CD4(+) regulatory T cells (T(reg) cells) characterized by expression of the transcription factor Foxp3 have a pivotal role in maintaining immunological tolerance. Here we show that mice with T cell-specific deletion of both the Foxo1 and Foxo3 transcription factors (collectively called 'Foxo proteins' here) developed a fatal multifocal inflammatory disorder due in part to T(reg) cell defects. Foxo proteins functioned in a T(reg) cell-intrinsic manner to regulate thymic and transforming growth factor-beta (TGF-beta)-induced Foxp3 expression, in line with the ability of Foxo proteins to bind to Foxp3 locus and control Foxp3 promoter activity. Transcriptome analyses showed that Foxo proteins regulated the expression of additional T(reg) cell-associated genes and were essential for inhibiting the acquisition of effector T cell characteristics by T(reg) cells. Thus, Foxo proteins have crucial roles in specifying the T(reg) cell lineage.

Regulatory T (Treg) cells, characterized by expression of the transcription factor forkhead box P3 (Foxp3), maintain immune homeostasis by suppressing self-destructive immune responses1–4. Foxp3 operates as a late-acting differentiation factor controlling Treg cell homeostasis and function5, whereas the early Treg-cell-lineage commitment is regulated by the Akt kinase and the forkhead box O (Foxo) family of transcription factors6–10. However, whether Foxo proteins act beyond the Treg-cell-commitment stage to control Treg cell homeostasis and function remains largely unexplored. Here we show that Foxo1 is a pivotal regulatorof Treg cell function. Treg cells express high amounts of Foxo1 and display reduced T-cell-receptor-induced Akt activation, Foxo1 phosphorylation and Foxo1 nuclear exclusion. Mice with Treg-cell-specific deletion of Foxo1 develop a fatal inflammatory disorder similar in severity to that seen in Foxp3-deficient mice, but without the loss of Treg cells. Genome-wide analysis of Foxo1 binding sites reveals ~300 Foxo1-bound target genes, including the pro-inflammatory cytokine Ifng, that do not seem to be directly regulated by Foxp3. These findings show that the evolutionarily ancient Akt–Foxo1 signalling module controls a novel genetic program indispensable for Treg cell function.

Summary TGFβ1 is a regulatory cytokine that has an important role in controlling T cell differentiation. T cell-produced TGFβ1 acts on T cells to promote Th17 cell differentiation and the development of experimental autoimmune encephalomyelitis (EAE). However, the exact TGFβ1-producing T cell subset required for Th17 cell generation and its cellular mechanism of action remain unknown. Here we showed that deletion of the Tgfb1 gene from activated T cells and Treg cells, but not Treg cells alone, abrogated Th17 cell differentiation resulting in almost complete protection from EAE. Furthermore, differentiation of T cells both in vitro and in vivo demonstrated that TGFβ1 was highly expressed by Th17 cells and acted in a predominantly autocrine manner to maintain Th17 cells in vivo. These findings reveal an essential role for activated T cell-produced TGFβ1 in promoting the differentiation of Th17 cells and controlling inflammatory diseases.

Summary Thymus-derived naturally occurring regulatory T cells (nTregs) are necessary for immunological self-tolerance. nTreg development is instructed by the T cell receptor, and can be induced by agonist antigens that trigger T cell negative selection. How T cell deletion is regulated so that nTregs are generated is unclear. Here we showed that transforming growth factor-β (TGF-β) signaling protected nTregs and antigen-stimulated conventional T cells from apoptosis. Enhanced apoptosis of TGF-β receptor-deficient nTregs was associated with high expression of pro-apoptotic proteins Bim, Bax, and Bak, and low expression of the anti-apoptotic protein Bcl-2. Ablation of Bim in mice corrected the Treg development and homeostasis defects. Our results suggest that nTreg commitment is independent of TGF-β signaling. Instead, TGF-β promotes nTreg survival by antagonizing T cell negative selection. These findings reveal a critical function for TGF-β in control of autoreactive T cell fates with important implications for understanding T cell self-tolerance mechanisms.

Background Dihydroartemisinin (DHA) has been shown to exert anticancer activity through iron-dependent reactive oxygen species (ROS) generation, which is similar to ferroptosis, a novel form of cell death. However, whether DHA causes ferroptosis in glioma cells and the potential regulatory mechanisms remain unclear. Methods Effects of DHA on the proliferation, cell death, ROS and lipid ROS generation as well as reduced gluthione consumption were assessed in glioma cells with or without ferroptosis inhibitor. The biological mechanisms by which glioma cells attenuate the pro-ferroptotic effects of DHA were assessed using molecular methods. Results DHA induced ferroptosis in glioma cells, as characterized by iron-dependent cell death accompanied with ROS generation and lipid peroxidation. However, DHA treatment simultaneously activated a feedback pathway of ferroptosis by increasing the expression of heat shock protein family A (Hsp70) member 5 (HSPA5). Mechanistically, DHA caused endoplasmic reticulum (ER) stress in glioma cells, which resulted in the induction of HSPA5 expression by protein kinase R-like ER kinase (PERK)-upregulated activating transcription factor 4 (ATF4). Subsequent HSPA5 upregulation increased the expression and activity of glutathione peroxidase 4 (GPX4), which neutralized DHA-induced lipid peroxidation and thus protected glioma cells from ferroptosis. Inhibition of the PERK-ATF4-HSPA5-GPX4 pathway using siRNA or small molecules increased DHA sensitivity of glioma cells by increasing ferroptosis both in vitro and in vivo. Conclusions Collectively, these data suggested that ferroptosis might be a novel anticancer mechanism of DHA in glioma and HSPA5 may serve as a negative regulator of DHA-induced ferroptosis. Therefore, inhibiting the negative feedback pathway would be a promising therapeutic strategy to strengthen the anti-glioma activity of DHA.