The in vivo inhibitory function of the MHC-I α3 domain–CD8α interaction

The interaction between the Major Histocompatibility Complex Class I (MHC-I) α3 domain and CD8α has classically been viewed as a positive coreceptor interaction that stabilizes TCR signaling during antigen recognition. However, its physiological function in mature peripheral CD8+ T cells in vivo remains incompletely understood. Here, we identify the MHC-I α3 domain–CD8α interaction as a previously unrecognized inhibitory pathway that tonically restrains peripheral CD8+ T-cell activation and maintains T-cell tolerance in vivo. Antibody-mediated disruption of the MHC-I α3 domain–CD8α interaction induced spontaneous activation of peripheral CD8+ T cells without impairing their survival, lowered the threshold for antigen-induced activation, and enhanced responsiveness to cognate peptide stimulation. In peptide-induced OT-I T-cell anergy models, blockade of either H-2D ^b or H-2K ^b α3 domain interactions with CD8α prevented the induction of anergy and restored responsiveness of previously anergic T cells. Notably, blockade of the H-2K ^b α3 domain enhanced OT-I responses despite simultaneously disrupting the classical positive coreceptor interaction within the TCR–peptide–MHC complex, indicating that tonic inhibitory signaling mediated by the MHC-I α3 domain predominates under these conditions. Together, these findings redefine the classical MHC-I–CD8α interaction as a bidirectional pathway that not only supports antigen recognition but also imposes tonic inhibitory control over peripheral CD8+ T cells. These results identify the MHC-I α3 domain–CD8α axis as a potential target for reversing T-cell tolerance and enhancing antitumor or antiviral immunity.