Regulation of immune responses by mTOR.

mTOR is an evolutionarily conserved serine/threonine kinase that plays a central role in integrating environmental cues in the form of growth factors, amino acids, and energy. In the study of the immune system, mTOR is emerging as a critical regulator of immune function because of its role in sensing and integrating cues from the immune microenvironment. With the greater appreciation of cellular metabolism as an important regulator of immune cell function, mTOR is proving to be a vital link between immune function and metabolism. In this review, we discuss the ability of mTOR to direct the adaptive immune response. Specifically, we focus on the role of mTOR in promoting differentiation, activation, and function in T cells, B cells, and antigen-presenting cells.

Acid Suspends the Circadian Clock in Hypoxia through Inhibition of mTOR.

Recent reports indicate that hypoxia influences the circadian clock through the transcriptional activities of hypoxia-inducible factors (HIFs) at clock genes. Unexpectedly, we uncover a profound disruption of the circadian clock and diurnal transcriptome when hypoxic cells are permitted to acidify to recapitulate the tumor microenvironment. Buffering against acidification or inhibiting lactic acid production fully rescues circadian oscillation. Acidification of several human and murine cell lines, as well as primary murine T cells, suppresses mechanistic target of rapamycin complex 1 (mTORC1) signaling, a key regulator of translation in response to metabolic status. We find that acid drives peripheral redistribution of normally perinuclear lysosomes away from perinuclear RHEB, thereby inhibiting the activity of lysosome-bound mTOR. Restoring mTORC1 signaling and the translation it governs rescues clock oscillation. Our findings thus reveal a model in which acid produced during the cellular metabolic response to hypoxia suppresses the circadian clock through diminished translation of clock constituents.

Asymmetric inheritance of mTORC1 kinase activity during division dictates CD8(+) T cell differentiation.

The asymmetric partitioning of fate-determining proteins has been shown to contribute to the generation of CD8(+) effector and memory T cell precursors. Here we demonstrate the asymmetric partitioning of mTORC1 activity after the activation of naive CD8(+) T cells. This results in the generation of two daughter T cells, one of which shows increased mTORC1 activity, increased glycolytic activity and increased expression of effector molecules. The other daughter T cell has relatively low mTORC1 activity and increased lipid metabolism, expresses increased amounts of anti-apoptotic molecules and subsequently displays enhanced long-term survival. Mechanistically, we demonstrate a link between T cell antigen receptor (TCR)-induced asymmetric expression of amino acid transporters and RagC-mediated translocation of mTOR to the lysosomes. Overall, our data provide important insight into how mTORC1-mediated metabolic reprogramming affects the fate decisions of T cells.

An engineered IL-2 partial agonist promotes CD8+ T cell stemness.

Adoptive transfer of antigen-specific T cells represents a major advance in cancer immunotherapy, with robust clinical outcomes in some patients1. Both the number of transferred T cells and their differentiation state are critical determinants of effective responses2,3. T cells can be expanded with T cell receptor (TCR)-mediated stimulation and interleukin-2, but this can lead to differentiation into effector T cells4,5 and lower therapeutic efficacy6, whereas maintenance of a more stem-cell-like state before adoptive transfer is beneficial7. Here we show that H9T, an engineered interleukin-2 partial agonist, promotes the expansion of CD8+ T cells without driving terminal differentiation. H9T led to altered STAT5 signalling and mediated distinctive downstream transcriptional, epigenetic and metabolic programs. In addition, H9T treatment sustained the expression of T cell transcription factor 1 (TCF-1) and promoted mitochondrial fitness, thereby facilitating the maintenance of a stem-cell-like state. Moreover, TCR-transgenic and chimeric antigen receptor-modified CD8+ T cells that were expanded with H9T showed robust anti-tumour activity in vivo in mouse models of melanoma and acute lymphoblastic leukaemia. Thus, engineering cytokine variants with distinctive properties is a promising strategy for creating new molecules with translational potential.

The AGC kinase SGK1 regulates TH1 and TH2 differentiation downstream of the mTORC2 complex.

SGK1 is an AGC kinase that regulates the expression of membrane sodium channels in renal tubular cells in a manner dependent on the metabolic checkpoint kinase complex mTORC2. We hypothesized that SGK1 might represent an additional mTORC2-dependent regulator of the differentiation and function of T cells. Here we found that after activation by mTORC2, SGK1 promoted T helper type 2 (TH2) differentiation by negatively regulating degradation of the transcription factor JunB mediated by the E3 ligase Nedd4-2. Simultaneously, SGK1 repressed the production of interferon-γ (IFN-γ) by controlling expression of the long isoform of the transcription factor TCF-1. Consistent with those findings, mice with selective deletion of SGK1 in T cells were resistant to experimentally induced asthma, generated substantial IFN-γ in response to viral infection and more readily rejected tumors.

