Reversal of New Onset Type 1 Diabetes by Oral Salmonella-Based Combination Therapy and Mediated by Regulatory T-Cells in NOD Mice

Abstract

Autoimmune diseases such as type 1 diabetes (T1D) involve the loss of regulatory mechanisms resulting in increased tissue-specific cytotoxicity. The result is destruction of pancreatic insulin-producing β-cells and loss of glucose homeostasis. We are developing a novel oral vaccine using live attenuated Salmonella to deliver TGFβ, IL10, and the diabetic autoantigen preproinsulin combined with low-doses of anti-CD3 mAb. Here we show that oral administration of Salmonella-based anti-CD3 mAb combined therapy reverses new-onset T1D in non-obese diabetic (NOD) mice. The therapeutic effect of the combined therapy was associated with induction of immune suppressive CD4⁺CD25⁺Foxp3⁺ Treg and CD4⁺CD49b⁺LAG3⁺ Tr1 cells. In adoptive transfer experiments, adding or depleting Treg or Tr1 cells indicated that both are important for preventing diabetes in combined therapy-treated mice, but that Tr1 cells may have a more central role. Furthermore, induced Tr1 cells were found to be antigen-specific responding to peptide stimulation by secreting tolerance inducing IL10. These preclinical data demonstrate a role for Treg and Tr1 cells in combined therapy-mediated induction of tolerance in NOD mice. These results also demonstrate the potential of oral Salmonella-based combined therapy in the treatment of early T1D.

Keywords: Salmonella; Tr1-cells; Treg-cells; immunomodulators; immunotherapy; oral vaccine; type 1 diabetes.

Figure 1

Salmonella-based combination therapy reverses diabetes in NOD mice. Female new onset diabetic NOD mice were treated via intraperitoneal (i.p.) injection with five once-a-day doses of anti-CD3 mAb (2.5 μg/mouse). Then mice were orally vaccinated with vehicle (A), anti-CD3 alone (B), TGFβ+IL10+anti-CD3 (C), PPI+TGFβ+IL10+anti-CD3 (D), PPI+TGFβ+anti-CD3 (E), PPI+IL10+anti-CD3 (F), and non-diabetogenic LLO+TGFβ+IL10+anti-CD3 (G) for 2 consecutive weeks starting one day after conclusion of anti-CD3 therapy. Data displayed as the blood glucose levels in NOD mice for 100 days after vaccine start.

Figure 2

Salmonella-based combination therapy induces suppressive Tregs. Female 7–8 week old NOD mice were treated with 2.5 μg/mouse of anti-CD3 mAb via i.p. injection for 5 days followed by 2 doses of oral combined vaccines. Splenocytes were harvested from the indicated group at day 30 post-vaccination. (A) Representative FACS plots gated on live CD4 T cells indicate the frequency of CD4⁺CD25⁺Foxp3⁺ T cells in the spleens of mice from each group. (B) Quantification the percentages of CD4⁺CD25⁺FoxP3⁺ Tregs in each treated group. Data is presented as means ± SD are from 2 independent experiments. Statistical analysis using one-way ANOVA shows the significance between combined therapy and vehicle group (*p < 0.05; **p < 0.01). (C) in vitro suppression assay of Treg in culture with CD4⁺CD25⁻ T responder cells and CD3/CD28 beads. Statistical analysis using two-way ANOVA shows the significance between combined therapy and vehicle group (****p < 0.0001).

