Differential effects of 2-deoxy-D-glucose on in vitro expanded human regulatory T cell subsets.

Regulatory T cells (Tregs) are required for the maintenance of immune tolerance and adoptive Treg infusion therapy has become a promising approach to suppress immune responses in diseases such as autoimmunity and transplant rejection. However, one critical challenge of Treg therapy is the requirement of in vitro expansion of functionally stable Tregs while preventing either the contamination of T effector and/or emergence of unstable pathogenic Tregs. Recent studies showing distinct metabolic requirements of T effectors and Tregs suggest that manipulation of cell metabolism may be an attractive strategy to achieve this goal. Here we show that human thymically derived Tregs (tTregs) and in vitro induced Tregs (iTregs) from naive T cells engage glycolysis equivalently upon activation. However, inhibiting glucose metabolism via 2-deoxy-D-glucose (2DG) has distinct effects on each of these subsets. While 2DG treatment at the onset of activation significantly reduced the proliferation and expression of suppressive molecules such as ICOS and CTLA-4 in tTregs, its effect on FOXP3 expression was small. In contrast, 2DG treatment during iTreg induction modestly decreased their proliferation but strongly reduced both ICOS and FOXP3 expression. Importantly, both Treg subsets became insensitive to 2DG after day 3 post activation with little effect on either proliferation or FOXP3 expression while T conventional Th0 cells showed reduced proliferation under the same conditions. Moreover, 2DG treatment at day 3 did not impair the suppressive capabilities of Treg subsets. Collectively, these findings suggest that there is a distinct temporal requirement of glycolysis in each of the activated human Treg subsets and T conventional cells. Furthermore, 2DG treatment at the onset as a strategy to impair contaminating T effector cell proliferation is unfavorable for optimal Treg generation as well.

Differential Roles of IL-2 Signaling in Developing versus Mature Tregs.

Although Foxp3+ regulatory T cells (Tregs) require interleukin-2 (IL-2) for their development, it has been unclear whether continuing IL-2 signals are needed to maintain lineage stability, survival, and suppressor function in mature Tregs. We generated mice in which CD25, the main ligand-binding subunit of the IL-2 receptor, can be inducibly deleted from Tregs after thymic development. In contrast to Treg development, we find that IL-2 is dispensable for maintaining lineage stability in mature Tregs. Although continuous IL-2 signaling is needed for long-term Treg survival, CD25-deleted Tregs may persist for several weeks in vivo using IL-7. We also observe defects in glycolytic metabolism and suppressor function following CD25 deletion. Thus, unlike developing Tregs in which the primary role of IL-2 is to initiate Foxp3 expression, mature Tregs require continuous IL-2 signaling to maintain survival and suppressor function, but not to maintain lineage stability.

Cutting Edge: TGF-ß and Phosphatidylinositol 3-Kinase Signals Modulate Distinct Metabolism of Regulatory T Cell Subsets.

Murine Foxp3+ regulatory T cells (Tregs) differentiated in vitro (induced Tregs [iTregs]) in the presence of anti-inflammatory cytokine TGF-ß rely predominantly upon lipid oxidation to fuel mitochondrial oxidative phosphorylation. Foxp3 expression underlies this metabolic preference, as it suppresses glycolysis and drives oxidative phosphorylation. In this study, we show that in contrast to iTregs, thymic-derived Tregs (tTregs), engage in glycolysis and glutaminolysis at levels comparable to effector T cells despite maintained Foxp3 expression. Interestingly, exposure of tTregs to the anti-inflammatory cytokine TGF-ß represses PI3K-mediated mTOR signaling, inhibits glucose transporter and Hk2 expression, and reprograms their metabolism to favor oxidative phosphorylation. Conversely, replicating the effects of inflammation via elevation of PI3K signaling has minimal effects on tTregs but dramatically enhances the glycolysis of normally oxidative iTregs, resulting in reduction of Foxp3 expression. Collectively, these findings suggest both extrinsic and intrinsic factors govern the unique metabolic signature of Treg subsets.

Navigating T-Cell Immunometabolism in Transplantation.

