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CD8+CD122+ regulatory T cells (CD8+CD122+ Treg) are naturally occurring Treg that effectively suppress the proliferation and IFN-γ production of both CD8+ and CD4+ target cells. This study investigated the molecular mechanisms of the recognition of target cells by CD8+CD122+ Treg using an in vitro culture system that reconstitutes the regulatory action of these cells. Naive CD8+CD122+ Treg co-cultured with pre-activated T cells became active Treg that produced IL-10 and suppressed IFN-γ production from the target T cells. CD8+CD122+ Treg effectively suppressed the IFN-γ production of the target cells of syngeneic mouse strains but not of allogeneic mouse strains with incompatible MHC. By using MHC-congeneic mouse strains, MHC-restricted suppression by CD8+CD122+ Treg was further confirmed. The blockade of cell surface molecules either on the Treg or on the target cells by specific blocking antibodies indicated that H-2K, H-2D, αβTCR and CD8 were involved in the regulatory action but I-A and Qa-1 were not. These results indicate that CD8+CD122+ Treg recognize already-activated T cells via the interaction of conventional MHC class I–αβTCR and become active regulatory cells that produce IL-10 and suppress the target cells. [ABSTRACT FROM PUBLISHER]