Adenovirus Early Region 3 (E3) Immunomodulatory Genes Decrease the Incidence of Autoimmune Diabetes in NOD Mice

The original RIP-E3 transgenic line was generated in B6D2/F2 mice and maintained by back-crossing into C57BL/6 (B6) background (10). To generate RIP-E3/NOD mice, the RIP-E3 B6 mice were backcrossed with NOD mice for 11 generations. The presence of the transgene was determined by polymerase chain reaction (PCR) analysis of tail DNA, as previously described (10). At the eighth backcross generation, RIP-E3 transgenic mice were typed by PCR for the previously described set of microsatellite markers (17) delineating all known genetic loci of NOD origin that contribute to diabetes susceptibility. All mice used in this study (from the 9th to 11th backcross) were derived from two 8th backcross females found to be homozygous for NOD alleles of all of these markers. Animals were followed until 30 weeks of age. Rates of type 1 diabetes development in the indicated experimental groups were assessed for statistically significant differences by Kaplan Meier life table analysis using the Statview 4.5 computer software program (Abacus Concepts, Berkeley, CA).

Starting at 2 months of age at the Albert Einstein College of Medicine (AECOM), RIP-E3/NOD and nontransgenic littermate female mice were monitored weekly for plasma glucose levels using Glucometer II strips. Values >400 mg/dl on two consecutive weekly determinations were considered to be diabetic. At The Jackson Laboratory (TJL), glycosuria was monitored with the Ames Diastix (supplied by Miles Diagnostics, Elkhart, IN). Values ≥3 were considered diagnostic for the onset of diabetes.

The pancreas was removed from killed animals, fixed in buffered formaldehyde, embedded in paraffin, and sectioned. Sections were stained with hematoxylin and eosin.

Splenic leukocytes were isolated as previously described (18) from the indicated donors. In some experiments, 1 × 10⁷ splenic leukocytes from prediabetic (6 weeks old) or overtly diabetic NOD female donors were injected intravenously into sublethally irradiated (700R) 6- to 8-week-old NOD or RIP-E3/NOD female recipients. In other experiments, 2 × 10⁷ splenic leukocytes isolated from 6-week-old NOD or RIP-E3/NOD female donors were injected intravenously into 4- to 6-week-old T- and B-cell–deficient NOD/scid female recipients. These experiments were terminated at 21 weeks posttransplantation. All animal studies were approved by the animal institute committees of AECOM and TJL.

To examine the effect of the E3 gene products on the development of diabetes in NOD mice, plasma glucose levels were monitored in 9th–11th backcross generation transgenic and nontransgenic littermate female mice. These animals were demonstrated to be homozygous for NOD alleles at markers delineating all known genetic loci that contribute to development of the disease (data not shown). The animals were evaluated for the protective effects of the Ad E3 genes in separate experiments either at AECOM or at TJL, where the incidence of diabetes in NOD controls was known to be higher. As seen in Fig. 1, by 21 weeks of age, 67% of the nontransgenic NOD females in the 11th backcross at AECOM developed hyperglycemia. In contrast, all of the RIP-E3/NOD females at AECOM remained euglycemic until 25 weeks of age, when 1 of 13 became diabetic. The incidence of diabetes at TJL was higher both in the NOD controls (93%) and in the RIP-E3/NOD animals (36%), but the delayed onset in the latter group was again noted. There was a statistically significant difference between RIP-E3/NOD and wild-type littermates at each institution (P < 0.0001 at TJL, P = 0.0012 at AECOM). Subsequent constructs of new transgenic NOD mice containing E3 genes have proven that the E3 effect on the incidence of diabetes is not attributable to a positional insertion effect of the transgene.

To determine whether the E3 gene products also retarded the development of insulitis, the pancreases from 14-week-old prediabetic female mice in the 11th backcross at AECOM were subjected to histologic analysis. The pancreases of both transgenic (Fig. 2) and nontransgenic littermates (data not shown) contained islets with varying degrees of insulitis as well as islets free of inflammatory cells. Quantitation of islets into four grades of insulitis revealed a lower insulitis score for the transgenic mice compared with the nontransgenic littermates (Table 1). Because these two groups developed insulitis at quantitatively different rates, a qualitative comparison by histochemistry of cell types comprising the insulitic lesions in age-matched standard and RIP-E3 transgenic NOD mice would not reveal mechanistic information about the basis of E3-induced type 1 diabetes resistance. Hence, alternative studies of mechanism were pursued.

Prevention of the development of diabetes in the RIP-E3/NOD mice might result from an aberrant stimulation of the immune system by the β-cells or from an increased resistance of β-cells to immune effector mechanisms. To evaluate both of these possibilities, several types of adoptive transfer experiments were performed. Splenocytes from 6-week-old RIP-E3/NOD or standard NOD female donors were transferred into NOD-severe combined immunodeficient (scid) female mice, which do not develop diabetes spontaneously due to their immunodeficiency (19). As seen in Fig. 3 data obtained in the TJL colony, ∼80% of recipients transplanted with splenocytes from euglycemic nontransgenic NOD donors developed hyperglycemia within 15–16 weeks. Among recipients transplanted with splenocytes from RIP-E3/NOD donors, 40% of the mice developed hyperglycemia within the same period of time. These differences, which are statistically significant (P = 0.042), demonstrate that immune effector cells that are primed and mature in animals expressing the E3 genes can transfer diabetes into the NOD-scid recipients, but they do so at the reduced rate characteristic of their endogenous function. These results support the conclusion that there are changes in the donor cell–mediated immune capacity as a result of expression of the RIP-E3 transgene. However, the following experiments suggest that there were also local effects of the RIP-E3 transgene in islet β-cells.

