Differentiation Of iTreg Cells Research Articles

GAB functions as a bioenergetic and signalling gatekeeper to control T cell inflammation

γ-Aminobutyrate (GAB), the biochemical form of (GABA) γ-aminobutyric acid, participates in shaping physiological processes, including the immune response. How GAB metabolism is controlled to mediate such functions remains elusive. Here we show that GAB is one of the most abundant metabolites in CD4+ T helper 17 (TH17) and induced T regulatory (iTreg) cells. GAB functions as a bioenergetic and signalling gatekeeper by reciprocally controlling pro-inflammatory TH17 cell and anti-inflammatory iTreg cell differentiation through distinct mechanisms. 4-Aminobutyrate aminotransferase (ABAT) funnels GAB into the tricarboxylic acid (TCA) cycle to maximize carbon allocation in promoting TH17 cell differentiation. By contrast, the absence of ABAT activity in iTreg cells enables GAB to be exported to the extracellular environment where it acts as an autocrine signalling metabolite that promotes iTreg cell differentiation. Accordingly, ablation of ABAT activity in T cells protects against experimental autoimmune encephalomyelitis (EAE) progression. Conversely, ablation of GABAA receptor in T cells worsens EAE. Our results suggest that the cell-autonomous control of GAB on CD4+ T cells is bimodal and consists of the sequential action of two processes, ABAT-dependent mitochondrial anaplerosis and the receptor-dependent signalling response, both of which are required for T cell-mediated inflammation.

A RORγt+ cell instructs gut microbiota-specific Treg cell differentiation.

The mutualistic relationship of gut-resident microbiota and the host immune system promotes homeostasis that ensures maintenance of the microbial community and of a largely non-aggressive immune cell compartment1,2. The consequences of disturbing this balance include proximal inflammatory conditions, such as Crohn's disease, and systemic illnesses. This equilibrium is achieved in part through the induction of both effector and suppressor arms of the adaptive immune system. Helicobacter species induce T regulatory (Treg) and T follicular helper (TFH) cells under homeostatic conditions, but induce inflammatory T helper 17 (TH17) cells when induced Treg (iTreg) cells are compromised3,4. How Helicobacter and other gut bacteria direct T cells to adopt distinct functions remains poorly understood. Here we investigated the cells and molecular components required for iTreg cell differentiation. We found that antigen presentation by cells expressing RORγt, rather than by classical dendritic cells, was required and sufficient for induction of Treg cells. These RORγt+ cells-probably type 3 innate lymphoid cells and/or Janus cells5-require the antigen-presentation machinery, the chemokine receptor CCR7 and the TGFβ activator αv integrin. In the absence of any of these factors, there was expansion of pathogenic TH17 cells instead of iTreg cells, induced by CCR7-independent antigen-presenting cells. Thus, intestinal commensal microbes and their products target multiple antigen-presenting cells with pre-determined features suited to directing appropriate T cell differentiation programmes, rather than a common antigen-presenting cell that they endow with appropriate functions.

Prostaglandin E2 directly inhibits the conversion of inducible regulatory T cells through EP2 and EP4 receptors via antagonizing TGF-β signalling.

Regulatory T (Treg) cells are essential for control of inflammatory processes by suppressing effector T‐cell functions. The actions of PGE2 on the development and function of Treg cells, particularly under inflammatory conditions, are debated. In this study, we employed pharmacological and genetic approaches to examine whether PGE2 had a direct action on T cells to modulate de novo differentiation of Treg cells. We found that TGF‐β‐induced Foxp3 expression and iTreg cell differentiation in vitro is markedly inhibited by PGE2, which was mediated by the receptors EP2 and EP4. Mechanistically, PGE2‐EP2/EP4 signalling interrupts TGF‐β signalling during iTreg differentiation. Moreover, EP4 deficiency in T cells impaired iTreg cell differentiation in vivo. Thus, our results demonstrate that PGE2 negatively regulates iTreg cell differentiation through a direct action on T cells, highlighting the potential for selectively targeting the PGE2‐EP2/EP4 pathway to control T cell‐mediated inflammation.

Arkadia-SKI/SnoN signaling differentially regulates TGF-β-induced iTreg and Th17 cell differentiation.

