SYSTEMIC TGF-BETA1 GENE THERAPY INDUCES CD4+CD25+ FOXP3+ REGULATORY T CELLS, RESTORES SELF-TOLERANCE AND FACILITATES ISLET REGENERATION IN OVERTLY DIABETIC NOD MICE

Abstract

O251* Aims: Type 1 diabetes results from autoaggressive T cell mediated destruction of insulin producing beta cells of the pancreas. Recent data support the idea that restoration of self-tolerance may facilitate islet cell regeneration/recovery. In view of the potent immunoregulatory activity of transforming growth factor beta1 (TGF-β1), we explored the hypothesis that systemic TGF-β1 therapy restores self-tolerance, facilitates islet regeneration and cures autoimmune diabetes. Methods: We designed and constructed a replication deficient adenovirus (Ad) vector encoding the active form of human TGF-β1 and tested the hypothesis that systemic TGF-β1 gene therapy restores self-tolerance and facilitates islet regeneration in the NOD model. Overtly diabetic NOD mice received intravenous injection of Ad.AH.TGF-β1 at a dose of 5x1010 particle units and seven to fourteen days after the injection, the mice were transplanted with 500-600 syngeneic islets under the kidney capsule. Results: Hyperglycemia occurred by day 17 in all untreated NOD mice whereas 50% of syngeneic grafts functioned until day 60 in the NOD mice treated with Ad.AH.TGF-β1. Analysis of retrieved islet grafts showed well-preserved islets with a peri-islet infiltrate primarily of CD4+ T cells and expression of CD25 and Foxp3. Staining for IFN-γ was negative whereas TGF-β1 and IL-10, and PD-L1 and PD-L2 all showed heightened expression. Having demonstrated that Ad.AH.TGF-β1 protects syngeneic islet grafts, we investigated whether systemic TGF-β1 gene therapy is associated with islet regeneration in the native pancreas. Thus, we removed the long-term functioning syngeneic islet grafts and examined whether the mice remained normoglycemic; we also removed the endogenous pancreas and examined the status of native islets. Normoglycemia was observed in 66% of the mice despite removal of the syngeneic islet grafts suggesting recovery of endogenous islet function upon treatment with Ad.AH.TGF-β1. Examination of the native pancreas revealed scattered islets, among pancreatic exocrine tissues, with intact islet structures (panel A). There were few intra-islet cellular infiltrates. However, similar to what was found in the syngeneic islet graft, there were well-demarcated lymphocytic infiltrates on the periphery of the islets without invasion of the islets themselves (Panel A. Peri-insulitis but not insulitis). Moreover, the intact islets stained strongly for insulin (Panel B) providing definitive evidence for functional restoration of endogenous beta cells.FigureConclusions: Our data demonstrate that systemic TGF-beta1 gene restores self-tolerance and facilitates islet regeneration in previously diabetic NOD mice. Our studies support the idea that in-vivo expansion of CD4+CD25+ Foxp3+ regulatory T cells is an effective strategy for constraining established islet directed autoimmunity and curing type 1 diabetes.