Natural and inducible TH17 cells are regulated differently by Akt and mTOR pathways.

Natural T helper 17 (nTH17) cells are a population of interleukin 17 (IL-17)-producing cells that acquire effector function in the thymus during development. Here we demonstrate that the serine/threonine kinase Akt has a critical role in regulating nTH17 cell development. Although Akt and the downstream mTORC1-ARNT-HIFα axis were required for generation of inducible TH17 (iTH17) cells, nTH17 cells developed independently of mTORC1. In contrast, mTORC2 and inhibition of Foxo proteins were critical for development of nTH17 cells. Moreover, distinct isoforms of Akt controlled the generation of TH17 cell subsets, as deletion of Akt2, but not of Akt1, led to defective generation of iTH17 cells. These findings define mechanisms regulating nTH17 cell development and reveal previously unknown roles of Akt and mTOR in shaping subsets of T cells.

SIKs Regulate HDAC7 Stabilization and Cytokine Recall in Late-Stage T Cell Effector Differentiation.

Understanding the mechanisms underlying the acquisition and maintenance of effector function during T cell differentiation is important to unraveling how these processes can be dysregulated in the context of disease and manipulated for therapeutic intervention. In this study, we report the identification of a previously unappreciated regulator of murine T cell differentiation through the evaluation of a previously unreported activity of the kinase inhibitor, BioE-1197. Specifically, we demonstrate that liver kinase B1 (LKB1)-mediated activation of salt-inducible kinases epigenetically regulates cytokine recall potential in effector CD8+ and Th1 cells. Evaluation of this phenotype revealed that salt-inducible kinase-mediated phosphorylation-dependent stabilization of histone deacetylase 7 (HDAC7) occurred during late-stage effector differentiation. HDAC7 stabilization increased nuclear HDAC7 levels, which correlated with total and cytokine loci-specific reductions in the activating transcription mark histone 3 lysine 27 acetylation (H3K27Ac). Accordingly, HDAC7 stabilization diminished transcriptional induction of cytokine genes upon restimulation. Inhibition of this pathway during differentiation produced effector T cells epigenetically poised for enhanced cytokine recall. This work identifies a previously unrecognized target for enhancing effector T cell functionality.

PKG1-modified TSC2 regulates mTORC1 activity to counter adverse cardiac stress.

The mechanistic target of rapamycin complex-1 (mTORC1) coordinates regulation of growth, metabolism, protein synthesis and autophagy1. Its hyperactivation contributes to disease in numerous organs, including the heart1,2, although broad inhibition of mTORC1 risks interference with its homeostatic roles. Tuberin (TSC2) is a GTPase-activating protein and prominent intrinsic regulator of mTORC1 that acts through modulation of RHEB (Ras homologue enriched in brain). TSC2 constitutively inhibits mTORC1; however, this activity is modified by phosphorylation from multiple signalling kinases that in turn inhibits (AMPK and GSK-3ß) or stimulates (AKT, ERK and RSK-1) mTORC1 activity3-9. Each kinase requires engagement of multiple serines, impeding analysis of their role in vivo. Here we show that phosphorylation or gain- or loss-of-function mutations at either of two adjacent serine residues in TSC2 (S1365 and S1366 in mice; S1364 and S1365 in humans) can bidirectionally control mTORC1 activity stimulated by growth factors or haemodynamic stress, and consequently modulate cell growth and autophagy. However, basal mTORC1 activity remains unchanged. In the heart, or in isolated cardiomyocytes or fibroblasts, protein kinase G1 (PKG1) phosphorylates these TSC2 sites. PKG1 is a primary effector of nitric oxide and natriuretic peptide signalling, and protects against heart disease10-13. Suppression of hypertrophy and stimulation of autophagy in cardiomyocytes by PKG1 requires TSC2 phosphorylation. Homozygous knock-in mice that express a phosphorylation-silencing mutation in TSC2 (TSC2(S1365A)) develop worse heart disease and have higher mortality after sustained pressure overload of the heart, owing to mTORC1 hyperactivity that cannot be rescued by PKG1 stimulation. However, cardiac disease is reduced and survival of heterozygote Tsc2S1365A knock-in mice subjected to the same stress is improved by PKG1 activation or expression of a phosphorylation-mimicking mutation (TSC2(S1365E)). Resting mTORC1 activity is not altered in either knock-in model. Therefore, TSC2 phosphorylation is both required and sufficient for PKG1-mediated cardiac protection against pressure overload. The serine residues identified here provide a genetic tool for bidirectional regulation of the amplitude of stress-stimulated mTORC1 activity.

Signatures of GVHD and relapse after posttransplant cyclophosphamide revealed by immune profiling and machine learning.

