Treg Cell Stability Research Articles

Defining mechanisms of semaphorin 4A stimulatory action on human Treg cells

Abstract We had shown previously that semaphorin 4A (Sema4A) suppresses allergic inflammation in mice (Mucosal Immunol., 2012, 5:409-19), regulates human Treg cell stability, and induces the generation of Treg cells in PBMC cultures acting through its Plexin B1 receptor (Immunohorizons, 2019, 3:78–87). Plexin B1 forms functional homodimers or heterodimers with other Plexin B molecules and co-precipitates with Neuropilins (NRPs) in transfected 293T cells. Furthermore, NRPs were shown to stabilize Sema/Plexin interactions. In this study, we sought to identify potential partner(s) for Plexin B1 in mediating Sema4A action. We examined surface expression of Plexin B2, B3, and NRPs 1, 2 in human Treg cells by flow cytometry, and observed that they express NRP-2 but not NRP-1, Plexin B2, or B3. Moreover, culture of PBMC with recombinant human Sema4A-Fc (rhSema4A) in vitro resulted in the downregulation of NRP-2 expression on human Treg cells suggesting a specific ligand-receptor interaction. We propose that Sema4A action on human Treg cells may be mediated by a Plexin B1-NRP-2 complex. Our ongoing research is focused on cryoEM studies of ligand-receptor and receptor-receptor interactions in conjunction with sequence/structure statistical analyses using the DARC (Deep Analysis of Residue Constraints) program (Scientific Reports, in press) with the goal of developing Sema4A-based therapies for allergic disease.

Modulation of regulatory T cell function and stability by co-inhibitory receptors.

Regulatory T (Treg) cells constitute a dynamic population that is essential for controlling immune responses in health and disease. Defects in Treg cell function and decreases in Treg cell numbers have been observed in patients with autoimmunity and the opposite effects on Treg cells occur in cancer settings. Current research on new therapies for these diseases is focused on modulating Treg cell function to increase or decrease suppressive activity in autoimmunity and cancer, respectively. In this regard, several co-inhibitory receptors that are preferentially expressed by Treg cells under homeostatic conditions have recently been shown to control Treg cell function and stability in different disease settings. These receptors could be amenable to therapeutic targeting aimed at modulating Treg cell function and plasticity. This Review summarizes recent data regarding the role of co-inhibitory molecules in the control of Treg cell function and stability, with a focus on their roles and potential therapeutic use in autoimmunity and cancer.

Stability of regulatory T cells in T helper 2-biased allergic airway diseases.

Regulatory T (Treg) cells potentially suppress the deleterious activities of effector T cells and maintain a state of tolerance against antigens in the airway mucosa. A decrease in the number and function of Treg cells is observed in T helper 2 (Th2)-biased allergic airway diseases. However, adoptive transfer of naturally occurring Treg (tTreg) cells or peripherally derived Treg (pTreg) cells in asthmatic mouse models did not yield satisfactory results in any previous studies. Here, we review the recent progress in the identification and plasticity of tTreg and pTreg cells in Th2-biased airway diseases and summarize the factors affecting the stability and function of Treg cells. This review may serve as foundation for understanding the molecular mechanisms underlying the stability of tTreg and pTreg cells and development of effective strategies for treating allergic airway diseases.

Involvement of Tumor Necrosis Factor Receptor Type II in FoxP3 Stability and as a Marker of Treg Cells Specifically Expanded by Anti-Tumor Necrosis Factor Treatments in Rheumatoid Arthritis.

