A Novel Role for Triglyceride Metabolism in Foxp3 Expression.

Lipid metabolism plays a key role in many cellular processes. We show here that regulatory T cells have enhanced lipid storage within subcellular lipid droplets (LD). They also express elevated amounts of both isoforms of diacylglycerol acyl transferase (DGAT1 & 2), enzymes required for the terminal step of triacylglycerol synthesis. In regulatory T-cells (Tregs), the conversion of diacylglycerols to triacylglycerols serves two additional purposes other than lipid storage. First, we demonstrate that it protects T cells from the toxic effects of saturated long chain fatty acids. Second, we show that Triglyceride formation is essential for limiting activation of protein kinase C via free diacyl glycerol moieties. Inhibition of DGAT1 resulted in elevated active PKC and nuclear NFKB, as well as impaired Foxp3 induction in response to TGFß. Thus, Tregs utilize a positive feedback mechanism to promote sustained expression of Foxp3 associated with control of LD formation.

CD4+ T Cell Fate Decisions Are Stochastic, Precede Cell Division, Depend on GITR Co-Stimulation, and Are Associated With Uropodium Development.

During an immune response, naïve CD4+ T cells proliferate and generate a range of effector, memory, and regulatory T cell subsets, but how these processes are co-ordinated remains unclear. A traditional model suggests that memory cells use mitochondrial respiration and are survivors from a pool of previously proliferating and glycolytic, but short-lived effector cells. A more recent model proposes a binary commitment to either a memory or effector cell lineage during a first, asymmetric cell division, with each lineage able to undergo subsequent proliferation and differentiation. We used improved fixation and staining methods with imaging flow cytometry in an optimized in vitro system that indicates a third model. We found that cell fates result from stochastic decisions that depend on GITR co-stimulation and which take place before any cell division. Effector cell commitment is associated with mTORC2 signaling leading to uropodium development, while developing memory cells lose mitochondria, have a nuclear localization of NFκB and depend on TGFß for their survival. Induced, T helper subsets and foxp3+ regulatory T cells were found in both the effector and memory cell lineages. This in vitro model of T cell differentiation is well suited to testing how manipulation of cytokine, nutrient, and other components of the microenvironment might be exploited for therapeutic purposes.

Foxp3 drives oxidative phosphorylation and protection from lipotoxicity.

Tregs can adopt a catabolic metabolic program with increased capacity for fatty acid oxidation-fueled oxidative phosphorylation (OXPHOS). It is unclear why this form of metabolism is favored in Tregs and, more specifically, whether this program represents an adaptation to the environment and developmental cues or is "hardwired" by Foxp3. Here we show, using metabolic analysis and an unbiased mass spectroscopy-based proteomics approach, that Foxp3 is both necessary and sufficient to program Treg-increased respiratory capacity and Tregs' increased ability to utilize fatty acids to fuel oxidative phosphorylation. Foxp3 drives upregulation of components of all the electron transport complexes, increasing their activity and ATP generation by oxidative phosphorylation. Increased fatty acid ß-oxidation also results in selective protection of Foxp3+ cells from fatty acid-induced cell death. This observation may provide novel targets for modulating Treg function or selection therapeutically.

The Role of Lipid Metabolism in T Lymphocyte Differentiation and Survival.

The differentiation and effector functions of both the innate and adaptive immune system are inextricably linked to cellular metabolism. The features of metabolism which affect both arms of the immune system include metabolic substrate availability, expression of enzymes, transport proteins, and transcription factors which control catabolism of these substrates, and the ability to perform anabolic metabolism. The control of lipid metabolism is central to the appropriate differentiation and functions of T lymphocytes, and ultimately to the maintenance of immune tolerance. This review will focus on the role of fatty acid (FA) metabolism in T cell differentiation, effector function, and survival. FAs are important sources of cellular energy, stored as triglycerides. They are also used as precursors to produce complex lipids such as cholesterol and membrane phospholipids. FA residues also become incorporated into hormones and signaling moieties. FAs signal via nuclear receptors and their channeling, between storage as triacyl glycerides or oxidation as fuel, may play a role in survival or death of the cell. In recent years, progress in the field of immunometabolism has highlighted diverse roles for FA metabolism in CD4 and CD8 T cell differentiation and function. This review will firstly describe the sensing and modulation of the environmental FAs and lipid intracellular signaling and will then explore the key role of lipid metabolism in regulating the balance between potentially damaging pro-inflammatory and anti-inflammatory regulatory responses. Finally the complex role of extracellular FAs in determining cell survival will be discussed.

Induction of Immunological Tolerance as a Therapeutic Procedure.

