Immunotherapy of spontaneous type 1 diabetes in nonobese ... - Nature

Gene Therapy (2000) 7, 1840–1846  2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt

ACQUIRED DISEASES

RESEARCH ARTICLE

Immunotherapy of spontaneous type 1 diabetes in nonobese diabetic mice by systemic interleukin-4 treatment employing adenovirus vector-mediated gene transfer MJ Cameron1,2, GA Arreaza1,2, L Waldhauser3, J Gauldie3 and TL Delovitch1,2,4 1

Autoimmunity/Diabetes Group, The John P Robarts Research Institute, London, Ontario; 2Department of Microbiology and Immunology, and 4Department of Medicine, University of Western Ontario, London, Ontario; and 3Department of Pathology, McMaster University, Hamilton, Ontario, Canada

We have previously shown that systemic injection of multiple low doses of recombinant murine interleukin-4 (mIL-4) can prevent type 1 diabetes (T1D) in nonobese diabetic (NOD) mice by activating regulatory T helper (Th) 2 cells in vivo. Here, we have developed a gene transfer approach to the prevention of T1D by testing the therapeutic potential of an adenovirus gene transfer vector engineered to express mIL4. We found that only two systemic injections of a recombinant adenovirus type 5 vector-expressing mIL-4 (Ad5mIL-4)

reduces destructive insulitis and protects NOD mice from the onset of diabetes by eliciting intrapancreatic Th2 cell responses. Host immune responses against the adenovirus vector were detectable; however, the levels of antibody production were insufficient to preclude Ad5mIL-4 treatment as a possible therapeutic agent against T1D. Thus, adenovirusmediated delivery of IL-4 provides protection of NOD mice from T1D and represents a clinically viable therapeutic approach. Gene Therapy (2000) 7, 1840–1846.

Keywords: type 1 diabetes; NOD mice; gene transfer; IL-4; t helper cells

Introduction Type 1 diabetes (T1D) is an organ-specific autoimmune disease that targets the insulin-producing ␤ cells in the islets of Langerhans. T1D patients can depend on insulin treatment for survival; however, this treatment does not prevent eventual complications such as blindness, nephropathy, atherosclerosis, and microvascular disease. An important and well-characterized experimental animal model of T1D is the nonobese diabetic (NOD) mouse. NOD mice spontaneously develop a form of T1D with a clinical phenotype that is similar to the human disease.1 The onset of T1D is the consequence of a progressive destruction of islet ␤ cells mediated by an imbalance between effector CD4+ T helper (Th)1 and regulatory CD4+ Th2 cell function.2–6 For example, during early pancreas inflammation, NOD islets are infiltrated by both antigen-presenting cells (APCs) and T cells, and this infiltration is accompanied by the subsequent expression of pro-inflammatory Th1 cytokines associated with an invasive insulitis.7 Consistent with the presence of a Th1enriched environment in the pancreas at diabetes onset, a higher interferon (IFN)-␥:interleukin (IL)-4 concentration ratio is evident in the pancreata of diabetic female NOD mice in comparison with non-diabetic NOD mice.8 Correspondence: TL Delovitch, Autoimmunity/Diabetes Group, The John P Robarts Research Institute, 1400 Western Road, London, Ontario N6G 2V4, Canada Received 27 March 2000; accepted 27 July 2000

NOD mice exhibit numerous T cell abnormalities at the time of onset of peri-insulitis. We have identified a T cell proliferative hyporesponsiveness upon TCR stimulation that is mediated by reduced IL-2 and IL-4 secretion in response to T cell activation.1,4,9 In addition, we have shown that systemic injection of female NOD mice with recombinant murine IL-4 (mIL-4) interferes with migration of T cells to pancreatic islets and induces the development of a predominant Th2-enriched environment in the pancreas. In this manner, IL-4 treatment limits damage to islet ␤ cells and prevents Th1-mediated destructive insulitis and T1D.5 There is a paucity of pharmacokinetic data on IL-4. Studies on the related cytokines IL-2 and IL-3 demonstrate that the serum half-lives of these cytokines are very short (⬍1 h) following intravenous bolus injection.10 Although there is a poor correlation between serum concentrations of cytokines and their biological or clinical effects,10 repeated injections of IL-4 are required to protect NOD mice from T1D.5 Our multiple low-dose approach did not elicit any undesirable effects in NOD mice; however, the application of such a protocol in humans may risk side-effect(s), eg allergic immune responses. Therefore, we developed a gene therapy strategy using a replication-deficient adenovirus (Ad) vector as a more efficient and optimal means of sustaining cytokine expression and reducing pancreas inflammation. Ad vectors are well suited to deliver efficiently transgenes as therapeutic agents for T1D.11 Replicationdeficient Ad vectors can be grown at very high density

