Hyperexpression of Foxp3 and IDO during acute rejection of islet allografts.

BACKGROUND: We investigated the hypothesis that Foxp3+ cells are an integral component of antiallograft immunity but are dominated by pathogenic effectors. METHODS: Wild-type H-2b C57BL/6 (B6) mice or B6 mice with a targeted disruption of c-Rel gene (c-Rel-/-) were used as recipients of islet grafts from allogeneic DBA/2 (H-2d) mice or syngeneic B6 mice. We developed kinetic quantitative polymerase chain reaction assays and measured intragraft expression of mRNA for Foxp3, IDO, cytolytic molecules, proinflammatory cytokines, and chemokines/receptors. RESULTS: Intraislet levels of mRNA for Foxp3, IDO, CD3, CD25, tumor necrosis factor-alpha, RANTES, IP-10, and CXCR3 were highest in DBA/2 islet allografts from WT B6 recipients compared to DBA/2 islet allografts from c-Rel-/- B6 recipients or syngeneic B6 islet grafts from WT B6 mice. The ratio of granzyme B or IFN-gamma to Foxp3 was higher with the DBA/2 islet allografts from the WT B6 recipients compared to DBA/2 islet allografts from c-Rel-/- B6 recipients or B6 islet grafts from WT B6 recipients. CONCLUSIONS: Foxp3+ cells are an integral component of acute rejection of allografts but may be dominated by pathogenic effectors.

Systemic transforming growth factor-beta1 gene therapy induces Foxp3+ regulatory cells, restores self-tolerance, and facilitates regeneration of beta cell function in overtly diabetic nonobese diabetic mice.

BACKGROUND: Type 1 diabetes results from auto-aggressive T-cell-mediated destruction of beta cells of the pancreas. Recent data suggest that restoration of self-tolerance may facilitate islet-cell regeneration/recovery. In view of the immunoregulatory activity of transforming growth factor (TGF)-beta1, we investigated whether systemic TGF-beta1 gene therapy blocks islet destructive autoimmunity and facilitates regeneration of beta-cell function in overtly diabetic nonobese diabetic (NOD) mice. METHODS: We used site-directed mutagenesis to create cysteine to serine mutation at sites 224 and 226 and constructed a replication deficient adenovirus (Ad) vector encoding active form of human TGF-beta1 (Ad-hTGF-beta1). Overtly diabetic NOD mice received intravenous injection of Ad-hTGF-beta1. Seven to 14 days after the injection, the mice received transplants with 500 syngeneic islets under the kidney capsule. Islet-graft survival and regeneration of endogenous beta-cell function were examined. RESULTS: Syngeneic islet grafts failed by day 17 in all untreated mice, whereas Ad-hTGF-beta1 therapy prolonged survival of islet grafts. Islet grafts from treated mice showed well-preserved islets with a peri-islet infiltrate primarily of CD4+ T cells and expression of CD25 and Foxp3. Systemic TGF-beta1 gene therapy was associated with islet regeneration in the native pancreas. Native pancreas of treated mice revealed islets staining strongly for insulin. Similar to what was found in the syngeneic islet graft, there were well-demarcated peri-islet infiltrates that were positive for CD4, TGF-beta1, and Foxp3. CONCLUSIONS: Our data demonstrate that systemic TGF-beta1 gene therapy blocks islet destructive autoimmunity, facilitates islet regeneration, and cures diabetes in diabetic NOD mice.

Mitochondrial targeting with antioxidant peptide SS-31 prevents mitochondrial depolarization, reduces islet cell apoptosis, increases islet cell yield, and improves posttransplantation function.

Apoptotic cell death is a defined pathway for islet cell demise, and mitochondrial dysfunction contributes to islet cell apoptosis. The hypothesis that the novel peptide D-Arg-2', 6'-dimethyltyrosine-Lys-Phe-NH2 (SS-31), previously shown to target inner mitochondrial membrane and prevent oxidative damage of neuronal cells and other cell types, optimizes pancreatic islet isolation and improves posttransplantation function in recipients with diabetes was investigated. Herein is demonstrated that SS-31 readily penetrates intact mouse islets, preserves mitochondrial polarization, reduces islet cell apoptosis, and increases islet cell yield. Optimization of islet isolation is demonstrable after SS-31 pretreatment of islet (pancreas) donor mice and with the addition of SS-31 to reagents that are used in the isolation of mouse islets. The supplementation of in vitro culture medium with SS-31 reduced islet cell apoptosis and increased the viability of human islets, as ascertained by dual-parameter flow cytometry analysis. In a stringent marginal islet cell mass transplantation model (200 mouse islets transplanted under the renal capsule of syngeneic diabetic mice) and using islets that were derived from old mice (>24 wk), SS-31 treatment was associated with prompt and sustained normoglycemia, whereas the untreated islet graft recipients remained diabetic. Our data suggest a novel strategy to optimize islet isolation and reduce the need for multiple pancreata to achieve insulin independence in the recipient with type 1 diabetes. Because SS-31 was effective with "extended" islet donors, it is hypothesized that the antioxidant SS-31 may serve to increase the pool of eligible organ donors.