Dendritic cells transduced to express interleukin 4 reduce diabetes onset in both normoglycemic and prediabetic nonobese diabetic mice

Abstract

Background: We and others have previously demonstrated that treatment with bone marrow derived DC genetically modified to express IL-4 reduce disease pathology in mouse models of collagen-induced arthritis and delayed-type hypersensitivity. Moreover, treatment of normoglycemic NOD mice with bone marrow derived DC, genetically modified to express interleukin 4 (IL-4), reduces the onset of hyperglycemia in a significant number of animals. However, the mechanism(s) through which DC expressing IL-4 function to prevent autoimmune diabetes and whether this treatment can reverse disease in pre-diabetic NOD mice are unknown.

Methodology/principal findings: DC were generated from the bone marrow of NOD mice and transduced with adenoviral vectors encoding soluble murine IL-4 (DC/sIL-4), a membrane-bound IL-4 construct, or empty vector control. Female NOD mice were segregated into normoglycemic (<150 mg/dL) and prediabetic groups (between 150 and 250 mg/dL) on the basis of blood glucose measurements, and randomized for adoptive transfer of 10(6) DC via a single i.v. injection. A single injection of DC/sIL-4, when administered to normoglycemic 12-week old NOD mice, significantly reduced the number of mice that developed diabetes. Furthermore, DC/sIL-4, but not control DC, decreased the number of mice progressing to diabetes when given to prediabetic NOD mice 12-16 weeks of age. DC/sIL-4 treatment also significantly reduced islet mononuclear infiltration and increased the expression of FoxP3 in the pancreatic lymph nodes of a subset of treated animals. Furthermore, DC/sIL-4 treatment altered the antigen-specific Th2:Th1 cytokine profiles as determined by ELISPOT of splenocytes in treated animals.

Conclusions: Adoptive transfer of DC transduced to express IL-4 into both normoglycemic and prediabetic NOD mice is an effective treatment for T1D.

Figure 1. Characterization of DC used for adoptive cellular therapy.

DC transduced with adenoviral vectors expressing soluble IL-4 (DC/sIL-4), membrane-bound IL-4 (DC/mbIL-4), or empty adenoviral vector (DC/ψ5) were compared to non-transduced DC (DC/Non) A.) After harvest on day 8, 1×10⁶ DC were plated per well in OPTI-MEM media a 96-well tissue-culture plate. Following 24 hours of culture, supernatants were analyzed by ELISA for levels of IL-4 secretion. B.) After harvest on day 8, DC were collected and analyzed by flow cytometry. Plots were based on live DC as determined by FSC vs. SSC profiles. Thin lines represent the isotype control stained samples, and bold lines represent staining with anti-B7-1, -B7-2, -PD-L1, or -PD-L2 antibodies, as shown. The percentage of cells in the sample with positive staining is displayed.

Figure 2. Incidence of diabetes in normoglycemic mice following DC therapy.

Mice were screened beginning at 11-weeks of age, and only mice with blood glucose measurements less than 150 mg/dL were randomized to receive a single tail vein injection of either 1×10⁶ DC/sIL-4, 1×10⁶ DC/mbIL-4, 1×10⁶ DC/ψ5, or saline. Combined results from 3 identical, independent experiments are shown. * denotes significance of p≤0.05 by log rank test of DC/sIL-4 compared to saline-treated group. The p-value of comparisons between the DC/mbIL-4, DC/ψ5, and saline-treated groups is greater than 0.05.

Figure 3. Mononuclear infiltration in mice following adoptive DC gene therapy.

Normoglycemic mice received a single tail vein injection of either 1×10⁶ DC/sIL-4, 1×10⁶ DC/ψ5, or saline at 12 weeks of age and were monitored. At 15 weeks of age, mice with normal blood glucose levels were sacrificed. Photomicrographs were taken of hematoxylin and eosin stained sections of pancreata collected from these mice. Mice treated with DC/sIL-4 show little or mild mononuclear infiltration surrounding the islets (A–B), whereas DC/ψ5 treated mice have moderate islet infiltration in the majority of islets (C–D), and the majority of mice treated with saline have severe insulitis (E–F). G.) The insulitis score was determined by a blinded investigator using the following scale: 0 = no lymphocytic infiltration; 1 = peri-insultits; 2 = insulitis affecting less than 33% of the islet area; 3 = insulitis affecting 33%–66% of the islet area; 4 = insluitis affecting greater than 66% of the islet area. A minimum of 30 islets were evaluated per mouse on a minimum of 3 slides at least 100 µm apart. Statistical comparison between the groups using Student's t-test demonstrates: DC/sIL4 vs. Saline p = 0.024, DC/sIL/4 vs. DC/ψ5 p = 0.167, DC/ψ5 vs. Saline p = 0.293.

