each group and at least 15 islets were surveyed per pancreas. As representatively shown in A, no BrDu incorporation was seen in any of the uninfiltrated or diabetic islets. As shown in B, 0–9 double-positive β-cells were observed in RIP-GPX SV129 N8 islets on average 5 double-positive β-cells. The insulitis score for diabetic and protected mice is displayed in Table 1. A: Islet from an uninfected RIP-LCMV-GP × SV129 N8 mouse. Insulin staining in red is evident, and no lymphocellular infiltration is seen. No incorporation of BrDU occurred. B: Islet from an LCMV-infected RIP-LCMV-GP × SV129 N8 mouse 2 months after infection without diabetes. Note the extensive remaining insulin staining and the incorporation of BrDU into nuclei of β-cells (arrows, double-positive staining) as well as infiltrating lymphocytes. Incorporation of BrDU into β-cells was never seen in RIP-LCMV mice crossed to other genetic backgrounds. However, it was also observed in RIP-GP × SV129 F1 intercrosses protected from diabetes (F). C: Islet from one of the few diabetic RIP-LCMV-GP × SV129 N8 mice 2 months after LCMV infection. Note that no incorporation of BrDU into nuclei of the remaining β-cells is seen. This type of histological finding is typical for RIP-LCMV mice that are crossed to genetic backgrounds other than SV129 and that are developing a high degree of diabetes. D: Islet of a nondiabetic SV129 (H-2b) mouse 6 days after low-dose streptozodozin treatment (see research design and methods) showing some β-cell proliferation. In comparison, an islet of a C57BL6/J (H-2b) mouse 6 days after low-dose streptozodozin treatment showed no β-cell proliferation and appeared similar to that seen in A. E: Islet from an LCMV-infected RIP-LCMV-GP × SV129 F1 mouse 1 month after infection without diabetes. Note the extensive remaining insulin staining and the incorporation of BrDU into the nuclei of β-cells, similar to the finding shown in B.

FIG. 5.

Islets from SV129 × RIP-GP N8 (back-cross) mice regenerate under attack, whereas islets from RIP-GP C57BL/6 mice do not. To selectively label dividing cells, all mice were injected 24 h before euthanasia with BrDU as previously described (36). LCMV infection was established with 1 × 105 pfu when indicated, and all mice were RIP-LCMV-GP transgenic and crossed for eight generations (N8) to the original C57BL/6 H-2Db background. Six mice were analyzed for each group and at least 15 islets were surveyed per pancreas. As representatively shown in A, no BrDu incorporation was seen in any of the uninfiltrated or diabetic islets. As shown in B, 0–9 double-positive β-cells were observed in RIP-GPX SV129 N8 islets on average 5 double-positive β-cells. The insulitis score for diabetic and protected mice is displayed in Table 1. A: Islet from an uninfected RIP-LCMV-GP × SV129 N8 mouse. Insulin staining in red is evident, and no lymphocellular infiltration is seen. No incorporation of BrDU occurred. B: Islet from an LCMV-infected RIP-LCMV-GP × SV129 N8 mouse 2 months after infection without diabetes. Note the extensive remaining insulin staining and the incorporation of BrDU into nuclei of β-cells (arrows, double-positive staining) as well as infiltrating lymphocytes. Incorporation of BrDU into β-cells was never seen in RIP-LCMV mice crossed to other genetic backgrounds. However, it was also observed in RIP-GP × SV129 F1 intercrosses protected from diabetes (F). C: Islet from one of the few diabetic RIP-LCMV-GP × SV129 N8 mice 2 months after LCMV infection. Note that no incorporation of BrDU into nuclei of the remaining β-cells is seen. This type of histological finding is typical for RIP-LCMV mice that are crossed to genetic backgrounds other than SV129 and that are developing a high degree of diabetes. D: Islet of a nondiabetic SV129 (H-2b) mouse 6 days after low-dose streptozodozin treatment (see research design and methods) showing some β-cell proliferation. In comparison, an islet of a C57BL6/J (H-2b) mouse 6 days after low-dose streptozodozin treatment showed no β-cell proliferation and appeared similar to that seen in A. E: Islet from an LCMV-infected RIP-LCMV-GP × SV129 F1 mouse 1 month after infection without diabetes. Note the extensive remaining insulin staining and the incorporation of BrDU into the nuclei of β-cells, similar to the finding shown in B.

FIG. 6.

RIP-LCMV-GP mice crossed with SV129 mice and protected from diabetes have lesser islet infiltration. A: Histological staining for CD8 lymphocytes in RIP-LCMV-GP × SV129-C57BL6/N8 mouse with no diabetes at day 60 postinfection. B: Histological staining for CD8 lymphocytes in RIP-LCMV-GP mouse with diabetes at day 14 post–LCMV infection (adopted from Oldstone et al. [43]).