To test whether the transgenes reduced the susceptibility of β-cells to immune destruction, the E3-expressing β-cells were exposed to immune effector cells that developed in the wild-type NOD environment. For this purpose, the RIP-E3/NOD transgenic mice and nontransgenic NOD controls were sublethally irradiated and transplanted with splenocytes harvested from either young prediabetic or overtly diabetic NOD female donors. As expected, splenocytes from overtly diabetic donors transferred disease more rapidly than those from prediabetic donors. However, in both cases, the onset of diabetes in RIP-E3 recipients was significantly delayed compared with standard NOD recipients (P = 0.0049 for prediabetic donor cells, and P = 0.035 for overtly diabetic donor cells). The results of these experiments, which were performed at TJL, are shown in Fig. 4.

These results demonstrate that expression of adenovirus E3 genes in β-cells in the NOD background decreases the incidence and delays the development of diabetes. These results were obtained with identical transgenic animals housed in two different institutions at which the rate of diabetes differed between 67 and 92% for the parental female NOD strain. The E3 gene products did not prevent the development of some insulitis in the RIP-E3/NOD animals, even when the rate of diabetes was reduced to <10% in this population. However, the amount of insulitis was significantly reduced in the presence of the E3 genes. Because it was unlikely that analysis of the quantitatively heterogeneous mononuclear infiltrates would yield an understanding of the Ad E3 regulatory process, we are performing other types of experiments to determine the nature of the effector cells that are altered in the presence of β-cell Ad E3 gene expression.

The mechanism of protection against diabetes by the Ad E3 gene products is not known, in part because of the multigenic nature and multiple pathways potentially altered by the E3 genes. In addition to the inhibitory effects on cell death by inhibiting class I MHC–mediated antigen presentation and TNF-α– and Fas-initiated apoptosis by various E3 proteins, the E3-14.7K protein has been shown to interact with important regulatory molecules on the nuclear factor-κB signal transduction pathway (20,21) and with a small guanosine 5′-triphosphatase that can complex to a component of dynein (22). Mapping studies are in progress to dissect the Ad E3 genes responsible for the inhibition of autoimmune diabetes in the LCMV-induced model as well as for the decrease of disease in the NOD mice. Animals that are transgenic for the Ad E3 cassette designed with various deletions have been constructed for these experiments, which are in progress. Although these experiments are not yet completed, it is clear that NOD transgenics expressing new constructs of E3 genes are also protected from diabetes. These recent results indicate that the effects of the E3 transgenes demonstrated in the current article are not attributable to insertional mutagenesis.

None of the Ad E3–coded proteins has been shown to be secreted. Thus, they should effect only cells in which they are synthesized or immunological reactions that depend on recognition of such cells. The insulin promoter is primarily active in pancreatic β-cells, but it has shown some activity in testis; the RIP II version used here has also been shown to be active at low levels in thymus (23,24). However, from adoptive transfer experiments, it appears that the RIP-driven E3 proteins affect both the activation and/or development of immune effector cells as well as the susceptibility of β-cell targets to destruction by fully activated immune effector cells. The two effects are likely to be interrelated, because the initial damage to β-cells may reinforce the immune response by providing further stimuli to immune effector cells. Adoptive transfer studies have been initiated to determine which immunologic cell types are altered during the reduction of the diabetogenic phenotype in NOD mice as a result of RIP-E3 transgene expression.

Whereas the reduction in diabetes by Ad E3 gene products is complete in the LCMV model and readily apparent in the NOD model, it is clear that the activity of the viral genes must be further enhanced before a useful prophylactic or therapeutic approach to diabetes in humans would be feasible using these principles. Although the system might be further optimized by increased expression of the E3 proteins, it also may be improved by deletion of E3 genes with potentially harmful effects. One example is an 11.6-kDa proapoptotic protein, also called the “adenovirus death protein,” that is thought to function in viral release from the nucleus at the end of the infectious cycle (25). Thus, viral genes evolving with an infectious agent to modify the host immune and cytokine responses to favor survival of the pathogen might prove to be useful for controlling the host response to endogenous stimuli that promote autoimmune diseases. Unlike immunomodulation approaches based on gene targeting, such as the β₂-microglobulin knockout animals (26,27), the E3 genes could be introduced into β-cells as dominant transgenes using viral vectors to modulate the immune response to human islets before transplantation.

This work was funded by Program Project grants from the Juvenile Diabetes Foundation International (JDFI) and the National Institutes of Health (NIH) (to M.H. and S.E.) and by NIH grants DK46266, DK51090, and AI41469 and JDFI grants (to D.S.).