The key immunologic signatures associated with clinical outcomes after posttransplant cyclophosphamide (PTCy)-based HLA-haploidentical (haplo) and HLA-matched bone marrow transplantation (BMT) are largely unknown. To address this gap in knowledge, we used machine learning to decipher clinically relevant signatures from immunophenotypic, proteomic, and clinical data and then examined transcriptome changes in the lymphocyte subsets that predicted major posttransplant outcomes. Kinetics of immune subset reconstitution after day 28 were similar for 70 patients undergoing haplo and 75 patients undergoing HLA-matched BMT. Machine learning based on 35 candidate factors (10 clinical, 18 cellular, and 7 proteomic) revealed that combined elevations in effector CD4+ conventional T cells (Tconv) and CXCL9 at day 28 predicted acute graft-versus-host disease (aGVHD). Furthermore, higher NK cell counts predicted improved overall survival (OS) due to a reduction in both nonrelapse mortality and relapse. Transcriptional and flow-cytometric analyses of recovering lymphocytes in patients with aGVHD identified preserved hallmarks of functional CD4+ regulatory T cells (Tregs) while highlighting a Tconv-driven inflammatory and metabolic axis distinct from that seen with conventional GVHD prophylaxis. Patients developing early relapse displayed a loss of inflammatory gene signatures in NK cells and a transcriptional exhaustion phenotype in CD8+ T cells. Using a multimodality approach, we highlight the utility of systems biology in BMT biomarker discovery and offer a novel understanding of how PTCy influences alloimmune responses. Our work charts future directions for novel therapeutic interventions after these increasingly used GVHD prophylaxis platforms. Specimens collected on NCT0079656226 and NCT0080927627 https://clinicaltrials.gov/.

Cutting Edge: mTORC2 Regulates CD8+ Effector and Memory T Cell Differentiation through Serum and Glucocorticoid Kinase 1.

The mechanistic target of rapamycin is an essential regulator of T cell metabolism and differentiation. In this study, we demonstrate that serum- and glucocorticoid-regulated kinase 1 (SGK1), a downstream node of mechanistic target of rapamycin complex 2 signaling, represses memory CD8+ T cell differentiation. During acute infections, murine SGK1-deficient CD8+ T cells adopt an early memory precursor phenotype leading to more long-lived memory T cells. Thus, SGK1-deficient CD8+ T cells demonstrate an enhanced recall capacity in response to reinfection and can readily reject tumors. Mechanistically, activation of SGK1-deficient CD8+ T cells results in decreased Foxo1 phosphorylation and increased nuclear translocation of Foxo1 to promote early memory development. Overall, SGK1 might prove to be a powerful target for enhancing the efficacy of vaccines and tumor immunotherapy.

Inhibition of the Adenosine Pathway to Potentiate Cancer Immunotherapy: Potential for Combinatorial Approaches.

Cancer immunotherapy has revolutionized the way that we think about treating cancer. Although checkpoint blockade therapy, including anti-PD-1/PD-L1 and anti-CTLA-4, has shown remarkable success, the responses are limited to only a subset of patients. This discrepancy highlights the many overlapping avenues for immune evasion or suppression that can be employed by a tumor. One such mechanism of immunosuppression is adenosinergic signaling within the tumor microenvironment. We provide an overview of the current status of clinical trials targeting the adenosine pathway, including CD73, CD39, and adenosine receptors. Additionally, we highlight several avenues that may be explored to further potentiate responses in the clinic by combining adenosine-targeting agents to target multiple arms of the pathway or by using conventional immunotherapy agents.

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.

mTORC1 Signaling Regulates Proinflammatory Macrophage Function and Metabolism.

Metabolic programming is integrally linked to immune cell function. Nowhere is this clearer than in the differentiation of macrophages. Proinflammatory M1 macrophages primarily use glycolysis as a rapid energy source but also to generate antimicrobial compounds, whereas alternatively activated M2 macrophages primarily rely on oxidative phosphorylation for the longevity required for proper wound healing. mTOR signaling has been demonstrated to be a key regulator of immune cell metabolism and function. mTORC2 signaling is required for the generation of M2 macrophages, whereas the role of mTORC1 signaling, a key regulator of glycolysis, has been controversial. By using genetic deletion of mTORC1 signaling in C57BL/6 mouse macrophages, we observed enhanced M1 macrophage function in vitro and in vivo. Surprisingly, this enhancement occurred despite a significant defect in M1 macrophage glycolytic metabolism. Mechanistically, enhanced M1 function occurred because of inhibition of the class III histone deacetylases the sirtuins, resulting in enhanced histone acetylation. Our findings provide a counterpoint to the paradigm that enhanced immune cell function must occur in the presence of increased cellular metabolism and identifies a potential, pharmacologic target for the regulation of inflammatory responses.

Fueling memories.

A hallmark of the adaptive immune response is rapid and robust activation upon rechallenge. In the current issue of Immunity, van der Windt et al. (2012) provide an important link between mitochondrial respiratory capacity and the development of CD8(+) T cell memory.

