Abstract Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is often the only curative treatment option for patients with hematologic malignancies. Its therapeutic effect is mediated by donor T cells, recognizing and eliminating residual malignant cells of the patient. However, the main adverse effect of allo-HSCT is also mediated by these T cells: the so-called graft-versus-host disease (GvHD). A promising approach to treat GvHD is the use of immunosuppressive T cell populations to inhibit uncontrolled T cell activation. A novel regulatory T cell population described in the setting of allo-HSCT is the subset of TCRαβ+ CD4- CD8- double-negative (DN) T cells. In murine models infusion and/or activation of DN T cells specifically suppressed alloreactive T cells and prevented development of GvHD after allo-HSCT. Of interest, clinical studies in patients who underwent allo-HSCT revealed an inverse correlation between the frequency of circulating DN T cells and the severity of GvHD. We have recently demonstrated that human DN T cells like their murine counterparts strongly inhibit proliferation of alloreactive CD4+ and CD8+ T-cells. Here we asked whether proinflammatory and homeostatic cytokines associated with incidence and severity of GvHD after allo-HSCT affect the suppressive activity of DN T cells. We found that the proinflammatory cytokines IL-6, IL-18, TNF and IFN-g do not affect the suppressive activity of human DN T cells nor render alloreactive T cells insensitive to inhibition. In contrast, the homeostatic cytokine IL-15 significantly diminished the immune regulatory function of DN T cells. Further analyses demonstrated that both conventional CD4+ T cells and DN T cells express the functional IL-15 receptor which elicits downstream receptor signaling. Of importance, IL-15 highly induced Akt/mTOR signaling cascade in DN T cells, a pathway reported to reverse suppressive activity of regulatory cells. Together, our findings indicate that the homeostatic cytokine IL-15 but not proinflammatory mediators impair DN T cell-mediated suppression of allogeneic T cell responses. 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.