DAPK1 (death associated protein kinase 1) mediates mTORC1 activation and antiviral activities in CD8+ T cells

Mechanistic target of rapamycin complex 1 (mTORC1) regulates CD8⁺ T-cell differentiation and function. Despite the links between PI3K-AKT and mTORC1 activation in CD8⁺ T cells, the molecular mechanism underlying mTORC1 activation remains unclear. Here, we show that both the kinase activity and the death domain of DAPK1 are required for maximal mTOR activation and CD8⁺ T-cell function. We found that TCR-induced activation of calcineurin activates DAPK1, which subsequently interacts with TSC2 via its death domain and phosphorylates TSC2 to mediate mTORC1 activation. Furthermore, both the kinase domain and death domain of DAPK1 are required for CD8⁺ T-cell antiviral responses in an LCMV infection model. Together, our data reveal a novel mechanism of mTORC1 activation that mediates optimal CD8⁺ T-cell function and antiviral activity.     

Sin1 regulates Treg-cell development but is not required for T-cell growth and proliferation

Mammalian Sin1 plays key roles in the regulation of mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) signaling. Sin1 is an essential component of mTOR complex 2 (mTORC2). The functions of Sin1 and mTORC2 remain largely unknown in T cells. Here, we investigate Sin1 function in T cells using mice that lack Sin1 in the hematopoietic system. Sin1 deficiency blocks the mTORC2-dependent Akt phosphorylation in T cells during development and activation. Sin1-deficient T cells exhibit normal thymic cellularity and percentages of double-negative, double-positive, and single-positive CD4(+) and CD8(+) thymocytes. Sin1 deficiency does not impair T-cell receptor (TCR) induced growth and proliferation. Sin1 appears dispensable for in vitro CD4(+) helper cell differentiation. However, Sin1 deficiency results in an increased proportion of Foxp3(+) natural T-regulatory (nTreg) cells in the thymus. The TGF-β-dependent differentiation of CD4(+) T cells in vitro is enhanced by the inhibition of mTOR but not by loss of Sin1 function. Our results reveal that Sin1 and mTORC2 are dispensable for the development and activation of T cells but play a role in nTreg-cell differentiation.     

mTOR signaling at the crossroads of environmental signals and T-cell fate decisions

The evolutionarily conserved serine/threonine kinase mTOR (mechanistic target of rapamycin) forms the distinct protein complexes mTORC1 and mTORC2 and integrates signals from the environment to coordinate downstream signaling events and various cellular processes. T cells rely on mTOR activity for their development and to establish their homeostasis and functional fitness. Here, we review recent progress in our understanding of the upstream signaling and downstream targets of mTOR. We also provide an updated overview of the roles of mTOR in T-cell development, homeostasis, activation, and effector-cell fate decisions, as well as its important impacts on the suppressive activity of regulatory T cells. Moreover, we summarize the emerging roles of mTOR in T-cell exhaustion and transdifferentiation. A better understanding of the contribution of mTOR to T-cell fate decisions will ultimately aid in the therapeutic targeting of mTOR in human disease.     

Role of Tumor Suppressor TSC1 in Regulating Antigen-Specific Primary and Memory CD8 T Cell Responses to Bacterial Infection

The serine/threonine kinase mammalian/mechanistic target of rapamycin (mTOR) integrates various environmental cues such as the presence of antigen, inflammation, and nutrients to regulate T cell growth, metabolism, and function. The tuberous sclerosis 1 (TSC1)/TSC2 complex negatively regulates the activity of an mTOR-containing multiprotein complex called mTOR complex 1. Recent studies have revealed an essential cell-intrinsic role for TSC1 in T cell survival, quiescence, and mitochondrial homeostasis. Given the emerging role of mTOR activity in the regulation of the quantity and quality of CD8 T cell responses, in this study, we examine the role of its suppressor, TSC1, in the regulation of antigen-specific primary and memory CD8 T cell responses to bacterial infection. Using an established model system of transgenic CD8 cell adoptive transfer and challenge with Listeria monocytogenes expressing a cognate antigen, we found that TSC1 deficiency impairs antigen-specific CD8 T cell responses, resulting in weak expansion, exaggerated contraction, and poor memory generation. Poor expansion of TSC1-deficient cells was associated with defects in survival and proliferation in vivo, while enhanced contraction was correlated with an increased ratio of short-lived effectors to memory precursors in the effector cell population. This perturbation of effector-memory differentiation was concomitant with decreased expression of eomesodermin among activated TSC1 knockout cells. Upon competitive adoptive transfer with wild-type counterparts and antigen rechallenge, TSC1-deficient memory cells showed moderate defects in expansion but not cytokine production. Taken together, these findings provide direct evidence of a CD8 T cell-intrinsic role for TSC1 in the regulation of antigen-specific primary and memory responses.     

