Activated T cells produce reactive oxygen species (ROS), which trigger the antioxidative glutathione (GSH) response necessary to buffer rising ROS and prevent cellular damage. We report that GSH is essential for T cell effector functions through its regulation of metabolic activity. Conditional gene targeting of the catalytic subunit of glutamate cysteine ligase (Gclc) blocked GSH production specifically in murine T cells. Gclc-deficient T cells initially underwent normal activation but could not meet their increased energy and biosynthetic requirements. GSH deficiency compromised the activation of mammalian target of rapamycin-1 (mTOR) and expression of NFAT and Myc transcription factors, abrogating the energy utilization and Myc-dependent metabolic reprogramming that allows activated T cells to switch to glycolysis and glutaminolysis. In vivo, T-cell-specific ablation of murine Gclc prevented autoimmune disease but blocked antiviral defense. The antioxidative GSH pathway thus plays an unexpected role in metabolic integration and reprogramming during inflammatory T cell responses.

Isocitrate dehydrogenase-1 (IDH1) R132 mutations occur in glioma, but their physiological significance is unknown. Here we describe the generation and characterization of brain-specific Idh1 R132H conditional knockin (KI) mice. Idh1 mutation results in hemorrhage and perinatal lethality. Surprisingly, intracellular reactive oxygen species (ROS) are attenuated in Idh1-KI brain cells despite an apparent increase in the NADP + /NADPH ratio. Idh1-KI cells also show high levels of D-2-hydroxyglutarate (D2HG) that are associated with inhibited prolyl-hydroxylation of hypoxia-inducible transcription factor-1a (Hif1a) and up-regulated Hif1a target gene transcription. Intriguingly, D2HG also blocks prolyl-hydroxylation of collagen, causing a defect in collagen protein maturation. An endoplasmic reticulum (ER) stress response induced by the accumulation of immature collagens may account for the embryonic lethality of these mutants. Importantly, D2HG-mediated impairment of collagen maturation also led to basement membrane (BM) aberrations that could play a part in glioma progression. Our study presents strong in vivo evidence that the D2HG produced by the mutant Idh1 enzyme is responsible for the above effects.

We previously reported that resident γδ T cells in the peritoneal cavity rapidly produced IL-17 in response to Escherichia coli infection to mobilize neutrophils. We found in this study that the IL-17-producing γδ T cells did not produce IFN-γ or IL-4, similar to Th17 cells. IL-17-producing γδ T cells specifically express CD25 but not CD122, whereas CD122+ γδ T cells produced IFN-γ. IL-17-producing γδ T cells were decreased but still present in IL-2- or CD25-deficient mice, suggesting a role of IL-2 for their maintenance. IFN-γ-producing CD122+ γδ T cells were selectively decreased in IL-15-deficient mice. Surprisingly, IL-17-producing γδ T cells were already detected in the thymus, although CD25 was not expressed on the intrathymic IL-17-producing γδ T cells. The number of thymic IL-17-producing γδ T cells was peaked at perinatal period and decreased thereafter, coincided with the developmental kinetics of Vγ6+Vδ1+ γδ T cells. The number of IL-17-producing γδ T cells was decreased in fetal thymus of Vδ1-deficient mice, whereas Vγ5+ fetal thymocytes in normal mice did not produce IL-17. Thus, it was revealed that the fetal thymus-derived Vγ6+Vδ1+ T cells functionally differentiate to produce IL-17 within thymus and thereafter express CD25 to be maintained in the periphery.

Tumorigenesis results from dysregulation of oncogenes and tumor suppressors that influence cellular proliferation, differentiation, apoptosis, and/or senescence. Many gene products involved in these processes are substrates of the E3 ubiquitin ligase Mule/Huwe1/Arf-BP1 (Mule), but whether Mule acts as an oncogene or tumor suppressor in vivo remains controversial. We generated K14Cre;Mule flox/flox(y) (Mule kKO) mice and subjected them to DMBA/PMAinduced skin carcinogenesis, which depends on oncogenic Ras signaling. Mule deficiency resulted in increased penetrance, number, and severity of skin tumors, which could be reversed by concomitant genetic knockout of c-Myc but not by knockout of p53 or p19Arf. Notably, in the absence of Mule, c-Myc/Miz1 transcriptional complexes accumulated, and levels of p21CDKN1A (p21) and p15INK4B (p15) were down-regulated. In vitro, Muledeficient primary keratinocytes exhibited increased proliferation that could be reversed by Miz1 knockdown. Transfer of Mule-deficient transformed cells to nude mice resulted in enhanced tumor growth that again could be abrogated by Miz1 knockdown. Our data demonstrate in vivo that Mule suppresses Ras-mediated tumorigenesis by preventing an accumulation of c-Myc/Miz1 complexes that mediates p21 and p15 down-regulation.