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Induction of specific immunological unresponsiveness by feeding protein antigens is termed oral tolerance and may be a potential therapy for autoimmune diseases. Whereas oral tolerance therapy may be both simple and effective, the requirement for large amounts of protein will limit clinical testing of autoantigens, which are difficult to produce. We have previously demonstrated transgenic plant production and direct oral delivery of a beta cell autoantigen murine GAD67 to prevent autoimmune diabetes in nonobese diabetic mice. Mucosal adjuvants such as cholera toxin B subunit may lower the level of autoantigen required, but the development of neutralizing mucosal antibody responses may limit usefulness in enhancing long-term oral tolerance. IL-4, being an endogenous protein, would avoid this result and possibly enhance oral tolerance but has not been tested as a mucosal adjuvant. In this study, human GAD65 (hGAD65), as well as murine IL-4, was expressed in transgenic plants for feeding trials. Both IL-4 and hGAD65 plant tissue were required to protect nonobese diabetic mice from diabetes, and no benefit was found if either was used alone. Combined therapy enhanced levels of IgG1 anti-GAD antibodies, increased splenocyte IL-4/IFN-gamma cytokine responses, and produced protective regulatory T cells. These results demonstrate that orally administered plant IL-4 remains biologically active and is synergistic when given with hGAD65 in inducing robust oral immune tolerance. Using transgenic plants expressing IL-4 and GAD65 may be a novel clinical approach to the prevention of human type 1 diabetes by oral tolerance.

Induction of oral tolerance to prevent diabetes with transgenic plants requires glutamic acid decarboxylase (GAD) and IL-4