TGF-β signaling is fundamental for both Th17 and regulatory T (Treg) cell differentiation. However, these cells differ in requirements for downstream signaling components, such as SMAD effectors. To further characterize mechanisms that distinguish TGF-β signaling requirements for Th17 and Treg cell differentiation, we investigated the role of Arkadia (RNF111), an E3 ubiquitin ligase that mediates TGF-β signaling during development. Inactivation of Arkadia in CD4+ T cells resulted in impaired Treg cell differentiation in vitro and loss of RORγt+FOXP3+ iTreg cells in the intestinal lamina propria, which increased susceptibility to microbiota-induced mucosal inflammation. In contrast, Arkadia was dispensable for Th17 cell responses. Furthermore, genetic ablation of two Arkadia substrates, the transcriptional corepressors SKI and SnoN, rescued Arkadia-deficient iTreg cell differentiation both in vitro and in vivo. These results reveal distinct TGF-β signaling modules governing Th17 and iTreg cell differentiation programs that could be targeted to selectively modulate T cell functions.

Whole-genome analysis of TET dioxygenase function in regulatory T cells

TET methylcytosine dioxygenases are essential for the stability and function of regulatory T cells (Treg cells), which maintain immune homeostasis and self-tolerance and express the lineage-determining transcription factor Foxp3. Here, we use whole-genome analyses to show that the transcriptional program and epigenetic features (DNA modification, chromatin accessibility) of Treg cells are attenuated in the absence of Tet2 and Tet3. Conversely, the addition of the TET activator vitamin C during TGFβ-induced iTreg cell differentiation in vitro potentiates the expression of Treg signature genes and alters the epigenetic landscape to better resemble that of Treg cells generated in vivo. Vitamin C enhances IL-2 responsiveness in iTreg cells by increasing IL2Rα expression, STAT5 phosphorylation, and STAT5 binding, mimicking the IL-2/STAT5 dependence of Treg cells generated in vivo. In summary, TET proteins play essential roles in maintaining Treg molecular features and promoting their dependence on IL-2. TET activity during endogenous Treg development and potentiation of TET activity by vitamin C during iTreg differentiation are necessary to maintain the transcriptional and epigenetic features of Treg cells.

Resolvin D1 Protects Against Ischemia/Reperfusion-Induced Acute Kidney Injury by Increasing Treg Percentages via the ALX/FPR2 Pathway.

AimsTo evaluate whether Resolvin D1 attenuates ischemia/reperfusion-induced (IRI) acute kidney injury (AKI) via affecting Tregs.Materials and MethodsThe IRI-AKI mouse model was established, and RvD1 was injected into the mouse tail vein. Further, the renal function, histological changes, injury markers and serum cytokines were detected at 24 and 72 h after IRI. Flow cytometry was used to categorize regulatory T cells (Tregs) in the spleen and kidney. Treg cells were stripped with the anti-CD25 antibody blocker PC61 to assess its role in the protective effect of RvD1 on IRI mice. CD4+ T cells were obtained from spleen monocytes by magnetic bead sorting and differentiated into induced Treg (iTreg) cells. The effect of RvD1 on iTreg cell differentiation was observed in vitro. In addition, neutralizing antibodies against the orphan receptor G-protein-coupled receptor 32 (anti-GPR32) and LXA4 receptor (anti-ALX/FPR2), both RvD1 receptor blockers, were used to evaluate the effect of RvD1 on iTreg cell differentiation. Boc-1, an ALX/FPR2 receptor inhibitor, was administered via the tail vein to observe its effects on the ameliorative efficacy of RvD1 in IRI-AKI mice in vivo.ResultsIn vivo, RvD1 increased Treg percentages, alleviated renal tubular injury and reduced the serum levels of IFN-γ, TNF-α and IL-6 in IRI-AKI mice, while PC61 depleted the number of Tregs and reversed the protective effects of RvD1. In vitro, RvD1 induced the generation of iTregs. Importantly, preincubation with anti-ALX/FPR2 neutralizing antibodies but not with anti-GPR32 neutralizing antibodies, abrogated the enhancement activity of RvD1 on iTregs. In addition, in vivo blockade of the receptor ALX/FPR2 by Boc-1 reversed the beneficial effects of RvD1 on the splenic and kidney Treg percentages, renal tubular injury and serum IFN-γ, TNF-α, and IL-6 levels.ConclusionOur study demonstrates that RvD1 protects against IRI-AKI by increasing the percentages of Tregs via the ALX/FPR2 pathway.