A major goal of immunosuppressive therapies is to harness immune tolerance mechanisms so as to minimize unwanted side effects associated with protracted immunosuppressive therapy. Antibody blockade of lymphocyte coreceptor and costimulatory pathways in mice has demonstrated the principle that both naive and primed immune systems can be reprogrammed toward immunological tolerance. Such tolerance can involve the amplification of activity of regulatory T cells, and is maintained through continuous recruitment of such cells through processes of infectious tolerance. We propose that regulatory T cells create around them microenvironments that are anti-inflammatory and endowed with enhanced protection against destructive damage. This acquired immune privilege involves the decommissioning of cells of the innate as well as adaptive immune systems. Evidence is presented that nutrient sensing by immune cells acting through the mammalian target of rapamycin (mTOR) pathway provides one route by which the immune system can be directed toward noninflammatory and regulatory behavior at the expense of destructive functions. Therapeutic control of immune cells so as to harness metabolic routes favoring dominant regulatory mechanisms has offered a new direction for immunosuppressive therapy, whereby short-term treatment may be sufficient for long-term benefit or even cure.

Epithelial-mesenchymal transition and nuclear ß-catenin induced by conditional intestinal disruption of Cdh1 with Apc is E-cadherin EC1 domain dependent.

Two important protein-protein interactions establish E-cadherin (Cdh1) in the adhesion complex; homophilic binding via the extra-cellular (EC1) domain and cytoplasmic tail binding to ß-catenin. Here, we evaluate whether E-cadherin binding can inhibit ß-catenin when there is loss of Adenomatous polyposis coli (APC) from the ß-catenin destruction complex. Combined conditional loss of Cdh1 and Apc were generated in the intestine, intestinal adenoma and adenoma organoids. Combined intestinal disruption (Cdh1fl/flApcfl/flVil-CreERT2) resulted in lethality, breakdown of the intestinal barrier, increased Wnt target gene expression and increased nuclear ß-catenin localization, suggesting that E-cadherin inhibits ß-catenin. Combination with an intestinal stem cell Cre (Lgr5CreERT2) resulted in ApcΔ/Δ recombination and adenoma, but intact Cdh1fl/fl alleles. Cultured ApcΔ/ΔCdh1fl/fl adenoma cells infected with adenovirus-Cre induced Cdh1fl/fl recombination (Cdh1Δ/Δ), disruption of organoid morphology, nuclear ß-catenin localization, and cells with an epithelial-mesenchymal phenotype. Complementation with adenovirus expressing wild-type Cdh1 (Cdh1-WT) rescued adhesion and ß-catenin membrane localization, yet an EC1 specific double mutant defective in homophilic adhesion (Cdh1-MutW2A, S78W) did not. These data suggest that E-cadherin inhibits ß-catenin in the context of disruption of the APC-destruction complex, and that this function is also EC1 domain dependent. Both binding functions of E-cadherin may be required for its tumour suppressor activity.

Induced Foxp3(+) T Cells Colonizing Tolerated Allografts Exhibit the Hypomethylation Pattern Typical of Mature Regulatory T Cells.

Regulatory T cells expressing the transcription factor Foxp3 require acquisition of a specific hypomethylation pattern to ensure optimal functional commitment, limited lineage plasticity, and long-term maintenance of tolerance. A better understanding of the molecular mechanisms involved in the generation of these epigenetic changes in vivo will contribute to the clinical exploitation of Foxp3(+) Treg. Here, we show that both in vitro and in vivo generated antigen-specific Foxp3(+) Treg can acquire Treg-specific epigenetic characteristics and prevent skin graft rejection in an animal model.

Nutrient Sensing via mTOR in T Cells Maintains a Tolerogenic Microenvironment.

We have proposed that tolerance can be maintained through the induction, by Treg cells, of a tolerogenic microenvironment within tolerated tissues that inhibits effector cell activity but which supports the generation of further Treg cells by "infectious tolerance." Two important components of this tolerogenic microenvironment depend on metabolism and nutrient sensing. The first is due to the up-regulation of multiple enzymes that consume essential amino acids, which are sensed in naïve T cells primarily via inhibition of the mechanistic target of rapamycin (mTOR) pathway, which in turn encourages their further differentiation into FOXP3(+) Treg cells. The second mechanism is the metabolism of extracellular ATP to adenosine by the ectoenzymes CD39 and CD73. These two enzymes are constitutively co-expressed on Treg cells, but can also be induced on a wide variety of cell types by TGFß and the adenosine generated can be shown to be a potent inhibitor of T cell proliferation. This review will focus on mechanisms of nutrient sensing in T cells, how these are integrated with TCR and cytokine signals via the mTOR pathway, and what impact this has on intracellular metabolism and subsequently the control of differentiation into different effector or regulatory T cell subsets.

