The mechanistic target of rapamycin (mTOR) pathway integrates diverse environmental inputs, including immune signals and metabolic cues, to direct T cell fate decisions1. Activation of mTOR, comprised of mTORC1 and mTORC2 complexes, delivers an obligatory signal for proper activation and differentiation of effector CD4+ T cells2,3, whereas in the regulatory T cell (Treg) compartment, the Akt-mTOR axis is widely acknowledged as a crucial negative regulator of Treg de novo differentiation4–8 and population expansion9. However, whether mTOR signaling affects the homeostasis and function of Tregs remains largely unexplored. Here we show that mTORC1 signaling is a pivotal positive determinant of Treg function. Tregs have elevated steady-state mTORC1 activity compared to naïve T cells. Signals via T cell receptor (TCR) and IL-2 provide major inputs for mTORC1 activation, which in turn programs suppressive function of Tregs. Disruption of mTORC1 through Treg-specific deletion of the essential component Raptor leads to a profound loss of Treg suppressive activity in vivo and development of a fatal early-onset inflammatory disorder. Mechanistically, Raptor/mTORC1 signaling in Tregs promotes cholesterol/lipid metabolism, with the mevalonate pathway particularly important for coordinating Treg proliferation and upregulation of suppressive molecules CTLA-4 and ICOS to establish Treg functional competency. In contrast, mTORC1 does not directly impact the expression of Foxp3 or anti- and pro-inflammatory cytokines in Tregs, suggesting a non-conventional mechanism for Treg functional regulation. Lastly, we provide evidence that mTORC1 maintains Treg function partly through inhibiting the mTORC2 pathway. Our results demonstrate that mTORC1 acts as a fundamental ‘rheostat’ in Tregs to link immunological signals from TCR and IL-2 to lipogenic pathways and functional fitness, and highlight a central role of metabolic programming of Treg suppressive activity in immune homeostasis and tolerance.

SUMMARY Naïve T cells respond to antigen stimulation by exiting from quiescence and initiating clonal expansion and functional differentiation, but the control mechanism is elusive. Here we describe that Raptor-mTORC1-dependent metabolic programming is a central determinant of this transitional process. Loss of Raptor abrogated T cell priming and Th2 cell differentiation, although Raptor function is less important for continuous proliferation of actively cycling cells. mTORC1 coordinated multiple metabolic programs in T cells including glycolysis, lipid synthesis and oxidative phosphorylation to mediate antigen-triggered exit from quiescence. mTORC1 further linked glucose metabolism to the initiation of Th2 cell differentiation by orchestrating cytokine receptor expression and cytokine responsiveness. Activation of Raptor-mTORC1 integrated T cell receptor and CD28 co-stimulatory signals in antigen-stimulated T cells. Our studies identify a Raptor-mTORC1-dependent pathway linking signal-dependent metabolic reprogramming to quiescence exit, and this in turn coordinates lymphocyte activation and fate decisions in adaptive immunity.

Regulatory T (Treg) cells respond to immune and inflammatory signals to mediate immunosuppression, but how functional integrity of Treg cells is maintained under activating environments remains elusive. Here we found that autophagy was active in Treg cells and supported their lineage stability and survival fitness. Treg cell-specific deletion of the essential autophagy gene Atg7 or Atg5 led to loss of Treg cells, increased tumor resistance, and development of inflammatory disorders. Atg7-deficient Treg cells had increased apoptosis and readily lost Foxp3 expression, especially after activation. Mechanistically, autophagy deficiency upregulated mTORC1 and c-Myc function and glycolytic metabolism that contributed to defective Treg function. Therefore, autophagy couples environmental signals and metabolic homeostasis to protect lineage and survival integrity of Treg cells in activating contexts.

The mechanisms that regulate T cell quiescence are poorly understood. We report that tuberous sclerosis complex 1 (Tsc1) establishes a quiescence program in naive T cells by controlling cell size, cell cycle entry, and responses to T cell receptor stimulation. Loss of quiescence predisposed Tsc1-deficient T cells to apoptosis that resulted in loss of conventional T cells and invariant natural killer T cells. Loss of Tsc1 function dampened in vivo immune responses to bacterial infection. Tsc1-deficient T cells exhibited increased mTORC1 but diminished mTORC2 activities, with mTORC1 activation essential for the disruption of immune homeostasis. Therefore, Tsc1-dependent control of mTOR is crucial in establishing naive T cell quiescence to facilitate adaptive immune function.