Adenovirus-based gene therapy of type 1 diabetes MJ Cameron et al

and maintain their genomes as episomal DNA in the nucleus of both proliferating and non-proliferating cells.12 Systemic transient Ad-based cytokine overexpression has been successful in the treatment of several inflammatory disease models, including collageninduced arthritis13 and experimental inflammatory bowel disease.14 The potential for Ad-based cytokine gene therapy of diabetes has been demonstrated only in islet studies. Ad-mediated expression of the immunoregulatory IL-12p40 homodimer in islets prolonged syngeneic islet graft survival in NOD mice.15 Also, the Ad gene transfer of the IL-1 receptor antagonist protein to human islets protects them against IL-1␤-induced ␤ cell impairment of glucose-stimulated insulin secretion and Fas-triggered activation of apoptosis.16 In this study, we demonstrate that systemic IL-4 gene transfer achieved by injection of a recombinant replication-deficient human Ad type 5 (Ad5) mIL-4-expressing vector (Ad5mIL-4) can protect NOD mice from the spontaneous development of T1D and thus establish proof of principle for the future development of Ad platform-based immunotherapy.

Results Ad5mIL-4 treatment elicits detectable levels of IL-4 in the sera of treated NOD mice We tested the ability of intraperitoneal (i.p.) injection of Ad5mIL-4 to yield detectable levels of serum mIL-4 production in female NOD mice. IL-4 (1.0–2.0 ng/ml) was transiently expressed in the serum of NOD mice for up to 3 days following each Ad5mIL-4 injection at 2 and 5 weeks of age (Table 1). IL-4 was undetectable in sera from DL70-3 control vector (no IL-4 cDNA)-treated NOD mice (Table 1) and naive age-matched female NOD mice (our unpublished observations). IL-4 was also undetectable in the sera of NOD mice treated with Ad5mIL-4 or DL70-3 at 5 and 7 weeks of age. Thus, Ad5mIL-4 treatment elicits measurable IL-4 in the sera of NOD mice providing treatment begins neonatally. Gene transfer of IL-4 reduces destructive insulitis and protects against T1D in female NOD mice The ability of Ad5mIL-4 to yield high levels of serum IL4 production in female NOD mice prompted us to deterTable 1 Serum IL-4 levels in female NOD mice treated with an IL-4-expressing Ad5 vectora IL-4 (ng/ml)b

Treatment

Control (2 Control (2 Control (5 Ad5mIL-4 Ad5mIL-4 Ad5mIL-4

weeks) and 5 weeks) and 7 weeks) (2 weeks) (2 and 5 weeks) (5 and 7 weeks)

3 days

5 days

− − − 1.5–2.0 1.0–2.0 −

− − − − − −

Serum pooled from female NOD mice (n = 3) following each inoculation with Ad5mIL-4 or the DL70–3 control vector was assayed for IL-4 content by ELISA. b Data represents the range of IL-4 concentrations (ng/ml) measured in sera at the given day following each injection. −, ⬍25 pg/ml, which is the threshold level of detection of the assay. a