MRI demonstrates glutamine antagonist-mediated reversal of cerebral malaria pathology in mice.

The deadliest complication of Plasmodium falciparum infection is cerebral malaria (CM), with a case fatality rate of 15 to 25% in African children despite effective antimalarial chemotherapy. No adjunctive treatments are yet available for this devastating disease. We previously reported that the glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) rescued mice from experimental CM (ECM) when administered late in the infection, a time by which mice had already suffered blood-brain barrier (BBB) dysfunction, brain swelling, and hemorrhaging. Herein, we used longitudinal MR imaging to visualize brain pathology in ECM and the impact of a new DON prodrug, JHU-083, on disease progression in mice. We demonstrate in vivo the reversal of disease markers in symptomatic, infected mice following treatment, including the resolution of edema and BBB disruption, findings usually associated with a fatal outcome in children and adults with CM. Our results support the premise that JHU-083 is a potential adjunctive treatment that could rescue children and adults from fatal CM.

A central role for mTOR kinase in homeostatic proliferation induced CD8+ T cell memory and tumor immunity.

The cell-intrinsic mechanisms guiding naive CD8+ T cells for clonal expansion and memory generation via homeostatic proliferation (HP) are unclear. Here, we have shown that HP of naive CD8+ T cells requires IL-7-, but not IL-15-induced mTOR kinase activation. HP-induced mTOR enhances transcription factor T-bet for functional maturation and CD122 expression, which sensitizes for an IL-15-dependent memory transition by favoring transcription factor Eomesodermin over T-bet. Inhibition of mTOR blocks T-bet and CD122 expression but preserves memory in an IL-15-independent manner by promoting Eomesodermin expression. The ability of rapamycin to augment HP-induced memory was cell-intrinsic given that silencing mTOR in CD8+ T cells generated identical outcomes. Strikingly, HP-induced CD8+ T cell memory generated by IL-15-dependent or -independent mechanisms demonstrated identical tumor efficacy. These results indicate a central role for mTOR in HP-induced CD8+ T cell responses and demonstrate the importance for CD8+ memory in HP-induced tumor efficacy.

mTOR Complex 1 Signaling Regulates the Generation and Function of Central and Effector Foxp3+ Regulatory T Cells.

The mechanistic/mammalian target of rapamycin (mTOR) has emerged as a critical integrator of signals from the immune microenvironment capable of regulating T cell activation, differentiation, and function. The precise role of mTOR in the control of regulatory T cell (Treg) differentiation and function is complex. Pharmacologic inhibition and genetic deletion of mTOR promotes the generation of Tregs even under conditions that would normally promote generation of effector T cells. Alternatively, mTOR activity has been observed to be increased in Tregs, and the genetic deletion of the mTOR complex 1 (mTORC1)-scaffold protein Raptor inhibits Treg function. In this study, by employing both pharmacologic inhibitors and genetically altered T cells, we seek to clarify the role of mTOR in Tregs. Our studies demonstrate that inhibition of mTOR during T cell activation promotes the generation of long-lived central Tregs with a memory-like phenotype in mice. Metabolically, these central memory Tregs possess enhanced spare respiratory capacity, similar to CD8+ memory cells. Alternatively, the generation of effector Tregs (eTregs) requires mTOR function. Indeed, genetic deletion of Rptor leads to the decreased expression of ICOS and PD-1 on the eTregs. Overall, our studies define a subset of mTORC1hi eTregs and mTORC1lo central Tregs.

Manipulation of Metabolic Pathways and Its Consequences for Anti-Tumor Immunity: A Clinical Perspective.

In the relatively short history of anti-tumor treatment, numerous medications have been developed against a variety of targets. Intriguingly, although many anti-tumor strategies have failed in their clinical trials, metformin, an anti-diabetic medication, demonstrated anti-tumor effects in observational studies and even showed its synergistic potential with immune checkpoint inhibitors (ICIs) in subsequent clinical studies. Looking back from bedside-to-bench, it may not be surprising that the anti-tumor effect of metformin derives largely from its ability to rewire aberrant metabolic pathways within the tumor microenvironment. As one of the most promising breakthroughs in oncology, ICIs were also found to exert their immune-stimulatory effects at least partly via rewiring metabolic pathways. These findings underscore the importance of correcting metabolic pathways to achieve sufficient anti-tumor immunity. Herein, we start by introducing the tumor microenvironment, and then we review the implications of metabolic syndrome and treatments for targeting metabolic pathways in anti-tumor therapies. We further summarize the close associations of certain aberrant metabolic pathways with impaired anti-tumor immunity and introduce the therapeutic effects of targeting these routes. Lastly, we go through the metabolic effects of ICIs and conclude an overall direction to manipulate metabolic pathways in favor of anti-tumor responses.