The interplay between effector and regulatory T (Treg) cells is crucial for adaptive immunity, but how Treg control diverse effector responses is elusive. We found that the phosphatase PTEN links Treg stability to repression of TH1 and TFH (follicular helper) responses. Depletion of PTEN in Treg resulted in excessive TFH and germinal center responses and spontaneous inflammatory disease. These defects are considerably blocked by deletion of Interferon-γ, indicating coordinated control of TH1 and TFH responses. Mechanistically, PTEN maintains Treg stability and metabolic balance between glycolysis and mitochondrial fitness. Moreover, PTEN deficiency upregulates mTORC2-Akt activity, and loss of this activity restores PTEN-deficient Treg function. Our studies establish a PTEN-mTORC2 axis that maintains Treg stability and coordinates Treg-mediated control of effector responses.

SUMMARY The molecular circuits by which antigens activate quiescent T cells remain poorly understood. We combined temporal profiling of the whole proteome and phosphoproteome via multiplexed isobaric labeling proteomics technology, computational pipelines for integrating multi-omics datasets, and functional perturbation to systemically reconstruct regulatory networks underlying T cell activation. T cell receptors activated the T cell proteome and phosphoproteome with discrete kinetics, marked by early dynamics of phosphorylation and delayed ribosome biogenesis and mitochondrial activation. Systems biology analyses identified multiple functional modules, active kinases, transcription factors and connectivity between them, and mitochondrial pathways including mitoribosomes and complex IV. Genetic perturbation revealed physiological roles for mitochondrial enzyme COX10-mediated oxidative phosphorylation in T cell quiescence exit. Our multi-layer proteomics profiling, integrative network analysis and functional studies define landscapes of the T cell proteome and phosphoproteome and reveal signaling and bioenergetics pathways that mediate lymphocyte exit from quiescence.

Naïve CD4+ T cells differentiate into diverse effector and regulatory lineages to orchestrate immunity and tolerance. The differentiation of pro-inflammatory TH1 and anti-inflammatory Foxp3+ regulatory T cells (Treg) was reciprocally regulated by S1P1, a receptor for the bioactive lipid sphingosine-1-phosphate. S1P1 inhibited extrathymic and natural Treg generation while driving TH1 cell development in a reciprocal manner and disrupted immune homeostasis. S1P1 signaled through mTOR and antagonized TGF-β function mainly by attenuating sustained Smad3 activity. S1P1 function was dependent upon endogenous sphingosine kinase activity. Remarkably, two seemingly unrelated immunosuppressants FTY720 and rapamycin targeted the same S1P1 and mTOR pathway to regulate the dichotomy between TH1 and Treg cells. Our studies establish an S1P1-mTOR axis that controls T cell lineage specification.

Regulatory T cells (Treg cells) play a pivotal role in the establishment and maintenance of immunological self-tolerance and homeostasis1,2. Transcriptional programming of regulatory mechanisms facilitates Treg cell functional activation in the prevention of diverse types of inflammatory responses3,4. How Treg cells orchestrate their homeostasis and interplay with environmental signals remains poorly understood. Here we show that liver kinase B1 (LKB1) programs proper metabolic and functional fitness of Treg cells in the control of immune tolerance and homeostasis. Mice with Treg-specific deletion of LKB1 developed a fatal inflammatory disease characterized by excessive TH2-dominant responses. LKB1 deficiency disrupted Treg cell survival and mitochondrial fitness and metabolism, but also induced aberrant expression of immune regulatory molecules including the negative co-receptor PD-1, and TNF receptor (TNRF) superfamily proteins GITR and OX40. Unexpectedly, LKB1 function in Treg cells was independent of conventional AMPK signaling or the mTORC1-HIF-1α axis, but contributed to the activation of β-catenin signaling for the proper control of PD-1 and TNFR proteins. Blockade of PD-1 activity reinvigorated the suppressive capability of LKB1-deficient Treg cells in the repression of TH2 responses and the interplay with thymic stromal lymphopoietin (TSLP)-primed dendritic cells (DCs). Thus, Treg cells employ LKB1 signaling to coordinate their metabolic and immunological homeostasis and to prevent apoptotic and functional exhaustion, thereby orchestrating the balance between immunity and tolerance.