To study the involvement of Treg cells expressing tumor necrosis factor receptor type II (TNFRII) in exerting control of inflammation in experimental models and in the response to anti-TNF treatments in patients with rheumatoid arthritis (RA) or spondyloarthritis (SpA). The role of TNFRII in Treg cells was explored using a multilevel translational approach. Treg cell stability was evaluated by analyzing the methylation status of the Foxp3 locus using bisulfite sequencing. Two models of inflammation (imiquimod-induced skin inflammation and delayed-type hypersensitivity arthritis [DTHA]) were induced in TNFRII-/- mice, with or without transfer of purified CD4+CD25+ cells from wild-type (WT) mice. In patients with RA and those with SpA, the evolution of the TNFRII+ Treg cell population before and after targeted treatment was monitored. Foxp3 gene methylation in Treg cells was greater in TNFRII-/- mice than in WT mice (50% versus 36.7%). In cultured Treg cells, TNF enhanced the expression, maintenance, and proliferation of Foxp3 through TNFRII signaling. Imiquimod-induced skin inflammation and DTHA were aggravated in TNFRII-/- mice (P < 0.05 for mice with skin inflammation and P < 0.0001 for mice with ankle swelling during DTHA compared to WT mice). Adoptive transfer of WT mouse Treg cells into TNFRII-/- mice prevented aggravation of arthritis. In patients with RA receiving anti-TNF treatments, but not those receiving tocilizumab, the frequency of TNFRII+ Treg cells was increased at 3 months of treatment compared to baseline (mean ± SEM 65.2 ± 3.1% versus 49.1 ± 5.5%; P < 0.01). In contrast, in anti-TNF-treated patients with SpA, the frequency of TNFRII+ Treg cells was not modified. TNFRII expression identifies a subset of Treg cells that are characterized by stable expression of Foxp3 via gene hypomethylation, and adoptive transfer of TNFRII-expressing Treg cells ameliorates inflammation in experimental models. Expansion and activation of TNFRII+ Treg cells may be one of the mechanisms by which anti-TNF agents control inflammation in RA, but not in SpA.

Molecular Mechanisms Controlling Foxp3 Expression in Health and Autoimmunity: From Epigenetic to Post-translational Regulation.

The discovery of the transcription factor Forkhead box-p3 (Foxp3) has shed fundamental insights into the understanding of the molecular determinants leading to generation and maintenance of T regulatory (Treg) cells, a cell population with a key immunoregulatory role. Work over the past few years has shown that fine-tuned transcriptional and epigenetic events are required to ensure stable expression of Foxp3 in Treg cells. The equilibrium between phenotypic plasticity and stability of Treg cells is controlled at the molecular level by networks of transcription factors that bind regulatory sequences, such as enhancers and promoters, to regulate Foxp3 expression. Recent reports have suggested that specific modifications of DNA and histones are required for the establishment of the chromatin structure in conventional CD4+ T (Tconv) cells for their future differentiation into the Treg cell lineage. In this review, we discuss the molecular events that control Foxp3 gene expression and address the associated alterations observed in human diseases. Also, we explore how Foxp3 influences the gene expression programs in Treg cells and how unique properties of Treg cell subsets are defined by other transcription factors.

Single-cell RNA-seq unveils critical regulators of human FOXP3+ regulatory T cell stability

The heterogeneity and plasticity of T lymphocytes is critical for determining immune response outcomes. Functional regulatory T (Treg) cells are commonly characterized by stable FOXP3 expression and have reported to exhibit heterogeneous phenotypes under inflammatory conditions. However, the interplay between inflammation and Treg cell suppressive activity still remains elusive. Here, we utilized single-cell RNA sequencing to investigate how human Treg cells respond to the pro-inflammatory cytokine interleukin-6 (IL-6). We observed that Treg cells divided into two subpopulations after IL-6 stimulation. TIGIT− unstable Treg cells lost FOXP3 expression and gained an effector-like T cell phenotype, whereas TIGIT+ Treg cells retained robust suppressive function. Single cell transcriptome analysis revealed a spectrum of cellular states of IL-6-stimulated Treg cells and how cytochrome P450 family 1 subfamily A member 1 (CYP1A1) is a crucial regulator of Treg cell suppressive capability and stability. CYP1A1-deficient human Treg cells developed a Th17-like phenotype after IL-6 stimulation. Our findings implicate CYP1A1 as a previously unidentified regulator of Treg cells that may have target potential for clinical application for biotherapies.

An intrinsic role of IL-33 in Treg cell-mediated tumor immunoevasion.

Regulatory T (Treg) cells accumulate into tumors hindering the success of cancer immunotherapy. Yet, therapeutic targeting of Treg cells shows limited efficacy or leads to autoimmunity. The molecular mechanisms that guide Treg cell stability in tumors, remain elusive. Herein, we identify a cell-intrinsic role of the alarmin IL-33 in the functional stability of Treg cells. Specifically, IL-33-deficient Treg cells demonstrated attenuated suppressive properties in vivo and facilitated tumor regression in an ST2 (IL-33 receptor)-independent fashion. Upon activation, Il33–/– Treg cells exhibited epigenetic reprogramming with increased chromatin accessibility of the Ifng locus leading to elevated interferon-γ (IFN-γ) production in an NF-κB–T-bet-dependent manner. IFN-γ was essential for Treg cell defective function since its ablation restored Il33–/– Treg cell suppressive properties. Importantly, genetic ablation of Il33 potentiated the therapeutic effect of immunotherapy. Our findings reveal a novel and therapeutically important intrinsic role of IL-33 in Treg cell stability in cancer.