Harnessing FOXP3+ regulatory T cells for transplantation tolerance.

Early demonstrations that mice could be tolerized to transplanted tissues with short courses of immunosuppressive therapy and that with regard to tolerance to self, CD4+FOXP3+ regulatory T cells (Tregs) appeared to play a critical role, have catalyzed strategies to harness FOXP3-dependent processes to control rejection in human transplantation. This review seeks to examine the scientific underpinning for this new approach to finesse immunosuppression.

The mTOR pathway and integrating immune regulation.

The mammalian target of rapamycin (mTOR) pathway is an important integrator of nutrient-sensing signals in all mammalian cells, and acts to coordinate the cell proliferation with the availability of nutrients such as glucose, amino acids and energy (oxygen and ATP). A large part of the immune response depends on the proliferation and clonal expansion of antigen-specific T cells, which depends on mTOR activation, and the pharmacological inhibition of this pathway by rapamycin is therefore potently immunosuppressive. It is only recently, however, that we have started to understand the more subtle details of how the mTOR pathway is involved in controlling the differentiation of effector versus memory CD8(+) T cells and the decision to generate different CD4(+) helper T-cell subsets. In particular, this review will focus on how nutrient sensing via mTOR controls the expression of the master transcription factor for regulatory T cells in order to maintain the balance between tolerance and inflammation.

Regulatory T cells and transplantation tolerance.

The success of clinical organ transplantation relies on life-long use of immunosuppressive drugs that target immune responses associated with graft rejection. Preclinical studies in mice have convincingly demonstrated that robust, long-term transplantation tolerance can be achieved after a short-term treatment with T-cell coreceptor and costimulation blockade even for a fully mismatched graft. Such therapeutically induced tolerance requires the induction of Foxp3⁺ Tregs, which are essential for both the development and maintenance of the tolerant state. Recent advances in understanding the molecular and epigenetic mechanisms underlying the induction and stabilization of Foxp3 expression, thus guiding Foxp3⁺ Treg differentiation, have revealed novel therapeutic targets in animal models that can be translated to harness Foxp3⁺ Tregs from within the patient. Such in vivo induced Foxp3⁺ Tregs can also induce the tolerant state. Pharmacological compounds are available to exploit these targets and their further development holds great promise for clinical translation.

Regulatory cells and transplantation tolerance.

Transplantation tolerance is a continuing therapeutic goal, and it is now clear that a subpopulation of T cells with regulatory activity (Treg) that express the transcription factor foxp3 are crucial to this aspiration. Although reprogramming of the immune system to donor-specific transplantation tolerance can be readily achieved in adult mouse models, it has yet to be successfully translated in human clinical practice. This requires that we understand the fundamental mechanisms by which donor antigen-specific Treg are induced and function to maintain tolerance, so that we can target therapies to enhance rather than impede these regulatory processes. Our current understanding is that Treg act via numerous molecular mechanisms, and critical underlying components such as mTOR inhibition, are only now emerging.

Foxp3 expression is required for the induction of therapeutic tissue tolerance.

CD4(+)Foxp3(+) regulatory T cells (Treg) are essential for immune homeostasis and maintenance of self-tolerance. They are produced in the thymus and also generated de novo in the periphery in a TGF-ß-dependent manner. Foxp3(+) Treg are also required to achieve tolerance to transplanted tissues when induced by coreceptor or costimulation blockade. Using TCR-transgenic mice to avoid issues of autoimmune pathology, we show that Foxp3 expression is both necessary and sufficient for tissue tolerance by coreceptor blockade. Moreover, the known need in tolerance induction for TGF-ß signaling to T cells can wholly be explained by its role in induction of Foxp3, as such signaling proved dispensable for the suppressive process. We analyzed the relative contribution of TGF-ß and Foxp3 to the transcriptome of TGF-ß-induced Treg and showed that TGF-ß elicited a large set of downregulated signature genes. The number of genes uniquely modulated due to the influence of Foxp3 alone was surprisingly limited. Retroviral-mediated conditional nuclear expression of Foxp3 proved sufficient to confer transplant-suppressive potency on CD4(+) T cells and was lost once nuclear Foxp3 expression was extinguished. These data support a dual role for TGF-ß and Foxp3 in induced tolerance, in which TGF-ß stimulates Foxp3 expression, for which sustained expression is then associated with acquisition of tolerance.

Sustained suppression by Foxp3+ regulatory T cells is vital for infectious transplantation tolerance.