Shedding new light on neurodegenerative diseases through the mammalian target of rapamycin

Neurodegenerative disorders affect a significant portion of the world's population leading to either disability or death for almost 30 million individuals worldwide. One novel therapeutic target that may offer promise for multiple disease entities that involve Alzheimer's disease, Parkinson's disease, epilepsy, trauma, stroke, and tumors of the nervous system is the mammalian target of rapamycin (mTOR). mTOR signaling is dependent upon the mTORC1 and mTORC2 complexes that are composed of mTOR and several regulatory proteins including the tuberous sclerosis complex (TSC1, hamartin/TSC2, tuberin). Through a number of integrated cell signaling pathways that involve those of mTORC1 and mTORC2 as well as more novel signaling tied to cytokines, Wnt, and forkhead, mTOR can foster stem cellular proliferation, tissue repair and longevity, and synaptic growth by modulating mechanisms that foster both apoptosis and autophagy. Yet, mTOR through its proliferative capacity may sometimes be detrimental to central nervous system recovery and even promote tumorigenesis. Further knowledge of mTOR and the critical pathways governed by this serine/threonine protein kinase can bring new light for neurodegeneration and other related diseases that currently require new and robust treatments.     

The Role and Regulation of mTOR in T-Lymphocyte Function

The conversion of naïve T cells into effector T cells is initiated by stimulation through the T-cell receptor (TCR). Upon activation, T cells undergo significant morphological and functional changes, putting new metabolic demands on the cell. Past research has identified the mammalian target of rapamycin (mTOR) as a critical regulator of cell metabolism, and the development of new genetic models has begun to reveal an important role for this pathway in the homeostasis and function of T lymphocytes. In this review, we focus on the most recent findings that demonstrate the ability of mTOR to regulate T-cell activation, CD8⁺ memory cell formation and function, and helper T lineage differentiation. Furthermore, we highlight the importance of tight control of mTOR signaling by tuberous sclerosis complex 1 for T-cell homeostasis, and the regulation of mTOR signaling by diacylglycerol kinases and the RasGRP1-Ras-Erk1/2 pathway in the context of TCR signaling.     

Bcat1 is controlled by Tsc2/mTORC1 pathway at expression levels and its deficiency together with Bcat2 inactivation suppresses the growth of a Tsc2−/− tumor cell line

The tuberous sclerosis complex (TSC) gene products (TSC1/TSC2) negatively regulate mTORC1. Although mTORC1 inhibitors are used for the treatment of TSC, incomplete tumor elimination and the adverse effects from long-term administration are problems that need to be solved. Branched-chain amino acid (BCAA) metabolism is involved in the growth of many tumor cells via the mTORC1 pathway. However, it remains unclear how BCAA metabolism affects the growth of mTORC1-dysregulated tumors. We show here that the expression of branched-chain amino transferase1 (Bcat1) was suppressed in Tsc2-deficient murine renal tumor cells either by treatment with rapamycin or restoration of Tsc2 expression suggesting that Bcat1 is located downstream of Tsc2-mTORC1 pathway. We also found that gabapentin, a Bcat1 inhibitor suppressed the growth of Tsc2-deficient tumor cells and increased efficacy when combined with rapamycin. We investigate the functional importance of Bcat1 and the mitochondrial isoform Bcat2 by inhibiting each enzyme separately or both together by genome editing and shRNA in Tsc2-deficient cells. We found that deficiency of both enzymes, but not either alone, inhibited cell growth, indicating that BCAA-metabolic reactions support Tsc2-deficient cell proliferation. Our results indicate that inhibition of Bcat1 and Bcat2 by specific drugs should be a useful method for TSC treatment.     

Receptor signaling in immune cell development and function

Immune cell development and function must be tightly regulated through cell surface receptors to ensure proper responses to pathogen and tolerance to self. In T cells, the signal from the T-cell receptor is essential for T-cell maturation, homeostasis, and activation. In mast cells, the high-affinity receptor for IgE transduces signal that promotes mast cell survival and induces mast cell activation. In dendritic cells and macrophages, the toll-like receptors recognize microbial pathogens and play critical roles for both innate and adaptive immunity against pathogens. Our research explores how signaling from these receptors is transduced and regulated to better understand these immune cells. Our recent studies have revealed diacylglycerol kinases and TSC1/2-mTOR as critical signaling molecules/regulators in T cells, mast cells, dendritic cells, and macrophages.     

Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex

Mammalian target of rapamycin (mTOR) is a central regulator of protein synthesis whose activity is modulated by a variety of signals. Energy depletion and hypoxia result in mTOR inhibition. While energy depletion inhibits mTOR through a process involving the activation of AMP-activated protein kinase (AMPK) by LKB1 and subsequent phosphorylation of TSC2, the mechanism of mTOR inhibition by hypoxia is not known. Here we show that mTOR inhibition by hypoxia requires the TSC1/TSC2 tumor suppressor complex and the hypoxia-inducible gene REDD1/RTP801. Disruption of the TSC1/TSC2 complex through loss of TSC1 or TSC2 blocks the effects of hypoxia on mTOR, as measured by changes in the mTOR targets S6K and 4E-BP1, and results in abnormal accumulation of Hypoxia-inducible factor (HIF). In contrast to energy depletion, mTOR inhibition by hypoxia does not require AMPK or LKB1. Down-regulation of mTOR activity by hypoxia requires de novo mRNA synthesis and correlates with increased expression of the hypoxia-inducible REDD1 gene. Disruption of REDD1 abrogates the hypoxia-induced inhibition of mTOR, and REDD1 overexpression is sufficient to down-regulate S6K phosphorylation in a TSC1/TSC2-dependent manner. Inhibition of mTOR function by hypoxia is likely to be important for tumor suppression as TSC2-deficient cells maintain abnormally high levels of cell proliferation under hypoxia.     

mTOR signaling in renal ion transport

The mammalian target of rapamycin (mTOR) signaling pathway is crucial in maintaining cell growth and metabolism. The mTOR protein kinase constitutes the catalytic subunit of two multimeric protein complexes called mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). As such, this pathway is indispensable for many organs, including the kidney. Since its discovery, mTOR has been associated with major renal disorders such as acute kidney injury, chronic kidney disease, and polycystic kidney disease. On top of that, emerging studies using pharmacological interventions and genetic disease models have unveiled mTOR role in renal tubular ion handling. Along the tubule, mTORC1 and mTORC2 subunits are ubiquitously expressed at mRNA level. Nevertheless, at the protein level, current studies suggest that a tubular segment-specific balance between mTORC1 and mTORC2 exists. In the proximal tubule, mTORC1 regulates nutrients transports through various transporters located in this segment. On the other hand, in the thick ascending limb of the loop of Henle, both complexes play a role in regulating NKCC2 expression and activity. Lastly, in the principal cells of the collecting duct, mTORC2 determines Na⁺ reabsorption and K⁺ excretion by regulating of SGK1 activation. Altogether, these studies establish the relevance of the mTOR signaling pathway in the pathophysiology of tubular solute transport. Despite extensive studies on the effectors of mTOR, the upstream activators of mTOR signaling remain elusive in most nephron segments. Further understanding of the role of growth factor signaling and nutrient sensing is essential to establish the exact role of mTOR in kidney physiology.     

CD8+ T cell survival in lethal fungal sepsis was ameliorated by T-cell-specific mTOR deletion

Lethal fungal sepsis causes high morbidity and mortality in intensive care patients. Fungal infections have an immunological basis, and it has been shown in recent studies that decreased CD8⁺ T-cell count in fungal infections is related to prognosis, while the underlying mechanism is still unclear. Here, a lethal fungal sepsis model induced by candidemia was created and we found a decreased CD8⁺ T-cell count and exaggerated apoptosis. Simultaneously, expression of light chain (LC)3B in CD8⁺ T cells increased, along with increased autophagosomes and accumulation of p62 in infected mice. We regulated the activity of the mammalian target of rapamycin (mTOR) pathway using T-cell-specific mTOR/ TSC1 deletion mice. We observed increased number of autophagosomes and expression of LC3B in CD8⁺T cells after T-cell-specific mTOR knockout, while accumulation of p62 was not ameliorated, and there was no increase in the number of autolysosomes. Apoptosis rate and expression of BIM, a pro-apoptotic gene, decreased in CD8⁺ T cells in mTOR-deletion mice but increased in TSC1-deletion mice. Our results showed increased CD8⁺ T-cell death in spleen of lethal fungal sepsis mice, and decreased expression of mTOR ameliorated CD8⁺ T-cell survival. mTOR may be a possible target to reverse CD8⁺ T-cell immune dysfunction in lethal fungal sepsis.     

