Abstract Regulatory T (Treg) cells have been shown to be involved in downregulating immune responses in autoimmunity, transplant rejection, and graft-versus-host disease (GvHD). The subpopulation of TCRαβ+ CD4- CD8- (double-negative, DN) T cells has been described to suppress immune responses in both mice and humans. Of particular interest, infusion and/or activation of murine DN T cells specifically suppressed alloreactive T cells and prevented development of GvHD after allogeneic hematopoietic stem cell transplantation (SCT). Moreover, clinical studies in patients after SCT revealed an inverse correlation between the frequency of circulating DN T cells and the severity of GvHD, suggesting a therapeutic potential of human DN T cells. However, the molecular mechanism of suppression still remains unclear. To gain a better understanding of DN T-cell functionality, we investigated whether human DN T cells modulate distinct TCR signaling processes in conventional T cells. We found that DN T cells selectively inhibit the mechanistic target of rapamycin (mTOR) signaling pathway but not activation of mitogen-activated protein kinases (MAPK). The crucial function of mTOR signaling was confirmed by treating effector T cells with a chemical activator of protein kinase Akt, which induces mTOR phosphorylation. Indeed, enforced activation of the mTOR pathway rendered conventional T cells unsusceptible to DN T cell-mediated suppression. Given that mTOR is a critical regulator of cellular metabolism, we further determined the impact of DN T cells on the metabolic framework of conventional T cells. Intriguingly, DN T cells significantly diminished upregulation of the glycolytic machinery, expression of glucose transporters and glucose uptake in conventional T cells. These findings indicate that DN T cells inhibit metabolic reprogramming of conventional T cells by abrogating mTOR signaling, thereby inducing a quiescent phenotype. Further understanding of the mechanisms involved in human DN T-cell suppression may have important implications for using them as a cellular-based therapy to limit alloreactive immune responses. Disclosures No relevant conflicts of interest to declare.