Metabolic Control of Treg Cell Stability, Plasticity, and Tissue-Specific Heterogeneity.

Regulatory T (Treg) cells are crucial for peripheral immune tolerance and prevention of autoimmunity and tissue damage. Treg cells are inherently defined by the expression of the transcription factor Foxp3, which enforces lineage development and immune suppressive function of these cells. Under various conditions as observed in autoimmunity, cancer and non-lymphoid tissues, a proportion of Treg cells respond to specific environmental signals and display altered stability, plasticity and tissue-specific heterogeneity, which further shape their context-dependent suppressive functions. Recent studies have revealed that metabolic programs play pivotal roles in controlling these processes in Treg cells, thereby considerably expanding our understanding of Treg cell biology. Here we summarize these recent advances that highlight how cell-extrinsic factors, such as nutrients, vitamins and metabolites, and cell-intrinsic metabolic programs, orchestrate Treg cell stability, plasticity, and tissue-specific heterogeneity. Understanding metabolic regulation of Treg cells should provide new insight into immune homeostasis and disease, with important therapeutic implications for autoimmunity, cancer, and other immune-mediated disorders.

Homeobox protein Hhex negatively regulates Treg cells by inhibiting Foxp3 expression and function

Regulatory T (Treg) cells play an essential role in maintaining immune homeostasis, but the suppressive function of Treg cells can be an obstacle in the treatment of cancer and chronic infectious diseases. Here, we identified the homeobox protein Hhex as a negative regulator of Treg cells. The expression of Hhex was lower in Treg cells than in conventional T (Tconv) cells. Hhex expression was repressed in Treg cells by TGF-β/Smad3 signaling. Retroviral overexpression of Hhex inhibited the differentiation of induced Treg (iTreg) cells and the stability of thymic Treg (tTreg) cells by significantly reducing Foxp3 expression. Moreover, Hhex-overexpressing Treg cells lost their immunosuppressive activity and failed to prevent colitis in a mouse model of inflammatory bowel disease (IBD). Hhex expression was increased; however, Foxp3 expression was decreased in Treg cells in a delayed-type hypersensitivity (DTH) reaction, a type I immune reaction. Hhex directly bound to the promoters of Foxp3 and other Treg signature genes, including Il2ra and Ctla4, and repressed their transactivation. The homeodomain and N-terminal repression domain of Hhex were critical for inhibiting Foxp3 and other Treg signature genes. Thus, Hhex plays an essential role in inhibiting Treg cell differentiation and function via inhibition of Foxp3.

Stability and inhibitory function of Treg cells under inflammatory conditions in vitro.

Immunotherapy with transplanted T-regulatory (Treg) cells is currently in use. However, patients have complex internal environments with confounding factors, including the presence of inflammatory cytokines. The present study aimed to detect Treg cell function under simulated inflammatory conditions to provide a foundation for Treg cell-based immunotherapy. CD4+CD25high Treg cells were sorted from peripheral blood mononuclear cells and cultured for 14 days in the presence of recombinant human interleukin-2 (rhIL-2) and anti-CD3/CD28 beads, with or without 25 ng/ml rhIL-6. Next, the absolute count of Treg cells was determined, the stability and activity were detected by measuring the expression levels of forkhead box (Fox)P3 and CD39, and the suppressive function of Treg cells was investigated by assessing the suppression of T-effector cell proliferation by Treg cells after co-culture for 5 days. The number of Treg cells cultured in the presence of 25 ng/ml rhIL-6 for 14 days was reduced by 49.7% when compared with that of cells cultured without rhIL-6. Of the Treg cells continually cultured for 14 days without or with 25 ng/ml rhIL-6, 56.15 and 24.7% expressed FoxP3, respectively. There was no difference in the activity of the FoxP3+ Treg cells after culture for 14 days without or with 25 ng/ml rhIL-6. The suppressive function of Treg cells tended to deteriorate in the presence of rhIL-6. In conclusion, IL-6 inhibited the proliferation and stability of Treg cells, suggesting that administration of increased numbers of Treg cells may be required during Treg cell-based immunotherapy.