Recently, a new discipline termed "immunometabolism" has transformed the field of immunology. It encompasses the study of the intrinsic metabolic pathways of different immune subsets and their impact on cellular fate and function. For instance, broadly speaking, proinflammatory cells depend on glycolysis and glutamine oxidation, whereas cells involved in anti-inflammatory response, such as Foxp3+ regulatory T (Treg) cells, use predominantly fatty acid oxidation. However, although a useful paradigm, this actually is a reductionist view, and the engagement of these metabolic pathways is not mutually exclusive between these subsets. Over the past several years, new insights and new methods to better dissect, define, and harness the metabolic properties of immune cells for immunotherapeutic purposes have come to the forefront. In this review, we will discuss the metabolic heterogeneity of different T-cell subsets as well as basic principles of integrative technologies, such as metabolomics, which can be used to better understand the metabolic signatures of immune responses. Given the interest of exploiting this information in the context of transplantation, we will highlight the scope of immunometabolism in unraveling novel mechanisms of immune regulation that can be manipulated to promote Treg cell stability and function while inhibiting T effectors to establish long-term transplantation tolerance.

Immunometabolism of regulatory T cells.

The bidirectional interaction between the immune system and whole-body metabolism has been well recognized for many years. Via effects on adipocytes and hepatocytes, immune cells can modulate whole-body metabolism (in metabolic syndromes such as type 2 diabetes and obesity) and, reciprocally, host nutrition and commensal-microbiota-derived metabolites modulate immunological homeostasis. Studies demonstrating the metabolic similarities of proliferating immune cells and cancer cells have helped give birth to the new field of immunometabolism, which focuses on how the cell-intrinsic metabolic properties of lymphocytes and macrophages can themselves dictate the fate and function of the cells and eventually shape an immune response. We focus on this aspect here, particularly as it relates to regulatory T cells.

T-cell energy metabolism as a controller of cell fate in transplantation.

PURPOSE OF REVIEW: To highlight some of the recent developments in the novel field of immunometabolism and the therapeutic potential of the many regulatory components of this immunometabolic network for transplantation. RECENT FINDINGS: In response to cytokines, changes in nutrients, and other alterations in the local milieu, immune cells are capable of changing their internal metabolic pathways to meet their energy demands. Recent studies demonstrate that activated T effectors (Th1 and Th17) are supported by aerobic glycolysis, whereas regulatory T cells and CD8 memory T cells favor fatty acid oxidation and lipid biosynthesis through mitochondrial oxidative phosphorylation. These bioenergetic processes are dependent upon the activation of metabolic sensors such as mammalian target of rapamycin and AMP-activated protein kinase, respectively, indicating that the cross-talk between immunity and metabolism can shape the fate and function of immune cells. Finally, exciting new studies suggest that differences in the bioenergetic mechanisms within the various immune subsets may selectively be exploited for regulating the immune responses. SUMMARY: In this review, we will discuss the metabolic signatures adopted by various immune cells during tolerance versus immunity and the promising avenues that can be modulated by targeting metabolic pathways with either nutrition or pharmacological intervention for establishing long-term transplantation tolerance.

Control of PI(3) kinase in Treg cells maintains homeostasis and lineage stability.

Foxp3(+) regulatory T cells (Treg cells) are required for immunological homeostasis. One notable distinction between conventional T cells (Tconv cells) and Treg cells is differences in the activity of phosphatidylinositol-3-OH kinase (PI(3)K); only Tconv cells downregulate PTEN, the main negative regulator of PI(3)K, upon activation. Here we found that control of PI(3)K in Treg cells was essential for lineage homeostasis and stability. Mice lacking Pten in Treg cells developed an autoimmune-lymphoproliferative disease characterized by excessive T helper type 1 (TH1) responses and B cell activation. Diminished control of PI(3)K activity in Treg cells led to reduced expression of the interleukin-2 (IL-2) receptor α subunit CD25, accumulation of Foxp3(+)CD25(-) cells and, ultimately, loss of expression of the transcription factor Foxp3 in these cells. Collectively, our data demonstrate that control of PI(3)K signaling by PTEN in Treg cells is critical for maintaining their homeostasis, function and stability.

Metabolic programming and PDHK1 control CD4+ T cell subsets and inflammation.