mine whether inoculation of this vector protects against insulitis and/or T1D. A comparison of histological sections of pancreata from 15- and 30-week-old non-diabetic NOD female mice treated with Ad5mIL-4 or DL70-3 control at 2 weeks of age only or at 5 and 7 weeks of age did not reveal any significant differences in the severity of insulitis (data not shown). In contrast, 40–45% of the islets were either normal or showed only peri-insulitis in the pancreas of 15- and 30-week-old NOD mice treated with Ad5mIL-4 at 2 and 5 weeks of age, whereas this low insulitis score was evident in 25–30% of islets from DL703 control NOD mice (Figure 1). The percentage of islets displaying severe insulitis in 15- and 30-week-old NOD mice treated with Ad5mIL-4 was 12–15% lower than that observed in DL70-3 control-treated NOD mice. Thus, delivery of mIL-4 using a replication-deficient Ad5 vector reduces destructive insulitis in NOD mice. One injection (i.p.) of Ad5mIL-4 at 2 weeks of age did not reduce the incidence of T1D in NOD mice; however, a 10 week delay in the appearance of 50% incidence of T1D was noted in Ad5mIL-4-treated compared with DL70-3 control-treated mice (Figure 2a). On the other hand, two injections of Ad5mIL-4, one administered neonatally at 2 weeks of age and the other at 5 weeks of age, delayed and reduced the incidence of diabetes from 80% (16/20) in DL70-3-treated mice to 20% (4/20) in Ad5mIL4-treated NOD mice (Figure 2b). In Ad5mIL-4-treated mice, diabetes did not occur until 28 weeks of age, while in DL70-3-treated mice diabetes was evident as early as 14 weeks of age. We did not detect serum IL-4 following the last injection in NOD mice that were treated with Ad5mIL-4 at 2, 5 and 7 weeks of age, nor was the incidence of T1D reduced any further in these mice (our unpublished observations). Mice treated with Ad5mIL-4 at 5 and 7 weeks of age were not significantly protected from diabetes onset (Figure 2c). Thus, neonatal Ad5mIL4-mediated gene therapy protects against the development of T1D in NOD mice.

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IL-4 gene therapy modifies intrapancreatic cytokine content We have previously demonstrated that systemic recombinant mIL-4 administration potentiates Th2 responses in the periphery and pancreas of NOD mice.5 Thus, we investigated whether a similar mechanism accounts for the protection afforded by systemic Ad5mIL-4-mediated gene delivery. The total intrapancreatic content of IL-4 and IFN-␥ was assayed in mIL-4-expressing and control vector-treated NOD mice at 10 weeks of age. Figure 3 shows that IL-4 was not detected in the pancreata of 10week-old DL70-3-treated NOD mice, however, IL-4 (15 pg/mg) was observed in the pancreata of Ad5mIL-4treated mice. Conversely, the expression of IFN-␥ in the pancreata of Ad5mIL-4-treated mice was decreased about 20-fold relative to the level found in DL70-3-treated NOD pancreata. These data indicate that treatment with Ad5mIL-4 potentiates Th2-type immune responses in the pancreas of vector-treated NOD mice. Neither vector-specific antibody responses nor alterations in IL-4-induced serum IgE levels contribute to protection of NOD mice from T1D One possible limitation to the use of viral vectors for gene therapy of disease is that antiviral immunity may ensue and give rise to adverse effects in the host under treatGene Therapy


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Figure 1 Ad5mIL-4 reduces islet inflammation in NOD mice. Pancreata from female NOD mice treated with either Ad5mIL-4 (n = 3) or DL70-3 control (n = 3) were killed at (a) 15 weeks and (b) 30 weeks of age. Following hematoxylin and eosin staining, a minimum of 20 islets from each NOD mouse was observed and the degree of mononuclear cell infiltration was graded independently by two observers as follows: 0 – normal; 1 – peri-insulitis (mononuclear cells surrounding islets and ducts but not infiltrating the architecture); 2 – moderate insulitis (mononuclear cells infiltrating ⬍50% of the islet architecture); 3 – severe insulitis (⬎50% of the islet tissue infiltrated by lymphocytes accompanied by a reduction in insulin staining). An example of an islet from a 30-week-old DL70-3 control-treated mouse (c) shows severe insulitis accompanied by a substantial mononuclear cell infiltrate. Examples of a normal islet from a 30-week-old Ad5mIL-4-treated NOD mouse (d, left of photo) that displays no evidence of islet infiltration and a moderately infiltrated islet (d, right of photo) are shown. These examples are representative of similarly scored islets from 15-week-old DL70-3 controland Ad5mIL-4-treated NOD mice.