The transcription factor NFIL3 controls regulatory T-cell function and stability

Regulatory T (Treg) cells are a CD4 T-cell subset with an important role in immune tolerance; however, the mechanisms underlying Treg cell differentiation and function are incompletely understood. Here, we show that NFIL3/E4BP4, a transcription factor, plays a key role in Treg cell differentiation and function. Microarray analysis showed that Treg cells had lower Nfil3 expression than all other CD4 T-cell subsets. Overexpression of Nfil3 in Treg cells led to diminished expression of Foxp3 and other signature Treg genes, including Il2ra, Icos, Tnfrsf18, and Ctla4. Furthermore, Nfil3-overexpressing Treg cells exhibited impaired immunosuppressive activity in vitro and in vivo. We discovered that NFIL3 directly binds to and negatively regulates the expression of Foxp3. In addition, bisulfite sequencing revealed that NFIL3 induces methylation at Foxp3 locus regulatory CpG sites, which contributes to the control of Treg cell stability. Together, these data indicate that NFIL3 impairs Treg cell function through the downregulation of Foxp3 expression.

Intratumoral regulatory T cells: markers, subsets and their impact on anti-tumor immunity.

Regulatory T (Treg) cells play a crucial role in maintaining self-tolerance and resolution of immune responses by employing multifaceted immunoregulatory mechanisms. However, Treg cells readily infiltrate into the tumor microenvironment (TME) and dampen anti-tumor immune responses, thereby becoming a barrier to effective cancer immunotherapy. There has been a substantial expansion in the development of novel immunotherapies targeting various inhibitory receptors (IRs), such as CTLA4, PD1 and LAG3, but these approaches have mechanistically focused on the elicitation of anti-tumor responses. However, enhanced inflammation in the TME could also play a detrimental role by facilitating the recruitment, stability and function of Treg cells by up-regulating chemokines that promote Treg cell migration, and/or increasing inhibitory cytokine production. Furthermore, IR blockade may enhance Treg cell function and survival, thereby serving as a resistance mechanism against effective immunotherapy. Given that Treg cells are comprised of functionally and phenotypically heterogeneous sub-populations that may alter their characteristics in a context-dependent manner, it is critical to identify unique molecular pathways that are preferentially used by intratumoral Treg cells. In this review, we discuss markers that serve to identify certain Treg cell subsets, distinguished by chemokine receptors, IRs and cytokines that facilitate their migration, stability and function in the TME. We also discuss how these Treg cell subsets correlate with the clinical outcome of patients with various types of cancer and how they may serve as potential TME-specific targets for novel cancer immunotherapies.

Activation of Mevalonate Pathway via LKB1 Is Essential for Stability of Treg Cells

The function of regulatory T (Treg) cells depends on lipid oxidation. However, the molecular mechanism by which Treg cells maintain lipid metabolism after activation remains elusive. Liver kinase B1 (LKB1) acts as a coordinator by linking cellular metabolism to substrate AMP-activated protein kinase (AMPK). We show that deletion of LKB1 in Treg cells exhibited reduced suppressive activity and developed fatal autoimmune inflammation. Mechanistically, LKB1 induced activation of the mevalonate pathway by upregulating mevalonate genes, which was essential for Treg cell functional competency and stability by inducing Treg cell proliferation and suppressing interferon-gamma and interleukin-17A expression independently of AMPK. Furthermore, LKB1 was found to regulate intracellular cholesterol homeostasis and to promote the mevalonate pathway. In agreement, mevalonate and its metabolite geranylgeranyl pyrophosphate inhibited conversion of Treg cells and enhanced survival of LKB1-deficient Treg mice. Thus, LKB1 is a key regulator of lipid metabolism in Treg cells, involved in optimal programming of suppressive activity, immune homeostasis, and tolerance.

Plexin B1 expression on human Treg cells is required for regulation of their phenotypic stability by semaphorin 4A

Abstract We previously reported that neuroimmune semaphorin (Sema) 4A regulates the severity of experimental allergic asthma and increases regulatory T-cell (Treg) numbers in vivo, however the mechanisms of Sema4A action remain unknown. It was also reported that Sema4A controls murine Treg cell function and survival acting through Neuropilin 1 (NRP-1) receptor. To clarify Sema4A action on human T cells, we employed T-cell lines, HuT78 and HuT102, as well as human PBMCs and CD4+ T cells in phenotypic and functional assays. We found that HuT78 demonstrated a T-effector-like phenotype (CD4+CD25lowFoxp3-), while HuT102 expressed a Treg-like phenotype (CD4+CD25hi Foxp3+). Neither cell line expressed NRP-1. However, HuT102 cells expressed high levels of the Sema4A-counter receptor, Plexin B1 whereas HuT78 cells demonstrated high cell-surface expression of Sema4A. All human peripheral blood CD4+ T cells, including Treg cells, expressed PlexinB1 and lacked both, NRP-1 and −2. Culture of HuT cells with soluble Sema4A led to an upregulation of CD25 and Foxp3 markers on HuT102 cells. Addition of soluble Sema4A increased the relative numbers of CD4+CD25+Foxp3+ cells in PBMC and CD4+ T cells which did not express NRP-1 but were PlexinB1+, suggesting the role of this receptor in Treg cell stability. The inclusion of anti-PlexinB1 blocking Ab in cultures before rSema4A addition significantly decreased Treg cell numbers as compared to cultures with rSema4A alone. Sema4A was as effective as TGF-b in iTreg induction from CD4+CD25depleted cells but did not affect Treg cell suppressive activity in vitro. These results suggest strategies for the development of new Sema4A-based therapeutic measures to combat allergic inflammatory diseases.