The kinase mTOR regulates the differentiation of helper T cells through the selective activation of signaling by mTORC1 and mTORC2

The kinase mTOR has emerged as an important regulator of the differentiation of helper T cells. Here we demonstrate that differentiation into the T(H)1 and T(H)17 subsets of helper T cells was selectively regulated by signaling from mTOR complex 1 (mTORC1) that was dependent on the small GTPase Rheb. Rheb-deficient T cells failed to generate T(H)1 and T(H)17 responses in vitro and in vivo and did not induce classical experimental autoimmune encephalomyelitis (EAE). However, they retained their ability to become T(H)2 cells. Alternatively, when mTORC2 signaling was deleted from T cells, they failed to generate T(H)2 cells in vitro and in vivo but preserved their ability to become T(H)1 and T(H)17 cells. Our data identify mechanisms by which two distinct signaling pathways downstream of mTOR regulate helper cell fate in different ways. These findings define a previously unknown paradigm that links T cell differentiation with selective metabolic signaling pathways.     

Systems biology of lupus: Mapping the impact of genomic and environmental factors on gene expression signatures,cellular signaling,metabolic pathways,hormonal and cytokine imbalance,and selecting targets for treatment

Systemic lupus erythematosus (SLE) is characterized by the dysfunction of T cells, B cells, and dendritic cells, the release of pro-inflammatory nuclear materials from necrotic cells, and the formation of antinuclear antibodies (ANA) and immune complexes of ANA with DNA, RNA, and nuclear proteins. Activation of the mammalian target of rapamycin (mTOR) has recently emerged as a key factor in abnormal activation of T and B cells in SLE. In T cells, increased production of nitric oxide and mitochondrial hyperpolarization (MHP) were identified as metabolic checkpoints upstream of mTOR activation. mTOR controls the expression T-cell receptor-associated signaling proteins CD4 and CD3ζ through increased expression of the endosome recycling regulator Rab5 and HRES-1/Rab4 genes, enhances Ca²⁺ fluxing and skews the expression of tyrosine kinases both in T and B cells, and blocks the expression of Foxp3 and the generation of regulatory T cells. MHP, increased activity of mTOR, Rab GTPases, and Syk kinases, and enhanced Ca²⁺ flux have emerged as common T and B cell biomarkers and targets for treatment in SLE.     

mTOR signaling mediates ILC3-driven immunopathology

Innate lymphoid cells (ILCs) have a protective immune function at mucosal tissues but can also contribute to immunopathology. Previous work has shown that the serine/threonine kinase mammalian target of rapamycin complex 1 (mTORC1) is involved in generating protective ILC3 cytokine responses during bacterial infection. However, whether mTORC1 also regulates IFN-γ-mediated immunopathology has not been investigated. In addition, the role of mTORC2 in ILC3s is unknown. Using mice specifically defective for either mTORC1 or mTORC2 in ILC3s, we show that both mTOR complexes regulate the maintenance of ILC3s at steady state and pathological immune response during colitis. mTORC1 and to a lesser extend mTORC2 promote the proliferation of ILC3s in the small intestine. Upon activation, intestinal ILC3s produce less IFN-γ in the absence of mTOR signaling. During colitis, loss of both mTOR complexes in colonic ILC3s results in the reduced production of inflammatory mediators, recruitment of neutrophils and immunopathology. Similarly, treatment with rapamycin after colitis induction ameliorates the disease. Collectively, our data show a critical role for both mTOR complexes in controlling ILC3 cell numbers and ILC3-driven inflammation in the intestine.     

Activation of the mTOR pathway in sporadic angiomyolipomas and other perivascular epithelioid cell neoplasms

Angiomyolipoma (AML) belong to a family of tumors known as perivascular epithelioid cell tumors (PEComas) that share a common immunophenotypic profile of muscle and melanocytic differentiation. These tumors are clonal in nature and have a strong association with tuberous sclerosis. Genetic analyses have reported allelic imbalance at the TSC2 locus on 16p13. In the context of non-tuberous sclerosis complex (TSC), non-lymphangioleiomyomatosis-associated AMLs, and non-renal PEComas, the functional status of the TSC2 signaling pathway has not been reported. Studies over the last several years have uncovered a critical role of the TSC1/2 genes in negatively regulating the Rheb/mTOR/p70S6K cascade. Here, we examined the activity of this pathway in sporadic AMLs and PEComas using immunohistochemical and biochemical analyses. We found increased levels of phospho-p70S6K, a marker of mTOR activity, in 15 of 15 non-TSC AMLs. This was accompanied by reduced phospho-AKT expression, a pattern that is consistent with the disruption of TSC1/2 function. Western blot analysis confirmed mTOR activation concurrent with the loss of TSC2 and not TSC1 in sporadic AMLs. Similarly, elevated phospho-p70S6K and reduced phospho-AKT expression was detected in 14 of 15 cases of extrarenal PEComas. These observations provide the first functional evidence that mTOR activation is common to sporadic, non-TSC-related AMLs and PEComas. This suggests the possibility that mTOR inhibitors such as rapamycin may be therapeutic for this class of disease.     