ment.11 For example, an immune response to viral capsid proteins in Ad5 vectors may reduce its therapeutic efficacy. We addressed this issue by assaying for anti-Ad5 antibodies in the sera of Ad5mIL4- and DL70-3-treated NOD mice. Titers of anti-Ad5 antibodies increased with age (4–25 weeks) in NOD mice treated with either Ad5mIL-4 or DL70-3 (Table 2). Anti-Ad5 IgG antibody titers differed only in 7-week-old NOD mice (2 weeks after the last injection of Ad5), whereas DL70-3-treated mice had a higher serum antibody titer (1/1350) than Ad5mIL-4-treated mice (1/150) at this time. Thus, while antiviral serum antibody responses were detectable in Ad5 vector-treated NOD mice, these responses did not compromise either the expression or therapeutic capacity of IL-4 to protect from T1D in Ad5mIL-4-treated NOD mice. Since IL-4 mediates immunoglobulin isotype class switch to IgE production,17 an undesirable side-effect such as the induction of an allergic response may be encountered with IL-4 immunotherapy. Therefore, we determined whether the Ad5-mediated IL-4 administration schedule used here to treat NOD mice elevated the level of IgE antibody production in these mice. Since Ad5mIL-4 administration at 2 and 5 weeks of age represents our most protective protocol for therapy of T1D, the sera from 10-week-old and 25-week-old NOD mice treated in this manner with Ad5mIL-4 and DL70-3 were assayed for their relative serum IgE concentrations. Gene Therapy

Serum IgE concentrations were not significantly altered in NOD mice treated with Ad5mIL-4 (Table 3).

Discussion Multiple low doses of recombinant mIL-4 administered i.p. protect NOD mice from T1D by limiting damage to islet ␤ cells and inducing a Th2-enriched environment in the pancreas.5 The short in vivo half-life of IL-4 necessitated that it be injected frequently, ie thrice weekly for 8–10 weeks. To enhance the potential efficacy of systemic IL-4 therapy of T1D, we devised an approach to reduce pancreas inflammation and onset of overt diabetes by replication-deficient Ad5 platform-based cytokine gene delivery. Here, we demonstrate that only two prophylactic injections of Ad5mIL-4 can transiently increase in vivo serum IL-4 to detectable levels and profoundly reduce the incidence of T1D in female NOD mice. The continuous presence of IL-4 provided during the expression of Ad5mIL-4 therefore offers a notable improvement over the short serum half-life of an injected cytokine.10 Concerns regarding the generation of allergic side-effects are minimal, as exemplified by our findings that NOD serum IgE levels are unaffected by our experimental intervention. As described above, several properties make replication-deficient Ad5 vectors good candidates for the expression of transgenes. However, the main problem


Figure 2 Ad5mIL-4 treatment protects female NOD mice from T1D. (a) Female NOD mice (n = 20, randomized from five litters) were injected i.p. at 2 weeks of age with 108 p.f.u. of either Ad5mIL-4 or the DL70-3 control virus. (b) NOD mice (n = 40, randomized from 10 litters) were injected i.p. at 2 weeks of age (108 p.f.u.) and 5 weeks of age (5 × 108 p.f.u.) with either Ad5mIL-4 or DL70-3 control. (c) Female NOD mice (n = 20, randomized from five litters) were injected i.p. at 5 and 7 weeks of age with 5 × 108 p.f.u. of either Ad5mIL-4 or DL70-3 control. Mice were screened weekly for the presence of hyperglycemia (BGL ⬎11.1 mmol/l) starting at 8 weeks of age. Diabetes was diagnosed when mice were hyperglycemic for two consecutive readings.