FIG. 6.

RIP-LCMV-GP mice crossed with SV129 mice and protected from diabetes have lesser islet infiltration. A: Histological staining for CD8 lymphocytes in RIP-LCMV-GP × SV129-C57BL6/N8 mouse with no diabetes at day 60 postinfection. B: Histological staining for CD8 lymphocytes in RIP-LCMV-GP mouse with diabetes at day 14 post–LCMV infection (adopted from Oldstone et al. [43]).

TABLE 1

Time course of islet infiltration comparing RIP-LCMV-GP C57BL/6 with SV129-crossed mice

           Insulitis (%)   
Time post–LCMV infection
 
LCMV CD4 CD8 F4/80 IDDM None Peri Intra Full 
Day 2 + −  −  +/− +/− no no 0 0 0 0 
Day 4/5 + −  −  +/− +/− no no 0 0 0 0 
Day 7 +/− +/− +/− +/− +/− +/− +/− +/− no no 80 80 18 17 3 0 
Day 10 −  +/− +/− +/− +/− no no 10 10 15 18 43 72 32 0 
Day 14 −  ++ + ++ + +/− +/− yes no 8 15 10 77 78 0 
Day 60 −  ∗ + ∗ + ∗ +/− yes no ∗ 8 ∗ 10 ∗ 82 ∗ 0 
           Insulitis (%)   
Time post–LCMV infection
 
LCMV CD4 CD8 F4/80 IDDM None Peri Intra Full 
Day 2 + −  −  +/− +/− no no 0 0 0 0 
Day 4/5 + −  −  +/− +/− no no 0 0 0 0 
Day 7 +/− +/− +/− +/− +/− +/− +/− +/− no no 80 80 18 17 3 0 
Day 10 −  +/− +/− +/− +/− no no 10 10 15 18 43 72 32 0 
Day 14 −  ++ + ++ + +/− +/− yes no 8 15 10 77 78 0 
Day 60 −  ∗ + ∗ + ∗ +/− yes no ∗ 8 ∗ 10 ∗ 82 ∗ 0 

Groups of 12 RIP-LCMV-GP and RIP-LCMS-GP × SV129 (N8 backcross to the B6 background) were infected with LCMV, and 2 mice were histologically analyzed at the indicated times, as described in research design and methods. At least 15 islets were surveyed per mouse; the percentages of noninfiltrated islets, infiltrated islets, and those with perinsulinitis are shown. Note that protected and diabetic mice only diverge immunopathologically after initiation of the local autoimmune process around day 10 post-LCMV infection, when systemic virus has already been cleared, and that the principal feature in SV129 crosses is that lymphocytes still enter into the islets, but insulinitis remains mild and never fills the whole islet. Values for RIP-LCMV-GP (C57BL/6) are shown in normal type. Values for RIP-LCMV-GP (SV129) are shown in bold. Insulinitis score: none = no lympocytes in or around islets; peri = lympocytes solely around the islets; intra = lympocytes found in the islets but do not fill out the whole islet; full = whole islet appears filled with lympocytes. −, no histological signal detectable; −/+, 8–10 positive cells per islet; +, 20–40 positive cells per islet; ++, too many cells to count.

This work was supported by National Institutes of Health Grants R01-AI4415 and R29-DK-51091 (to M.G.V.). M.G.V. is also supported by Juvenile Diabetes Foundation International Career Development Award 296120.

This report is publication no. 13440-NP from the Department of Neuropharmacology, The Scripps Research Institute (La Jolla, CA).

We thank Diana Frye for assistance with the manuscript preparation.

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Address correspondence and reprint requests to Matthias G. von Herrath, La Jolla Institute for Allergy and Immunology, 10355 Science Park Dr., San Diego, CA 92121. E-mail: matthias@liai.org.

Received for publication 2 November 2000 and accepted in revised form 5 September 2001.

APC, antigen-presenting cell; BrDU, 5′-bromo-2′-deoxyuridine; CTL, cytotoxic T-cell; DPBS, Dulbecco’s phosphate-buffered saline; FACS, fluorescence-activated cell sorter; FCS, fetal calf serum; GP, glycoprotein; HBSS, Hank’s balanced salt solution; IFN-γ, interferon-γ; IL, interleukin; LCMV, lymphocytic choriomeningitis virus; mAb, monoclonal antibody; MHC, major histocompatibility complex; NP, nucleoprotein; RIP, rat insulin promoter; TNF-α, tumor necrosis factor-α.