A paradigm shift in immunology has been the recent discovery of regulatory T cells (T reg cells), of which CD4(+)Foxp3(+) cells are proven as essential to self-tolerance. Using transgenic B6.Foxp3(hCD2) mice to isolate and ablate Foxp3(+) T reg cells with an anti-hCD2 antibody, we show for the first time that CD4(+)Foxp3(+) cells are crucial for infectious tolerance induced by nonablative anti-T cell antibodies. In tolerant animals, Foxp3(+) T reg cells are constantly required to suppress effector T cells still capable of causing tissue damage. Tolerated tissue contains T cells that are capable of rejecting it, but are prevented from doing so by therapeutically induced Foxp3(+) T reg cells. Finally, Foxp3(+) cells have been confirmed as the critical missing link through which infectious tolerance operates in vivo. Peripherally induced Foxp3(+) cells sustain tolerance by converting naive T cells into the next generation of Foxp3(+) cells. Empowering Foxp3(+) regulatory T cells in vivo offers a tractable route to avoid and correct tissue immunopathology.

TGF-ß in transplantation tolerance.

TGF-ß is a cytokine required for the induction and maintenance of transplantation tolerance in animal models. TGF-ß mediates anti-inflammatory effects by acting on many immune cell-types. Central for transplantation tolerance is the role for TGF-ß in the induction of Foxp3 and regulatory capacity in CD4(+) T cells. Recently, however, the general anti-inflammatory role of TGF-ß in CD4(+) T cell polarization was questioned by the discovery that, in the presence of inflammatory cytokines such as IL-6 or IL-1, TGF-ß drives the differentiation of Th17 cells associated with transplant rejection. A better understanding of the factors determining TGF-ß production and activation, Foxp3 induction and Treg stability is vital for the development of tolerogenic strategies in transplantation.

Generation of anti-inflammatory adenosine by leukocytes is regulated by TGF-ß.

Levels of anti-inflammatory extracellular adenosine are controlled by the sequential action of the ectonucleotidases CD39 and CD73, whose expression in CD4(+) T cells has been associated with natural regulatory T cells (nTregs). We here show that CD73 expression on activated murine CD4(+) T cells is induced by TGF-ß independently of Foxp3 expression, operates at the transcriptional level and translates into gain of functional capacity to generate adenosine. In the presence of AMP, CD73 induced by TGF-ß generates adenosine able to suppress proliferation of activated CD4(+) T cells in vitro. These effects are contextual and opposed by proinflammatory cytokines. CD73 is also upregulated by TGF-ß in CD8(+) T cells, DCs and macrophages, so providing an amplification mechanism for adenosine generation in tissue microenvironments. Together, these findings expose a novel anti-inflammatory role for TGF-ß.

Biomarkers of transplantation tolerance: more hopeful than helpful?

A major limitation to the translation of tolerogenic therapies to clinical transplantation is a lack of biomarkers that can be used as surrogate measures for predicting the successful induction of immune tolerance which would allow for the safe withdrawal of immunosuppression. We have used three different mouse models of donor specific tolerance to skin grafts together with quantitative RT-PCR to search for potential biomarkers of tolerance using criteria based on the presence or activity of regulatory T cells and antigen presenting cells (APCs) within grafts or lymphoid organs. We find that significant differences in gene expression between tolerated and rejecting grafts are observed primarily within the grafted skin and not systemically or in the draining lymph node. The pattern of gene expression within long-term surviving tolerated grafts appear very similar to syngeneic grafts, with both having low levels of T cell and APC infiltration and a bias toward relative over-expression of "regulatory-associated" genes, while allografts destined for rejection show an overall increase in both "regulatory" and "effector" cell associated transcripts. We also, however, find an increase in a large number of regulatory genes, of both innate and T cell origin, even after grafting syngeneic skin. Taken together, these findings suggest that there may be no tissue biomarkers uniquely able to predict donor antigen specific tolerance per se, but that patterns of gene expression within tolerated grafts may be similar to those found in self tissues recovering from an inflammatory insult.

mTOR signalling and metabolic regulation of T cell differentiation.

T cells constantly monitor energy status and nutrient levels in order to adjust metabolic pathways according to their nutritional status and other environmental stimuli. It is increasingly evident that the regulation of cellular metabolism is tightly coupled to T cell differentiation that ultimately determines the cellular fate. The mammalian target of Rapamycin (mTOR) pathway has emerged as a key player in sensing these nutritional/energetic signals and in addition, acts as a major integrator of growth factor induced signals, so placing mTOR at the core of a signalling network controlling metabolism and cellular fate. The mTOR pathway has been shown to play an important role in determining the differentiation of CD4(+) T cells into inflammatory and regulatory subsets, in the induction of anergy, in the development of CD8(+) memory T cells and the regulation of T cell trafficking.