A critical role for IL-37 in regulatory T cell maintenance and CD4+ T cell inhibition

Abstract Regulatory T (Treg) cells express the transcription factor Foxp3 and play a vital role in suppressing immune responses and maintaining tolerance and homeostasis. Foxp3 expression is induced and maintained in Treg cells during thymic development and adaptive immune insults through three signaling pathways including TCR signaling and costimulation, IL-2 signaling, and TGF-β signaling, as well as Foxp3 expression itself. Recent studies, however, have suggested that these signaling pathways involved in the induction and maintenance of Foxp3 expression in the thymus and during activation are more dispensable in a steady state. To identify additional intracellular signaling molecules that induce/maintain Foxp3 expression and support Treg cell homeostasis and stability, we have screened proteins interacting with the three Foxp3 transcription factors NFAT, Stat5 and Smad3 for their expression in Treg cells and regulation of Foxp3 transcription. Using this protein screen we discovered IL-1 family member IL-37 plays a significant role in Foxp3 expression. By using human T cells from healthy donors and transgenic mice expressing IL-37, we uncovered a novel role for endogenous (expressed) and extracellular (secreted) IL-37 in Foxp3 expression and CD4+ T cells immunity. Treg cells that expressed IL-37 had elevated Foxp3 expression and were more suppressive in vitro and in vivo compared to Treg cells lacking IL-37. Extracellular IL-37, secreted by Treg cells, directly inhibited the canonical T cell signaling pathways through surface receptors. These results characterize an important mechanism for Treg directed immune suppression and Foxp3 expression.

Loss of TET2 and TET3 in regulatory T cells unleashes effector function

TET enzymes oxidize 5-methylcytosine to 5-hydroxymethylcytosine and other oxidized methylcytosines in DNA. Here we examine the role of TET proteins in regulatory T (Treg) cells. Tet2/3fl/flFoxp3Cre mice lacking Tet2 and Tet3 in Treg cells develop inflammatory disease, and Treg cells from these mice show altered expression of Treg signature genes and upregulation of genes involved in cell cycle, DNA damage and cancer. In littermate mice with severe inflammation, both CD4+Foxp3+ and CD4+Foxp3− cells show strong skewing towards Tfh/Th17 phenotypes. Wild-type Treg cells in mixed bone marrow chimeras and in Tet2/3fl/flFoxp3WT/Cre heterozygous female mice are unable to rescue the aberrant properties of Tet2/3fl/flFoxp3Cre Treg cells. Treg cells from Tet2/3fl/flFoxp3Cre mice tend to lose Foxp3 expression, and transfer of total CD4+ T cells isolated from Tet2/3fl/flFoxp3Cre mice could elicit inflammatory disease in fully immunocompetent mice. Together, these data indicate that Tet2 and Tet3 are guardians of Treg cell stability and immune homeostasis.

Treg cells in autoimmunity: from identification to Treg-based therapies.

Regulatory (Treg) cells are key regulators of inflammation and important for immune tolerance and homeostasis. A major progress has been made in the identification and classification of Treg cells. Due to technological advances, we have gained deep insights in the epigenetic regulation of Treg cells. The use of fate reporter mice allowed addressing the functional consequences of loss of Foxp3 expression. Depending on the environment Treg cells gain effector functions upon loss of Foxp3 expression. However, the traditional view that Treg cells become necessarily pathogenic by gaining effector functions was challenged by recent findings and supports the notion of Treg cell lineage plasticity. Treg cell stability is also a major issue for Treg cell therapies. Clinical trials are designed to use polyclonal Treg cells as therapeutic tools. Here, we summarize the role of Treg cells in selected autoimmune diseases and recent advances in the field of Treg targeted therapies.