Activation of CD4+ T cells results in rapid proliferation and differentiation into effector and regulatory subsets. CD4+ effector T cell (Teff) (Th1 and Th17) and Treg subsets are metabolically distinct, yet the specific metabolic differences that modify T cell populations are uncertain. Here, we evaluated CD4+ T cell populations in murine models and determined that inflammatory Teffs maintain high expression of glycolytic genes and rely on high glycolytic rates, while Tregs are oxidative and require mitochondrial electron transport to proliferate, differentiate, and survive. Metabolic profiling revealed that pyruvate dehydrogenase (PDH) is a key bifurcation point between T cell glycolytic and oxidative metabolism. PDH function is inhibited by PDH kinases (PDHKs). PDHK1 was expressed in Th17 cells, but not Th1 cells, and at low levels in Tregs, and inhibition or knockdown of PDHK1 selectively suppressed Th17 cells and increased Tregs. This alteration in the CD4+ T cell populations was mediated in part through ROS, as N-acetyl cysteine (NAC) treatment restored Th17 cell generation. Moreover, inhibition of PDHK1 modulated immunity and protected animals against experimental autoimmune encephalomyelitis, decreasing Th17 cells and increasing Tregs. Together, these data show that CD4+ subsets utilize and require distinct metabolic programs that can be targeted to control specific T cell populations in autoimmune and inflammatory diseases.

Alloreactive CD8 T cells rescued from apoptosis during co-stimulation blockade by Toll-like receptor stimulation remain susceptible to Fas-induced cell death.

Blockade of co-stimulatory signals to T cells is extremely effective for the induction of transplantation tolerance in immunologically naive rodents. However, infections and inflammation compromise the efficacy of co-stimulation blockade regimens for the induction of tolerance, thereby stimulating the rejection of allografts. Previous studies have shown that stimulation of innate immunity abrogates tolerance induction by preventing the deletion of alloreactive CD8(+) T cells that normally occurs during co-stimulation blockade. Although inflammation prevents the deletion of alloreactive T cells during co-stimulation blockade, it is not known if this resistance to cell death is the result of a mechanism intrinsic to the T cell. Here, we used syngeneic bone marrow chimeric mice that contain a trace population of T-cell receptor transgenic alloreactive CD8(+) T cells to investigate the early apoptotic signature and activation status of alloreactive T cells following exposure to inflammatory signals during co-stimulation blockade with an antibody specific for CD154. Our findings revealed that the presence of bacterial lipopolysaccharide during co-stimulation blockade enhanced the early activation of alloreactive CD8(+) T cells, as indicated by the up-regulation of CD25 and CD69, suppressed Fas ligand expression, and prevented apoptotic cell death. However, alloreactive CD8(+) T cells from lipopolysaccharide-treated mice remained sensitive to Fas-mediated apoptosis in vitro. These findings suggest that alloreactive T cells rescued from deletion during co-stimulation blockade by inflammation are still sensitive to pro-apoptotic signals and that stimulating these apoptotic pathways during co-stimulation blockade may augment the induction of tolerance.

T-cell activation and transplantation tolerance.

Transplantation of allogeneic or "nonself" tissues stimulates a robust immune response leading to graft rejection, and therefore, most recipients of allogeneic organ transplants require the lifelong use of immune suppressive agents. Excellent outcomes notwithstanding, contemporary immunosuppressive medications are toxic, are often not taken by patients, and pose long-term risks of infection and malignancy. The ultimate goal in transplantation is to develop new treatments that will supplant the need for general immunosuppression. Here, we will describe the development and application of costimulation blockade to induce transplantation tolerance and discuss how the diverse array of signals that act on T cells will determine the balance between graft survival and rejection.

Maturation-dependent licensing of naive T cells for rapid TNF production.

The peripheral naïve T cell pool is comprised of a heterogeneous population of cells at various stages of development, which is a process that begins in the thymus and is completed after a post-thymic maturation phase in the periphery. One hallmark of naïve T cells in secondary lymphoid organs is their unique ability to produce TNF rapidly after activation and prior to acquiring other effector functions. To determine how maturation influences the licensing of naïve T cells to produce TNF, we compared cytokine profiles of CD4(+) and CD8(+) single positive (SP) thymocytes, recent thymic emigrants (RTEs) and mature-naïve (MN) T cells during TCR activation. SP thymocytes exhibited a poor ability to produce TNF when compared to splenic T cells despite expressing similar TCR levels and possessing comparable activation kinetics (upregulation of CD25 and CD69). Provision of optimal antigen presenting cells from the spleen did not fully enable SP thymocytes to produce TNF, suggesting an intrinsic defect in their ability to produce TNF efficiently. Using a thymocyte adoptive transfer model, we demonstrate that the ability of T cells to produce TNF increases progressively with time in the periphery as a function of their maturation state. RTEs that were identified in NG-BAC transgenic mice by the expression of GFP showed a significantly enhanced ability to express TNF relative to SP thymocytes but not to the extent of fully MN T cells. Together, these findings suggest that TNF expression by naïve T cells is regulated via a gradual licensing process that requires functional maturation in peripheral lymphoid organs.