Shikonin Controls the Differentiation of CD4+CD25+ Regulatory T Cells by Inhibiting AKT/mTOR Pathway.

CD4+CD25+ regulatory T (Treg) cells maintain the function of immune tolerance and the balance of immune cells. Defects in the number and function of Treg cells can induce the development and progression of inflammatory disease. Shikonin, the main active ingredient of Lithospermum, has anti-inflammatory and anti-tumor effects. Shikonin is also an effective drug for the treatment of psoriasis, which is a chronic inflammatory skin disease. However, the underlying mechanism is not yet clear. To evaluate the role of shikonin on the induction of Treg cells, we tested the number and function of Treg cells in vivo and in vitro. Shikonin can effectively promote the differentiation of iTreg cells by inhibiting the AKT/mTOR pathway in vitro. Moreover, in vivo, intragastrically administered shikonin effectively improved lesions in mice with imiquimod-induced psoriasis and increased the number of iTreg cells in the spleen and their secretion. Shikonin significantly increases the expression of Foxp3mRNA in skin of the psorisic mice. Therefore, we expect that shikonin can prevent the development of inflammation and treat psoriasis by regulating iTreg cells. Novel ideas for the treatment of psoriasis are also proposed.

Tetrandrine inhibits differentiation of proinflammatory subsets of T helper cells but spares de novo differentiation of iTreg cells

Tetrandrine (TET) is an anti-inflammatory compound isolated from Chinese herb Stephania tetrandra S. Moore. It was reported recently that the differentiation of Th17 cells was inhibited, while the generation of induced Treg cells (iTregs) was promoted, by TET treatment. We therefore carefully examined the effect of TET on the differentiation of four major subsets of T helper cells. The results showed that in vitro treatment with TET potently inhibited the differentiation of Th1, Th2 and Th17 cells. Administration of LPS resulted in a mixed Th1, Th2 and Th17 responses in normal mice, and such effect of LPS was inhibited by in vivo TET treatment as well. In contrast, TET did not promote or inhibit the in vitro generation of iTregs from naïve CD4+CD25−Foxp3/gfp− T cells. Furthermore, spontaneous and rapamycin-induced conversion of naïve CD4+CD25−Foxp3/gfp− T cells into Foxp3-expressing iTregs in congenic mice was not affected by TET treatment. Thus, TET had the capacity to inhibit the differentiation of proinflammatory Th1, Th2 and Th17 cells, while sparing the generation of Tregs. As a Treg-friendly and broad spectrum anti-inflammatory agent, the molecular mechanism and the therapeutic potential of TET in various human inflammatory diseases should be further studied.

Contribution of neuraminidase 3 to the differentiation of induced regulatory T cells.

Neuraminidase family enzymes that hydrolyze the terminal sialic acid linkage in biomolecules are involved in various immune responses. We previously showed that Th1 and Th2 cells differentially express several neuraminidases. Herein, the expression of neuraminidases in induced regulatory T (iTreg) cells was investigated in comparison with that in other T-cell subsets. Contrary to the tendency toward higher neuraminidase 1 mRNA expression in in vitro-differentiated Th2 cells, compared to Th1, Th17 and iTreg cells, we observed significantly higher expression of neuraminidase 3 (Neu3) in iTreg cells. Furthermore, the expression of Neu3 in FoxP3+ CD62L- spleen cells was higher than that in FoxP3+ CD62L+ and FoxP3- cells. Lentiviral expression of Neu3 in naïve CD4+ T cells during the stimulation culture led to upregulation of FoxP3 expression. On the basis of these findings, we conclude that Neu3 contributes to the differentiation of iTreg cells by upregulation of FoxP3.

Roquin Suppresses the PI3K-mTOR Signaling Pathway to Inhibit T Helper Cell Differentiation and Conversion of Treg to Tfr Cells

Roquin proteins preclude spontaneous Tcell activation and aberrant differentiation of T follicular helper (Tfh) or T helper 17 (Th17) cells. Here we showed that deletion of Roquin-encoding alleles specifically in regulatory T (Treg) cells also caused the activation of conventional Tcells. Roquin-deficient Treg cells downregulated CD25, acquired a follicular Treg (Tfr) cell phenotype, and suppressed germinal center reactions but could not protect from colitis. Roquin inhibited the PI3K-mTOR signaling pathway by upregulation of Pten through interfering with miR-17∼92 binding to an overlapping cis-element in the Pten 3'UTR, and downregulated the Foxo1-specific E3 ubiquitin ligase Itch. Loss of Roquin enhanced Akt-mTOR signaling and protein synthesis, whereas inhibition of PI3K or mTOR in Roquin-deficient Tcells corrected enhanced Tfh and Th17 or reduced iTreg cell differentiation. Thereby, Roquin-mediated control of PI3K-mTOR signaling prevents autoimmunity by restraining activation and differentiation of conventional Tcells and specialization of Treg cells.

