The recently discovered population of TCRαβ+ CD4–/CD8– (double-negative, DN) T-cells are highly potent suppressor cells in mice and humans. In preclinical transplantation models, adoptive transfer of DN T-cells specifically inhibits alloreactive T-cells and prevents transplant rejection or graft-vs.-host disease (GvHD). Interestingly, clinical studies in patients who underwent allogeneic stem cell transplantation reveal 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, their exact mode of action has not been elucidated yet. Investigating the impact of DN T-cells on conventional T-cells, we found that human DN T-cells selectively inhibit mTOR signaling in CD4 T-cells. Given that mTOR is a critical regulator of cellular metabolism, we further determined the impact of DN T-cells on the metabolic framework of T-cells. Intriguingly, DN T-cells diminished expression of glucose transporters and glucose uptake, whereas fatty acid uptake was not modified, indicating that DN T-cells prevent metabolic adaptation of CD4 T-cells upon activation (i.e., glycolytic switch) thereby contributing to their suppression. Further analyses demonstrated that CD4 T-cells also do not upregulate homing receptors associated with inflammatory processes. In contrast, expression of central memory-cell associated cell surface markers and transcription factors were increased by DN T-cells. Moreover, CD4 T-cells failed to produce inflammatory cytokines after co-culture with DN T-cells, whereas IL-2 secretion was enhanced. Taken together DN T-cells impair metabolic reprogramming of conventional CD4 T-cells by abrogating mTOR signaling, thereby modulating CD4 T-cell functionality. These results uncover a new mechanism of DN T-cell-mediated suppression, pointing out that DN T-cells could serve as cell-based therapy to limit alloreactive immune response.
Regulatory T cells play an important role in the maintenance of immune tolerance to self-antigens and are involved in modulating immune responses to promote resolution of inflammation. The population of TCRαβ+ CD4-/CD8- (double-negative, DN) T cells has attracted growing attention as a result of their potent immune regulatory function. In murine models, DN T cells were able to prevent rejection of allogeneic and xenogeneic organ grafts by effectively suppressing reactive T cells. In addition, DN T cells possess the capacity to resolve various inflammatory conditions, including graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation. Notably, first clinical studies in patients after stem cell transplantation demonstrated an inverse correlation between the frequency of circulating DN T cells and the severity of acute GvHD, suggesting a therapeutic potential of human DN T cells. To gain a better understanding of the molecular mechanism of suppression, we investigated whether human DN T cells modulate distinct signaling processes in conventional T cells. We found that DN T cells selectively block mTOR signaling but not activation of mitogen-activated protein kinases. Enforced activation of the mTOR pathway by a chemical activator rendered conventional T cells unsusceptible to DN T cell-mediated suppression, confirming the critical function of mTOR signaling. Given that mTOR is a major regulator of cellular metabolism, we further determined the impact of DN T cells on the metabolic framework of conventional T cells. Of interest, DN T cells diminished upregulation of the glycolytic machinery and glucose uptake in conventional T cells, whereas fatty acid uptake was not modified. Next, we investigated the fate and function of effector cells after DN T cell co-culture. Of importance, DN T cells suppressed proliferation but also altered expression of differentiation markers, transcription factors and homing receptors. Further analyses demonstrated that CD4+ T cells failed to produce effector cytokines IL-17 and IFN-γ after coculture with DN T cells, whereas IL-2 secretion was amplified. The selective modification was induced by a direct cell-cell contact dependent mechanism between CD4 and DN T cells and not as a consequence of competition for nutrients or growth factors. Together, our findings expand the understanding of DN T cell functionality and support that human DN T cells represent an interesting opportunity to limit and modulate T-cell reactivity. Disclosures No relevant conflicts of interest to declare.
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.
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