associated with the therapeutic use of Ad vectors is their immunogenicity.11 Anti-viral cellular and humoral immune responses may preclude the stable gene expression and repeated dosing that treatment of chronic diseases may require. Indeed, a third Ad5mIL-4 injection at 7 weeks of age did not elicit detectable levels of serum IL-4 nor did it further reduce diabetes incidence. Therefore, we treated NOD mice with only two Ad5mIL-4 injections, both accompanied by a relatively short burst of detectable serum IL-4. While increasing levels of antiviral capsid IgG antibodies in sera from both DL70-3- and Ad5mIL-4-treated NOD mice were detected, the levels of antibody production detected appeared to be insufficient to preclude Ad5mIL-4 treatment as an effective therapeutic agent against diabetes. However, antibody responses against the virus likely limited the ability of

Ad5mIL-4 to interfere with the migration of diabetogenic T cells to the pancreatic islets of Ad5mIL-4-treated mice as significantly as we previously reported in NOD mice treated i.p. with multiple doses of recombinant mIL-4.5 Rather, Ad5mIL-4 treatment appears to induce regulatory T cells in the pancreas to suppress islet ␤ cell destruction and progression to overt T1D. Evidence in support of this notion is derived from analyses of the levels of expression of these cytokines in the pancreas of Ad5mIL-4-treated NOD mice at 10 weeks of age. The detectable amounts of IL-4 and significant downregulation of IFN-␥ expression elicited in the pancreas by Ad5mIL-4 treatment may explain its ability to protect NOD mice from diabetes. Committed autoreactive cells, including Th1 cells, may accumulate in pancreatic islets, but the function of IL-4 predominates to inhibit any IFN␥-mediated ␤ cell damage. The persistent effect of an apparent Th2 class shift noted here is consistent with reports demonstrating that the presence of specific cytokines at the initiation of an immune response can lead to the generation of both effector and long-lived memory T cell populations that produce restricted patterns of cytokines.18 Future experiments may benefit from the use of less immunogenic Ad-based gene transfer vehicles.11 For example, second-generation vectors have been used in which the E2 or E4 regions are deleted or inactivated to cripple the virus biology further. These vectors persist longer in transduced cells, are associated with a decreased inflammatory response and extend the duration of transgene expression.19 Helper-dependent Ad vector systems that have reduced immunogenicity and rely on a complementing virus to provide the necessary proteins in trans for packaging are also available. This system utilizes a helper virus that has packaging sequences flanked by loxP sites so that in transduced cells that stably express the Cre recombinase, the packaging signal is efficiently excised rendering the helper virus unpackagable.20 In addition, adeno-associated virus (AAV) vectors possess low immunogenicity, but generally afford low-level gene transfer. Transduction by AAV vectors can be enhanced in the presence of Ad gene products through the formation of double-stranded, nonintegrated AAV genomes, which elicit high-level and stable transgene expression in mice after intramuscular injection of recombinant AAV.21 Also, HIV-based lentivirus vectors represent promising candidate gene transfer vehicles. Indeed, lentivirus-mediated IL-4 transduced islet grafts are protected from insulitis.22 However, safety concerns preclude clinical consideration until non-human lentivirus vectors are developed.11 The objective of this study was to induce immune deviation and modify the pathologic mechanisms occurring in the development of T1D by gene transfer of IL-4. To our knowledge, this is the first report to demonstrate that IL-4 can protect against spontaneous diabetes in NOD mice in an Ad-based systemic gene therapy approach. Our experimental approach, making use of transient IL4 gene transfer by means of a replication-deficient human Ad5 vector, offers the prospect of studies of effects of a range of cytokines and approaches that may favorably modify autoimmune responses with minimal intervention. Second-generation Ad vectors may be engineered to reduce their inherent immunogenicity. Until then, caution must be taken in the utility of Ad5-based gene

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Figure 3 A Th2-type cytokine profile is observed in the pancreas of NOD mice after treatment with an IL-4-expressing Ad5 vector. NOD mice treated at 2 and 5 weeks of age with either Ad5mIL-4 (n = 3) or DL70-3 control (n = 3) were killed at 10 weeks of age, and the intrapancreatic IL-4 and IFN␥ concentrations were determined by ELISA. Values are expressed as mean pg/mg of total protein extracted from pancreatic tissue samples. Comparison between means was performed by the Student’s t test (**P ⬍0.05).

