FOXP3(+) regulatory T cells (T-reg cells) prevent autoimmunity by limiting the effector activity of T cells that have escaped thymic negative selection or peripheral inactivation. Despite the information available about molecular factors mediating the suppressive function of T-reg cells, the relevant cellular events in intact tissues remain largely unexplored, and whether Treg cells prevent activation of self-specific T cells or primarily limit damage from such cells has not been determined. Here we use multiplex, quantitative imaging in mice to show that, within secondary lymphoid tissues, highly suppressive Treg cells expressing phosphorylated STAT5 exist in discrete clusters with rare IL-2-positive T cells that are activated by self-antigens. This local IL-2 induction of STAT5 phosphorylation in T-reg cells is part of a feedback circuit that limits further autoimmune responses. Inducible ablation of T cell receptor expression by T-reg cells reduces their regulatory capacity and disrupts their localization in clusters, resulting in uncontrolled effector T cell responses. Our data thus reveal that autoreactive T cells are activated to cytokine production on a regular basis, with physically co-clustering T cell receptor-stimulated T-reg cells responding in a negative feedback manner to suppress incipient autoimmunity and maintain immune homeostasis.