Reciprocal regulation of Th17 and Treg cell differentiation by RhoA

Abstract Th17 cells are important for protective immunity as well as tissue inflammation and autoimmune disorders. Treg cells, including induced Treg (iTreg) and natural Treg (nTreg), play a critical role in immune tolerance. RhoA of the Rho small GTPase family is an intracellular signal transducer that cycles between an inactive GDP-bound form and an active GTP-bound form. We have previously reported that RhoA plays an important role in Th2 cell differentiation by regulating glycolysis. In this study, we further examined the role of RhoA in Th17 and Treg cell differentiation. Genetic deletion of RhoA in T cells or pharmacologic inhibition of RhoA by a RhoA-specific inhibitor Y16 led to reduced Th17 differentiation in vitro as evidenced by reduction of Th17 effector cytokines, transcriptional downregulation of RORγT and reduced phosphorylation of Stat3. Accordingly, RhoA deficiency ameliorated house dust mites (HDM)-induced allergic airway inflammation and dextran sodium sulphate (DSS)-induced colitis in vivo where Th17 cells plays an important role. Inhibition of ROCK, an immediate downstream effector of RhoA, recapitulated the effect of RhoA deficiency or inhibition on Th17 differentiation, suggesting that RhoA regulates Th17 cell differentiation through ROCK. On the other hand, ablation of RhoA had no effect on iTreg cell differentiation. However, inhibition of ROCK enhanced iTreg differentiation, suggesting that ROCK has RhoA-independent role in iTreg differentiation. In contrast to its in-effect on iTreg differentiation, RhoA deficiency increased nTreg differentiation. Thus, our data have provided new insights into Th17 and Treg differentiation and identified RhoA as a potential therapeutic target for tissue inflammation.

Obesity Drives Th17 Cell Differentiation by Inducing the Lipid Metabolic Kinase, ACC1

Chronic inflammation due to obesity contributes to the development of metabolic diseases, autoimmune diseases, and cancer. Reciprocal interactions between metabolic systems and immune cells have pivotal roles in the pathogenesis of obesity-associated diseases, although the mechanisms regulating obesity-associated inflammatory diseases are still unclear. In the present study, we performed transcriptional profiling of memory phenotype CD4 T cells in high-fat-fed mice and identified acetyl-CoA carboxylase 1 (ACC1, the gene product of Acaca) as an essential regulator of Th17 cell differentiation in vitro and of the pathogenicity of Th17 cells in vivo. ACC1 modulates the DNA binding of RORγt to target genes in differentiating Th17 cells. In addition, we found a strong correlation between IL-17A-producing CD45RO(+)CD4 T cells and the expression of ACACA in obese subjects. Thus, ACC1 confers the appropriate function of RORγt through fatty acid synthesis and regulates the obesity-related pathology of Th17 cells.

An epigenetic mechanism of Dll4-facilitated Foxp3+ regulatory T cells differentiation (LYM8P.637)

Abstract Notch signaling facilitates inducible Foxp3+ regulatory T cells (iTreg) differentiation. Epigenetic modifications, which include histone modifications, are critical in cell differentiation. Recent genome-wide studies showed that iTreg have increased trimethylation on histone H3 at lysine 4 (H3K4me3) around Foxp3 loci, but the role of Notch signaling and its underlying epigenetic mechanisms remain elusive. Here we report that Delta-like ligand 4 (Dll4) but not Jagged1 can activate Notch signaling, facilitate Foxp3+ iTreg differentiation, and increased H3K4me3 around Foxp3 promoter and conserved noncoding sequences (CNS). We performed epigenetic enzyme array analysis and found one histone methyltransferase—SET and MYND domain containing protein 3 (Smyd3) that is abundant in iTreg. Dll4/Notch signaling amplified and accelerated Smyd3 expression in vitro during iTreg cell differentiation via an RBPJ-mediated transcription activation. Inhibition of Notch signaling decreased Smyd3 expression. When Smyd3 was genetically knocked out in CD4 T cells, the Dll4-enhanced H3K4me3 around the Foxp3 loci and Foxp3+ Tregs were decreased in vitro and in vivo. These results demonstrate that Notch ligand Dll4 can epigenetically regulate Foxp3+ Tregs through Smyd3.

