This study describes a robust method for expanding antigen-specific regulatory T cells (Tregs) from autoimmune-prone nonobese diabetic (NOD) mice. The method involves in vitro expansion using anti-CD3, anti-CD28, and interleukin-2, resulting in a 200-fold increase in Treg numbers within two weeks. The expanded Tregs retain their classical cell surface phenotype and function both in vitro and in vivo to suppress effector T cell functions. Importantly, small numbers of antigen-specific Tregs can reverse diabetes after disease onset, suggesting a novel approach to cellular immunotherapy for autoimmunity. The study highlights the importance of antigen-specific Tregs in regulating autoimmunity. It shows that antigen-specific Tregs are more effective than polyclonal Tregs in suppressing autoimmune responses in vivo. The expanded antigen-specific Tregs were able to reverse diabetes in new-onset diabetic NOD mice, indicating their potential for therapeutic use. The study also demonstrates that the expanded Tregs can survive and function in vivo, even in the presence of a fully functional pathogenic T cell response. The results suggest that antigen-specific Tregs are more effective in regulating autoimmune responses in vivo compared to polyclonal Tregs. This is likely due to the fully functional immune system in these animals and the requirement for effective antigen-mediated reactivation of Tregs at the inflammatory site. The study also shows that Tregs can reverse diabetes in an ongoing autoimmune setting, highlighting their potential for clinical therapy. The study emphasizes the importance of antigen specificity in Treg function. It suggests that the efficacy of Treg-based immune therapy is critically dependent on the antigen specificity of the Tregs, at least in the autoimmune diabetes setting. Therefore, it is important to develop a procedure to selectively and reproducibly expand antigen-specific Tregs from polyclonal populations for therapeutic use. The study also highlights the potential of using MHC multimers coupled with antigenic epitopes to expand islet-specific Tregs. Current efforts are underway to identify antigen-specific Tregs in the polyclonal population and develop a protocol to selectively expand islet antigen-specific Tregs from normal NOD mice using immobilized antigenic peptide-linked MHC multimers. Additionally, similar techniques are being developed for purifying and expanding human Tregs for clinical use in these settings.This study describes a robust method for expanding antigen-specific regulatory T cells (Tregs) from autoimmune-prone nonobese diabetic (NOD) mice. The method involves in vitro expansion using anti-CD3, anti-CD28, and interleukin-2, resulting in a 200-fold increase in Treg numbers within two weeks. The expanded Tregs retain their classical cell surface phenotype and function both in vitro and in vivo to suppress effector T cell functions. Importantly, small numbers of antigen-specific Tregs can reverse diabetes after disease onset, suggesting a novel approach to cellular immunotherapy for autoimmunity. The study highlights the importance of antigen-specific Tregs in regulating autoimmunity. It shows that antigen-specific Tregs are more effective than polyclonal Tregs in suppressing autoimmune responses in vivo. The expanded antigen-specific Tregs were able to reverse diabetes in new-onset diabetic NOD mice, indicating their potential for therapeutic use. The study also demonstrates that the expanded Tregs can survive and function in vivo, even in the presence of a fully functional pathogenic T cell response. The results suggest that antigen-specific Tregs are more effective in regulating autoimmune responses in vivo compared to polyclonal Tregs. This is likely due to the fully functional immune system in these animals and the requirement for effective antigen-mediated reactivation of Tregs at the inflammatory site. The study also shows that Tregs can reverse diabetes in an ongoing autoimmune setting, highlighting their potential for clinical therapy. The study emphasizes the importance of antigen specificity in Treg function. It suggests that the efficacy of Treg-based immune therapy is critically dependent on the antigen specificity of the Tregs, at least in the autoimmune diabetes setting. Therefore, it is important to develop a procedure to selectively and reproducibly expand antigen-specific Tregs from polyclonal populations for therapeutic use. The study also highlights the potential of using MHC multimers coupled with antigenic epitopes to expand islet-specific Tregs. Current efforts are underway to identify antigen-specific Tregs in the polyclonal population and develop a protocol to selectively expand islet antigen-specific Tregs from normal NOD mice using immobilized antigenic peptide-linked MHC multimers. Additionally, similar techniques are being developed for purifying and expanding human Tregs for clinical use in these settings.