Semaphorin 4A Stabilizes Human Regulatory T Cell Phenotype via Plexin B1.

We previously reported that neuroimmune semaphorin (Sema) 4A regulates the severity of experimental allergic asthma and increases regulatory T (Treg) cell numbers in vivo; however, the mechanisms of Sema4A action remain unknown. It was also reported that Sema4A controls murine Treg cell function and survival acting through neuropilin 1 (NRP-1) receptor. To clarify Sema4A action on human T cells, we employed T cell lines (HuT78 and HuT102), human PBMCs, and CD4+ T cells in phenotypic and functional assays. We found that HuT78 demonstrated a T effector-like phenotype (CD4+CD25lowFoxp3-), whereas HuT102 expressed a Treg-like phenotype (CD4+CD25hi Foxp3+). Neither cell line expressed NRP-1. HuT102 cells expressed Sema4A counter receptor Plexin B1, whereas HuT78 cells were Sema4A+. All human peripheral blood CD4+ T cells, including Treg cells, expressed PlexinB1 and lacked both NRP-1 and -2. However, NRP-1 and Sema4A were detected on CD3negativeCD4intermediate human monocytes. Culture of HuT cells with soluble Sema4A led to an upregulation of CD25 and Foxp3 markers on HuT102 cells. Addition of Sema4A increased the relative numbers of CD4+CD25+Foxp3+ cells in PBMCs and CD4+ T cells, which were NRP-1negative but PlexinB1+, suggesting the role of this receptor in Treg cell stability. The inclusion of anti-PlexinB1 blocking Ab in cultures before recombinant Sema4A addition significantly decreased Treg cell numbers as compared with cultures with recombinant Sema4A alone. Sema4A was as effective as TGF-β in inducible Treg cell induction from CD4+CD25depleted cells but did not enhance Treg cell suppressive activity in vitro. These results suggest strategies for the development of new Sema4A-based therapeutic measures to combat allergic inflammatory diseases. ImmunoHorizons, 2019, 3: 71-87.

Blimp1 Prevents Methylation of Foxp3 and Loss of Regulatory T Cell Identity at Sites of Inflammation

SummaryFoxp3+ regulatory T (Treg) cells restrict immune pathology in inflamed tissues; however, an inflammatory environment presents a threat to Treg cell identity and function. Here, we establish a transcriptional signature of central nervous system (CNS) Treg cells that accumulate during experimental autoimmune encephalitis (EAE) and identify a pathway that maintains Treg cell function and identity during severe inflammation. This pathway is dependent on the transcriptional regulator Blimp1, which prevents downregulation of Foxp3 expression and “toxic” gain-of-function of Treg cells in the inflamed CNS. Blimp1 negatively regulates IL-6- and STAT3-dependent Dnmt3a expression and function restraining methylation of Treg cell-specific conserved non-coding sequence 2 (CNS2) in the Foxp3 locus. Consequently, CNS2 is heavily methylated when Blimp1 is ablated, leading to a loss of Foxp3 expression and severe disease. These findings identify a Blimp1-dependent pathway that preserves Treg cell stability in inflamed non-lymphoid tissues.

Activation of Mevalonate Pathway Via LKB1 is Essential for Stability of Treg Cells

The function of Foxp3+ regulatory T cells (Treg cells) depends on lipid oxidation. However, the molecular mechanism by which Treg cells maintain lipid metabolism after activation remains elusive. LKB1 serine/threonine kinase acts as a coordinator of metabolism by linking cellular metabolism to the substrate AMP-activated protein kinase (AMPK). We found that mice with a conditional deletion of LKB1 in Treg cells developed a fatal autoimmune inflammation. LKB1-deficient Treg cells exhibited a reduced suppressive activity associated with effector T cell-like phenotypes. Mechanistically, LKB1 induced the activation of mevalonate pathway by up-regulating mevalonate genes in Treg cells independently of AMPK. LKB1-mediated mevalonate pathway was essential for Treg cell functional competency and stability by inducing Treg cell proliferation and by suppressing their IFN-γ and IL-17A expression. In agreement, mevalonate and its metabolite geranylgeranyl pyrophosphate (GGPP) not only inhibited the conversion of Treg cells to Th1- and Th17-like cells, but also enhanced the survival of LKB1-deficient Treg cells. Our results demonstrate that LKB1 is a key regulator of lipid metabolisms in Treg cells, involved in optimal programming of suppressive activity, immune homeostasis, and tolerance.