Translational control of TGF-β-mediated Treg cell differentiation by hnRNP E1 (IRM15P.460)

Abstract CD4+Foxp3+ T cells are generated either in the thymus for natural regulatory T cells, or in periphery for inducible Treg cells (iTregs), which differentiate from naïve T cell precursors. Both Tregs express Foxp3 albeit they differ in their plasticity and in their expression of certain genes. Trans­forming growth factor-β (TGF-β) and interleukin-2 are important for iTreg generation and maintenance, whereas proinflammatory cytokines such as IL-4 and IL-6 impair Foxp3 induction. Much has been learned regarding the transcriptional roles of TGF-β in determining the fate of iTregs; however, the limited focus if any on a translational mechanism by which TGF-β regulates the fate of functional T cells is somewhat myopic. We previously showed a novel translational mechanism for TGF-β regulation of EMT in mouse mammary gland epithelial cells whereby hnRNP E1 (E1) binds to a structural, 33-nucleotide TGF-β-activated translation element in the 3’ UTR of target transcripts and represses their translation. In this study, we found that E1 knockdown (E1KD) in naïve CD4+ T cells induces TGF-β signaling and upregulates expression of multiple TGF-β target molecules implicated in iTreg cell differentiation. Furthermore, E1KD CD4+ T cells bypass the need for TGF-β and express Foxp3. This study adds to our knowledge on a hitherto unrecognized translational mechanism by which TGF-β controls iTreg cell fate.

Hypoxic culture conditions enhance the generation of regulatory T cells.

The generation of large amounts of induced CD4+ CD25+ Foxp3+ regulatory T (iTreg) cells is of great interest for several immunotherapy applications, therefore a better understanding of signals controlling iTreg cell differentiation and expansion is required. There is evidence that oxidative metabolism may regulate several key signalling pathways in T cells. This prompted us to investigate the effects of oxygenation on iTreg cell generation by comparing the effects of atmospheric (21%) or of low (5%) O2 concentrations on the phenotype of bead-stimulated murine splenic CD4+ T cells from Foxp3-KI-GFP T-cell receptor transgenic mice. The production of intracellular reactive oxygen species was shown to play a major role in the generation of iTreg cells, a process characterized by increased levels of Sirt1, PTEN and Glut1 on the committed cells, independently of the level of oxygenation. The suppressive function of iTreg cells generated either in atmospheric or low oxygen levels was equivalent. However, greater yields of iTreg cells were obtained under low oxygenation, resulting from a higher proliferative rate of the committed Treg cells and higher levels of Foxp3, suggesting a better stability of the differentiation process. Higher expression of Glut1 detected on iTreg cells generated under hypoxic culture conditions provides a likely explanation for the enhanced proliferation of these cells as compared to those cultured under ambient oxygen. Such results have important implications for understanding Treg cell homeostasis and developing in vitro protocols for the generation of Treg cells from naive T lymphocytes.

Ctla-4 modulates the differentiation of inducible Foxp3+ Treg cells but IL-10 mediates their function in experimental autoimmune encephalomyelitis.

In vitro induced Foxp3+ T regulatory (iTreg) cells form a novel and promising target for therapeutic tolerance induction. However, the potential of these cells as a target for the treatment of various immune diseases, as well as the factors involved in their development and function, remain debated. Here, we demonstrate in a myelin basic protein (MBP)-specific murine model of CNS autoimmune disease that adoptive transfer of antigen-specific iTreg cells ameliorates disease progression. Moreover, we show that the co-stimulatory molecule CTLA-4 mediates in vitro differentiation of iTreg cells. Finally, we demonstrate that the secreted, immunosuppressive cytokine IL-10 controls the ability of antigen-specific iTreg cells to suppress autoimmune disease. Overall, we conclude that antigen-specific iTreg cells, which depend on various immune regulatory molecules for their differentiation and function, represent a major target for effective immunotherapy of autoimmune disease.