Table 2 Anti-Ad IgG antibody responses in the serum of Ad5treated NOD micea Titerb Treatment

4 weeks

7 weeks

25 weeks

Control Ad5mIL-4

1/150 1/150

1/1350 1/150

1/4050 1/4050

Sera collected and pooled from female NOD mice (n = 3) treated with Ad5mIL-4 or DL70-3 control at 2 weeks of age (108 p.f.u.) and 5 weeks of age (5 × 108 p.f.u.) were assayed for levels of anti-Ad5 IgG antibodies by ELISA. b Titers were recorded as the highest serum dilution where the difference in the OD454 between an Ad5-infected HeLa cell extractcoated well and an uninfected HeLa cell extract-coated well was ⬍0.4. a

Table 3 Serum IgE levels in NOD mice treated with an IL-4expressing Ad vectora IgEb Treatment

7 weeks

25 weeks

Control Ad5mIL-4

0.50 ± 0.1 0.60 ± 0.2

0.30 ± 0.1 0.30 ± 0.1

Sera from 7 and 25-week-old NOD mice (n = 3) treated with Ad5mIL-4 or DL70-3 control vector at 2 weeks of age (108 p.f.u.) and 5 weeks of age (5 × 108 p.f.u.) were monitored for their relative IgE content by ELISA. b Optical densities at 405 nm were determined to quantify the relative amount of IgE. a

transfer in the long- or short-term modification of cellular physiology. Indeed, the recent ill-fated attempt at Adbased gene therapy has forced researchers back to the bench to understand better the nature of gene therapy vectors and to develop safer approaches.23 None the less, we anticipate that our studies will aid in the future development of therapies for several Th1-mediated autoimmune diseases. Specifically, gene transfer-based strategies may become available to modify the intra-islet environment in order to restore and preserve islet ␤ cell function and prevent T1D. Gene Therapy

Materials and methods Ad5 vectors Construction, propagation, and characterization of the replication-deficient recombinant Ad5 vector that has a deletion of the E1 and E3 sequences and expresses mIL4 cDNA under the control of the human cytomegalovirus immediate–early (hCMV IE) promoter has been previously described.24 This vector stimulates the production of a high level of bioactive IL-4 protein in transfected human and murine cells and modulates tumorigenicity in a murine model of breast cancer.24 The control DL703 Ad5 variant is also deleted in the E1 region and does not carry a transgene.25 Mice NOD/Del mice were bred in a specific pathogen-free barrier facility at The John P Robarts Research Institute (London, ON, Canada). Islet infiltration begins at 4–6 weeks of age in our colony of female NOD mice, and progression to destructive insulitis and overt diabetes occurs by 4–6 months of age. The incidence of diabetes in female NOD mice in our colony is 40–50% at 15 weeks of age and 80–90% by 25 weeks. Treatment of NOD mice with Ad5mIL-4 The treatment protocol was based on the parameters of dose, timing, and period optimized in adult NOD mice in our previous publications.5,24 The dose of IL-4 administered to neonatal mice was five-fold lower than that given to adult mice to compensate for the difference in body mass. NOD female mice were injected i.p. at either: (1) 2 weeks of age (108 p.f.u.), (2) 2 weeks (108 p.f.u.) and 5 weeks (5 × 108 p.f.u.) of age, or (3) 5 weeks (5 × 108 p.f.u.) and 7 weeks (5 × 108 p.f.u.) of age with either Ad5mIL4 or DL70-3. The number of mice (n values) in each of these groups were: (1) 10 Ad5mIL-4- and 10 DL70-3treated, (2) 20 Ad5mIL-4- and 20 DL70-3-treated, and (3) 10 Ad5mIL-4- and 10 DL70-3-treated for a total of 80 mice randomized from 20 different litters. Blood glucose levels (BGL) were monitored weekly with a Glucometer Encore (Miles/Bayer, Toronto, ON, Canada) as previously described.5 Mice with a BGL ⬎11.1 mmol/l (200 mg/dl) for 2 consecutive weeks were considered diabetic. Histopathology analysis Pancreatic tissue was removed, fixed with 10% buffered formalin, embedded in paraffin and sectioned at 5 ␮m