Figure 4. Antigen-specific IL-4 secretion by splenic T cells following treatment with DC overexpressing IL-4.

Normoglycemic mice received a single tail vein injection of either 1×10⁶ DC/sIL-4, 1×10⁶ DC/ψ5, or saline at 12 weeks of age and were monitored until 15 weeks of age, when they were sacrificed. Splenocytes were obtained from mice and assessed by ELISPOT for the frequency of splenic T cells secreting IL-4 (panel A) or IFN-γ (panel B) in response to stimulation with GAD65_206–220, insulin, and NIT-1 cell lysate. Results shown are representative of four mice per group. Using student's t-test, ** denotes significance at p<0.05 of DC/sIL-4 treated group compared to both DC/ψ5 and saline treated groups, * denotes significance at p<0.05 of DC/sIL-4 group compared to saline alone.

Figure 5. Analysis of regulatory T cell induction by DC/sIL-4 treatment.

Normoglycemic mice received a single tail vein injection of either 1×10⁶ DC/sIL-4, 1×10⁶ DC/ψ5, or saline at 12 weeks of age and were monitored until 15 or 16 weeks of age, when they were sacrificed. A.) Splenocytes were obtained from mice at 15- or 16-weeks of age and analyzed by FACS for expansion of the CD4+CD25+FoxP3+ regulatory T cell compartment. Splenocytes were stained using isotype control and monoclonal antibodies to CD4, CD25 and FoxP3. Gates were set around live cells using FSC vs. SSC, then around CD4+ cells. Isotype control staining was used to set the FoxP3 gating, and the number of CD4+CD25+FoxP3+ cells per sample were collected and are represented as a ratio over the number of total CD4+ cells counted in each sample. B.) FoxP3 gene expression from pancreatic (PLN) and inguinal lymph nodes (ILN) obtained from mice at 15 weeks of age was analyzed using real-time PCR. Mice had been treated with 1×10⁶ DC/sIL-4 (n = 4), 1×10⁶ DC/ψ5 (n = 4), or saline (n = 6) at 12 weeks of age by a single tail vein injection. Expression of FoxP3 was normalized to the expression of beta-actin to give the relative expression, and samples from all mice were then normalized to the mean of the saline-treated ILN group.

Figure 6. Incidence of diabetes in prediabetic mice following DC therapy.

Mice were screened beginning at 11-weeks of age, and mice with blood glucose measurements between 150 and 250 mg/dL were considered prediabetic while mice with blood glucose measurements below 150 mg/dL were considered normoglycemic. A.) Normoglycemic and prediabetic mice were injected with 2mg of L-Dextrose per gram of body weight, and their blood glucose measurements were measured at 15, 30, 60, and 90 minutes post injection. B.) Mice were randomized into groups in order to avoid treatment bias based on the initial blood glucose. There were no significant differences in the blood glucose of mice prior to receiving treatment. By Student t-test: DC/sIL-4 vs DC/Ψ5 p = 0.703; DC/Ψ5 vs. Saline p = 0.8937; DC/sIL-4 vs Saline p = 0.8183. C.) Female prediabetic mice between 12 and 16 weeks of age were randomized to receive a single tail vein injection of either 1×10⁶ DC/sIL-4, 1×10⁶ DC/ψ5, or saline, and were then monitored for the development of hyperglycemia. The results represent the individual responses of each animal treated. D.) Kaplein-Meier survival analysis was performed to examine differences in response to treatment by group. * denotes significance of p≤0.05 by log-rank test of DC/sIL-4 compared to DC/ψ5, and saline-treated groups. The p-value of comparisons between the DC/ψ5 and saline-treated groups is greater than 0.05.