Figure 3

The role of Salmonella-based combination therapy induced Tregs in suppression of autoimmune diabetes. Female 7–8 week old NOD mice were treated with 2.5 μg/mouse of anti-CD3 mAb via i.p. injection for 5 days followed by 2 doses of oral combined vaccine. Splenocyte pools were prepared from the indicated treated groups at day 30 post-vaccination. (A) Adoptive transfer of splenocytes from overtly diabetic NOD mice into recipient NSG mice. (B,E) Adoptive transfer of splenocyte pools from NOD mice treated with vehicle or combined therapy into NSG mice. (C,F) Co-transfer of splenocyte Tregs from vehicle or vaccinated mice with diabetic splenocytes into NSG recipient mice. (D,G) Adoptive transfer of Treg-depleted splenocytes isolated from vehicle or vaccinated NOD mice into NSG mice. (A–D) Data display the blood glucose levels for individual mice over the time. (E–G) Data presented as the Log-rank plot of the percentage of NSG mice that remained diabetes-free over the time after transfer. The differences between the group of mice vaccinated with combined therapy and vehicle was significant (*p < 0.05, ****p < 0.0001) by the log-rank (Mantel-Cox) test.

Figure 4

Salmonella-based combination therapy induces Tr1 cells in NOD mice. Female 7–8 weeks old NOD mice were treated orally with 2 doses of combined vaccine and with 2.5 μg/mouse of anti-CD3 mAb as described. Splenocytes were prepared from the indicated treated groups at day 30 post-vaccination. (A) Representative FACS plots gated on live CD4 T cells indicate the frequency of CD4⁺CD49b⁺LAG3⁺ T cells in the spleens of mice from each treatment group. (B) Quantification the percentages of CD4⁺CD49b⁺LAG3⁺ T cells in each treated group (10 mice each). Data presented as means ± SD from 2 independent experiments. Statistical analysis using one-way ANOVA shows the significance between combined therapy and control group (**p < 0.01).

Figure 5

IL10 is required for long-term Tr1 induction in NOD mice following Salmonella-based combination therapy. Female 7–8 weeks old NOD mice were treated orally with 2 doses of combined vaccine with 2.5 μg/mouse of anti-CD3 mAb as described. Splenocytes were prepared from the indicated treated groups at 7 weeks and 15 weeks post-vaccination. (A) Representative FACS plots gated on live CD4 T cells indicate the frequency of CD4⁺CD49b⁺LAG3⁺ T cells in the spleens of mice from the indicated groups. (B) Scatter plots of the percentages of CD4⁺CD49b⁺LAG3⁺ Tr1 expression across the 3 groups at 7 weeks and 15 weeks post-vaccination. Data presented as means ± SD from 2 independent experiments. Statistical analysis using one-way ANOVA shows the significance between combined therapy and control group (*p < 0.05: **p < 0.01; ****p < 0.0001). (C) in vitro stimulation assays of vaccine induced Tr1. Pooled splenocytes CD4⁺CD49b⁺LAG3⁺ Tr1 cells were isolated from vaccinated and vehicle-treated mice 3 weeks post-vaccination by FACS sorting. Secretion of IL10 was determined by ELISA of culture supernatants obtained from CD4⁺CD49b⁺LAG3⁺ Tr1 cells after peptide stimulation for 3 days. Data presented as means ± SD and obtained from 4 replicas. The statistical significance was calculated with two-way ANOVA and the significance level indicated by asterisks (****p < 0.0001).

Figure 6

Tr1 cells are crucial for Salmonella-based combined therapy of diabetes in NOD mice. Female 7–8 weeks old NOD mice were orally vaccinated as described. Splenocytes were prepared from the indicated treated groups at day 30 post-vaccination. (A) Adoptive transfer of splenocytes from overtly diabetic NOD mice into recipient NSG mice. (B,D) Co-transfer of splenocyte Tr1cells from vehicle or vaccinated mice with diabetic splenocytes into NSG recipient mice. (C,E) Adoptive transfer of Tr1 depleted splenocytes isolated from vehicle or vaccinated NOD into NSG mice. (A–C) Data display the blood glucose levels for individual mice over the time. (D,E) Data presented as the Log-rank plot of the percentage of NSG mice that remained diabetes-free over the time after transfer. The differences between the group of mice vaccinated with combined therapy and vehicle was significant (**p < 0.01) by the log-rank (Mantel-Cox) test.