Antigen-specific immunotherapy, an approach to selectively block autoimmune diabetes, generally declines in nonobese diabetic (NOD) mice as disease progresses. To define the parameters influencing the efficacy of antigenspecific immunotherapy once diabetes is established, plasmid DNA (pDNA) vaccination was used to suppress autoimmune-mediated destruction of syngeneic islet grafts in diabetic NOD recipients. pDNAs encoding a glutamic acid decarboxylase 65 (GAD65)-Ig molecule (pGAD65),

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... (IL)-4 (pIL4), and IL-10 (pIL10) significantly delayed the onset of recurrent diabetes compared with pGAD65؉pIL10-vaccinated recipients. Despite differences in efficacy, a similar frequency of GAD65-specific CD4 ؉ T-cells secreting IL-4, IL-10, or interferon-␥ were detected in mice treated with pGAD65؉pIL4؉pIL10 and pGAD65؉pIL10. However, the frequency of FoxP3-expressing CD4 ؉ CD25 ؉ CD62L hi T-cells was increased in the renal and pancreatic lymph nodes of diabetic recipients vaccinated with pGAD65؉pIL4؉pIL10. These immunoregulatory CD4 ؉ CD25 ؉ T-cells (CD4 ؉ CD25 ؉ Treg) exhibited enhanced in vivo and in vitro suppressor activity that partially was transforming growth factor-␤ dependent. Furthermore, duration of islet graft protection in pGAD65؉pIL4؉pIL10-vaccinated diabetic recipients correlated with the persistence of CD4 ؉ CD25 ؉ Treg. These data demonstrate that the frequency and maintenance of FoxP3-expressing CD4 ؉ CD25 ؉ Treg influence antigen-induced suppression of ongoing ␤-cell autoimmunity in diabetic recipients.