Current interventions for arresting autoimmune diabetes have yet to strike the balance between sufficient efficacy, minimal side effects, and lack of generalized immunosuppression. Introduction of antigen via the gut represents an appealing method for induction of antigen-specific tolerance. Here, we developed a strategy for tolerance restoration using mucosal delivery in mice of biologically contained Lactococcus lactis genetically modified to secrete the whole proinsulin autoantigen along with the immunomodulatory cytokine IL-10. We show that combination therapy with low-dose systemic anti-CD3 stably reverted diabetes in NOD mice and increased frequencies of local Tregs, which not only accumulated in the pancreatic islets, but also suppressed immune response in an autoantigen-specific way. Cured mice remained responsive to disease-unrelated antigens, which argues against excessive immunosuppression. Application of this therapeutic tool achieved gut mucosal delivery of a diabetes-relevant autoantigen and a biologically active immunomodulatory cytokine, IL-10, and, when combined with a low dose of systemic anti-CD3, was well tolerated and induced autoantigenspecific long-term tolerance, allowing reversal of established autoimmune diabetes. Therefore, we believe this method could be an effective treatment strategy for type 1 diabetes in humans. IntroductionType 1 diabetes (T1D) is an autoimmune disease characterized by breach in tolerance toward pancreatic insulin-producing β cells (1, 2). Interventions aimed at arresting β cell destruction once the autoimmune attack has set in, as is the case in antibody-positive or newly diagnosed patients, have not been successful, with the exception of trials using frank immunosuppression such as cyclosporine or high doses of nonspecific immune modulators such as anti-CD3 mAbs. High doses of these Abs reverted disease in mouse models of T1D (3, 4) and preserved C-peptide production in recently diagnosed patients for more than 1 year in two phase II studies (5-7). However, the high doses of anti-CD3 required for efficacy were accompanied by moderate cytokine release-related symptoms at the time of administration and transient Epstein-Barr virus reactivation (8), whereas recent studies such as DEFEND-1 (9) and Protégé (10) demonstrate that low doses of anti-CD3 by themselves do not robustly influence the course of β cell destruction in T1D in humans. Post hoc analysis in the recent Protégé trial of patient subgroups treated

In conclusion, we have shown that miR-326 is expressed at higher levels in T1D subjects with ongoing islet autoimmunity, similar to what has been observed in multiple sclerosis, in which levels of this microRNA were highly correlated with disease severity. Interestingly, an online search of miR-326 predicted targets revealed vitamin D receptor and Erythroblastosis virus E26 oncogene homologue 1, two molecules highly involved in immune regulation.

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