Blockade of LFA-1 augments in vitro differentiation of antigen-induced Foxp3+ Treg cells

Adoptive transfer of antigen-specific, in vitro-induced Foxp3+ Treg (iTreg) cells protects against autoimmune disease. To generate antigen-specific iTreg cells at high purity, however, remains a challenge. Whereas polyclonal T cell stimulation with anti-CD3 and anti-CD28 antibody yields Foxp3+ iTreg cells at a purity of 90–95%, antigen-induced iTreg cells typically do not exceed a purity of 65–75%, even in a TCR-transgenic model. In a similar vein to thymic Treg cell selection, iTreg cell differentiation is influenced not only by antigen recognition and the availability of TGF-β but also by co-factors including costimulation and adhesion molecules. In this study, we demonstrate that blockade of the T cell integrin Leukocyte Function-associated Antigen-1 (LFA-1) during antigen-mediated iTreg cell differentiation augments Foxp3 induction, leading to approximately 90% purity of Foxp3+ iTreg cells. This increased efficacy not only boosts the yield of Foxp3+ iTreg cells, it also reduces contamination with activated effector T cells, thus improving the safety of adoptive transfer immunotherapy.

Galectin-9-CD44 Interaction Enhances Stability and Function of Adaptive Regulatory T Cells

The β-galactoside-binding protein galectin-9 is critical in regulating the immune response, but the mechanism by which it functions remains unclear. We have demonstrated that galectin-9 is highly expressed by induced regulatory T cells (iTreg) and was crucial for the generation and function of iTreg cells, but not natural regulatory T (nTreg) cells. Galectin-9 expression within iTreg cells was driven by the transcription factor Smad3, forming a feed-forward loop, which further promoted Foxp3 expression. Galectin-9 increased iTreg cell stability and function by directly binding to its receptor CD44, which formed a complex with transforming growth factor-β (TGF-β) receptor I (TGF-βRI), and activated Smad3. Galectin-9 signaling was further found to regulate iTreg cell induction by dominantly acting through the CNS1 region of the Foxp3 locus. Our data suggest that exogenous galectin-9, in addition to being an effector molecule for Treg cells, acts synergistically with TGF-β to enforce iTreg cell differentiation and maintenance.

Inhibition of CDK2 promotes inducible regulatory T-cell differentiation through TGFβ-Smad3 signaling pathway

Inducible regulatory T-cells (iTReg) can be generated from CD4+Foxp3− naïve conventional T-cells by a combination of TGF-β and T-cell receptor (TCR) signaling. It is of enormous clinical importance to identify agents that can promote the generation and differentiation of functional iTreg cells. We have established a phenotypic screening platform to identify new compounds that can promote the TGFβ-mediated iTreg differentiation. We have found Kenpaullone, a potent CDK1, CDK2 and CDK5 inhibitor, as new enhancer for iTreg cell differentiation. Kenpaullone promotes iTreg cell differentiation through increased and prolonged transcription of foxp3 gene by enhancing TGFβ-Smad3 signaling pathway. Thus, we have demonstrated that CDK2 is the biological target of Kenpaullone and proven that CDK2 is a novel negative regulator of iTreg cell differentiation.

Loss of β‐arrestin 2 exacerbates experimental autoimmune encephalomyelitis with reduced number of Foxp3+ CD4+ regulatory T cells

β-Arrestins are well-known regulators and mediators of G protein-coupled receptor signalling, and accumulating evidence reveals that they are functionally involved in inflammation and autoimmune diseases. Of the two β-arrestins, β-arrestin 1 is documented to play regulatory roles in an animal model of multiple sclerosis (MS), whereas the role of β-arrestin 2 is less clear. Here, we show that β-arrestin 2-deficient mice displayed the exacerbated and sustained symptoms of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. At the cellular level, deficiency of β-arrestin 2 led to a decreased number of Foxp3(+) CD4(+) regulatory T (Treg) cells in peripheral lymphoid organs of EAE mice. Consistently, our in vitro observations also revealed that loss of β-arrestin 2 impaired the conversion of Foxp3(-) CD4(+) T cells into Foxp3(+) CD4(+) inducible Treg cells. Taken together, our data suggest that β-arrestin 2 plays a regulatory role in MS, that is opposite to that of β-arrestin 1, in autoimmune diseases such as MS, which is at least partially through regulation of iTreg cell differentiation.