Metabolic control of T cell activation and death in SLE

Systemic lupus erythematosus (SLE) is characterized by abnormal T cell activation and death, processes which are crucially dependent on the controlled production of reactive oxygen intermediates (ROI) and of ATP in mitochondria. The mitochondrial transmembrane potential (Deltapsi(m)) has conclusively emerged as a critical checkpoint of ATP synthesis and cell death. Lupus T cells exhibit persistent elevation of Deltapsi(m) or mitochondrial hyperpolarization (MHP) as well as depletion of ATP and glutathione which decrease activation-induced apoptosis and instead predispose T cells for necrosis, thus stimulating inflammation in SLE. NO-induced mitochondrial biogenesis in normal T cells accelerates the rapid phase and reduces the plateau of Ca(2+) influx upon CD3/CD28 co-stimulation, thus mimicking the Ca(2+) signaling profile of lupus T cells. Treatment of SLE patients with rapamycin improves disease activity, normalizes CD3/CD28-induced Ca(2+) fluxing but fails to affect MHP, suggesting that altered Ca(2+) fluxing is downstream or independent of mitochondrial dysfunction. Understanding the molecular basis and consequences of MHP is essential for controlling T cell activation and death signaling in SLE.     

Resistance exercise, but not endurance exercise, induces IKKβ phosphorylation in human skeletal muscle of training-accustomed individuals

The mammalian target of rapamycin complex 1 (mTORC1) is considered an important role in the muscular adaptations to exercise. It has been proposed that exercise-induced signaling to mTORC1 do not require classic growth factor PI3K/Akt signaling. Activation of IKKβ and the mitogen-activated protein kinases (MAPKs) Erk1/2 and p38 has been suggested to link inflammation and cellular stress to activation of mTORC1 through the tuberous sclerosis 1 (TSC1)/tuberous sclerosis 2 (TSC2) complex. Consequently, activation of these proteins constitutes potential alternative mechanisms of mTORC1 activation following exercise. Previously, we demonstrated that mTOR is preferentially activated in response to resistance exercise compared to endurance exercise in trained individuals without concomitant activation of Akt. In the present study, we extended this investigation by examining IκB kinase complex (IKK), TSC1, MAPK, and upstream Akt activators, along with gene expression of selected cytokines, in skeletal muscles from these subjects. Biopsies were sampled prior to, immediately after, and in the recovery period following resistance exercise, endurance exercise, and control interventions. The major finding was that IKKβ phosphorylation increased exclusively after resistance exercise. No changes in TSC1, Erk1/2, insulin receptor, or insulin receptor substrate 1 phosphorylation were observed in any of the groups, while p38 phosphorylation was higher in the resistance exercise group compared to both other groups immediately after the intervention. Resistance and endurance exercise increased IL6, IL8, and TNFα gene expression immediately after exercise. The non-exercise control group demonstrated that cytokine gene expression is also sensitive to repeated biopsy sampling, whereas no effect of repeated biopsy sampling on protein expression and phosphorylation was observed. In conclusion, resistance exercise, but not endurance exercise, increases IKKβ phosphorylation in trained human subjects, which support the idea that IKKβ can influence the activation of mTORC1 in human skeletal muscle.     

mTOR抑制剂一雷帕霉素对T细胞功能调控的研究进展

哺乳动物雷帕霉素靶蛋白（mTOR）作为信号通路的调节分子参与多条重要的信号转导通．路,能形成细胞对多种刺激的应答,mTOR至少存在两种功能性多蛋白复合物形式：mTORCl和mTORC2,其可发挥不同的生物学作用。雷帕霉素作为mTOR的特异性抑制剂可阻断mTOR信号通路信息的传导,调节T细胞的分化、发育、失能以及调节性T细胞（Treg）的增殖和功能,影响生长因子和细胞因子等生物活性物质的分泌,表现出有效的免疫抑制作用。