Regulatory T cells (Treg cells) play a critical role in controlling autoimmune responses in nonobese diabetic (NOD) mice. Using two-photon laser-scanning microscopy, researchers observed that Treg cells inhibit the priming of diabetogenic T helper (Th) cells in pancreatic lymph nodes. Treg cells interact with antigen-bearing dendritic cells (DCs), which then suppress Th cell activation. This interaction precedes the inhibition of Th cell activation, suggesting that DCs are central to Treg function in vivo. Treg cells prevent the formation of stable interactions with Th cells, instead interacting with DCs to block Th cell priming. In Treg cell-deficient NOD.Cd28⁻/⁻ mice, Th cells exhibit increased proliferation and activation, highlighting the importance of Treg cells in maintaining self-tolerance. Treg cells can suppress Th cell proliferation and differentiation, even when Th cells recognize different antigens. Treg cells also alter Th cell dynamics, promoting static clustering and dynamic swarming in the presence of antigen. However, no stable interactions between Treg and Th cells were observed during suppression. Instead, Treg cells interact with DCs to prevent Th cell priming. These findings suggest that Treg cells regulate Th cell activation through interactions with DCs, rather than direct cell-cell contact with Th cells. The study provides insights into the mechanisms by which Treg cells control autoimmune responses in vivo.Regulatory T cells (Treg cells) play a critical role in controlling autoimmune responses in nonobese diabetic (NOD) mice. Using two-photon laser-scanning microscopy, researchers observed that Treg cells inhibit the priming of diabetogenic T helper (Th) cells in pancreatic lymph nodes. Treg cells interact with antigen-bearing dendritic cells (DCs), which then suppress Th cell activation. This interaction precedes the inhibition of Th cell activation, suggesting that DCs are central to Treg function in vivo. Treg cells prevent the formation of stable interactions with Th cells, instead interacting with DCs to block Th cell priming. In Treg cell-deficient NOD.Cd28⁻/⁻ mice, Th cells exhibit increased proliferation and activation, highlighting the importance of Treg cells in maintaining self-tolerance. Treg cells can suppress Th cell proliferation and differentiation, even when Th cells recognize different antigens. Treg cells also alter Th cell dynamics, promoting static clustering and dynamic swarming in the presence of antigen. However, no stable interactions between Treg and Th cells were observed during suppression. Instead, Treg cells interact with DCs to prevent Th cell priming. These findings suggest that Treg cells regulate Th cell activation through interactions with DCs, rather than direct cell-cell contact with Th cells. The study provides insights into the mechanisms by which Treg cells control autoimmune responses in vivo.