intervals. The incidence and severity of insulitis was examined by hematoxylin and eosin staining as well as insulin immunostaining. A minimum of 30 islets from each mouse was observed, and the degree of mononuclear cell infiltration was scored independently and blindly by two observers using the following ranking: 0 – normal; 1 – peri-insulitis (mononuclear cells surrounding islets and ducts, but no infiltration of the islet architecture); 2 – moderate insulitis (mononuclear cells infiltrating ⬍50% of the islet architecture); and 3 – severe insulitis (⬎50% of the islet tissue infiltrated by lymphocytes accompanied by a reduction in insulin staining). The immunohistochemical identification of insulin was performed using a porcine anti-insulin antibody and avidin–biotin peroxidase detection system (Dako, Carpinteria, CA, USA).

Intrapancreatic cytokine analysis Intrapancreatic concentrations of IL-4 and IFN-␥ were quantified in tissue samples, as previously described.5,26 Briefly, pancreata were isolated and snap-frozen in liquid nitrogen. Immediately before analysis, tissues were homogenized and sonicated in an antiprotease buffer (Boehringer Mannheim, Laval, QC, Canada). Homogenates were centrifuged to remove debris and then passed through 1.2 ␮ filters (Gelman Sciences, Ann Arbor, MI, USA). The filtrates were analyzed for IL-4 and IFN-␥ concentrations by ELISA. ELISA results were normalized relative to the total protein (Bradford dye-binding protein assay; Bio-Rad, Hercules, CA, USA) derived from each tissue. Serum IL-4, IgE antibody and antiviral antibody measurement Serum pooled from three female NOD mice was collected after each inoculation in vivo with either Ad5mIL-4 or the DL70-3 control vector. It was necessary to pool the sera to meet the volume requirements of the multiple assays described below. Previously, we reported that the variability in such serum assays between mice is ⬍15%.5 Sera were appropriately diluted and assayed for IL-4 content by ELISA as described.5 The presence of IgE antibodies in sera from selected treatment groups was also determined by ELISA. Briefly, serum samples were added at appropriate dilutions to plates coated with 2 ␮g/ml purified anti-mouse IgE capture mAb (clone R35–72; Pharmingen, Mississauga, ON, Canada). Biotinylated antimouse IgE mAb (2 ␮g/ml, clone R35–118; Pharmingen) together with avidin-peroxidase and 2,2⬘-Azino-bis-(3ethylbenzthiazoline-6-sulfonic acid) (ABTS) substrate (both from Sigma, Oakville, ON, Canada) were used for detection. Optical densities were read at 405 nm to determine the relative amounts of IgE in the sera. Sera collected from NOD mice treated with Ad5mIL-4 or DL70-3 were assayed by ELISA for their levels of antiAd5 IgG antibodies. Appropriate dilutions of NOD sera were added to Ad5-infected or uninfected HeLa cell extract-coated ELISA plates. Following detection with an alkaline-phosphatase-conjugated rat anti-mouse IgG mAb (Sigma), titers were recorded as the highest serum dilution where the difference in the OD454 between an Ad5-infected HeLa cell extract and an uninfected HeLa cell extract was ⬍0.4.

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Acknowledgements We thank Dr Subrata Chakrabarti and Kollol Mukerjee for their assistance with the pancreas histology, Ms Carol Richardson and her staff for maintaining our mouse colony, Ms Anne Leaist for her cheerful assistance with the preparation of this manuscript, and all members of our laboratory for their advice and encouragement. Supported by grants from the Juvenile Diabetes Foundation International (JDFI), Medical Research Council (MRC) of Canada (MT-5729), MRC/JDFI Diabetes Centre of Excellence and Canadian Diabetes Association (CDA) (TLD), a postdoctoral fellowship from the CDA and Fundayacucho (Venezuela) (GAA), and a Doctoral Research Award from the MRC of Canada (MJC).

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