Regulatory T Cells and Human Disease.

Naturally occurring CD4+ regulatory T cells (Tregs), which specifically express the transcription factor FoxP3 in the nucleus and CD25 and CTLA-4 on the cell surface, are a functionally distinct T cell subpopulation actively engaged in the maintenance of immunological self-tolerance and homeostasis. Recent studies have facilitated our understanding of the cellular and molecular basis of their generation, function, phenotypic and functional stability, and adaptability. It is under investigation in humans how functional or numerical Treg anomalies, whether genetically determined or environmentally induced, contribute to immunological diseases such as autoimmune diseases. Also being addressed is how Tregs can be targeted to control physiological and pathological immune responses, for example, by depleting them to enhance tumor immunity or by expanding them to treat immunological diseases. This review discusses our current understanding of Treg immunobiology in normal and disease states, with a perspective on the realization of Treg-targeting therapies in the clinic.

The role of miR-183 cluster in immunity.

MicroRNAs (miRNAs) are essential factors of an extensively conserved post-transcriptional process to regulate gene expression. MiRNAs play a pivotal role in immunity, including controlling the differentiation of various immune cells as well as their immunological functions. The miR-183 cluster, which is comprised of miR-183, -96 and -182, is a miRNA family with sequence homology. These miRNAs are usually transcribed together as a polycistronic miRNA cluster during development and are required for maturation of sensory organs. In comparison to defined sensory-specific role of these miRNAs in normal development, they are frequently over-expressed in several non-sensory diseases, including autoimmune diseases and cancers. Because individual miRNAs of miR-183 cluster have both common and unique targets within functionally interrelated pathways, they can show cooperative or opposing effects on biological processes, implying the complexity of this miR cluster-mediated gene regulation. Therefore, a better understanding of the molecular regulation of miR-183 cluster expression and its downstream networks is important for the therapeutic applications. In this review, we will discuss the characteristics of miR-183 cluster and a wide variety of evidence on its function in immune system. Newer knowledge summarized here will help readers understand the versatile role of miR-183 cluster in this field.

Regulation of Pathogenic T Helper 17 Cell Differentiation by Steroid Receptor Coactivator-3.

T helper 17 (Th17) cell development is programmed by the orphan nuclear receptor RORγt, but the underlying mechanism is not well understood. Nuclear receptor-mediated transcriptional activation depends on coactivators. Here, we show that steroid receptor coactivator-3 (SRC-3) critically regulates Th17 cell differentiation. Reduced incidence of experimental autoimmune encephalitis (EAE) associated with decreased Th17 cell generation in vivo was observed in mice with SRC-3 deletion specifically in T cells. In vitro, SRC-3 deficiency did not affect TGF-ß/IL-6-induced Th17 cell generation but severely impaired pathogenic Th17 differentiation induced by IL-1/IL-6/IL-23. Microarray analysis revealed that SRC-3 not only regulates IL-17A but also IL-1R1 expression. SRC-3 bound to Il17a and Il1r1 loci in a RORγt-dependent manner and was required for recruitment of the p300 acetyltransferase. Thus, SRC-3 is critical for RORγt-dependent gene expression in Th17 cell-driven autoimmune diseases.

Generation of RORγt+ Antigen-Specific T Regulatory 17 Cells from Foxp3+ Precursors in Autoimmunity.

Th17 cells are potent mediators in autoimmune diseases, and RORγt is required for their development. Recent studies have shown that RORγt+ Treg cells in the gut regulate intestinal inflammation by inhibiting effector T cell function. In the current study, we report that RORγt+ Treg cells were also found in lymph nodes following immunization. Not only distinct from intestinal RORγt+ Treg cells in their transcriptomes, peripheral RORγt+ Treg cells were derived from Foxp3+ thymic Treg cells in an antigen-specific manner. Development of these RORγt+ Treg cells, coined T regulatory 17 (Tr17) cells, depended on IL-6/Stat3 signaling. Tr17 cells showed suppressive activity against antigen-specific effector T cells in vitro. In addition, Tr17 cells efficiently inhibited myelin-specific Th17-cell-mediated CNS auto-inflammation in a passive EAE model. Collectively, our study demonstrates that Tr17 cells are effector Treg cells that potentially restrict autoimmunity.

The MicroRNA-183-96-182 Cluster Promotes T Helper 17 Cell Pathogenicity by Negatively Regulating Transcription Factor Foxo1 Expression.

T helper 17 (Th17) cells are key players in autoimmune diseases. However, the roles of non-coding RNAs in Th17 cell development and function are largely unknown. We found that deletion of the endoribonuclease-encoding Dicer1 specifically in Th17 cells protected mice from experimental autoimmune encephalomyelitis. We found that the Dicer1-regulated microRNA (miR)-183-96-182 cluster (miR-183C) was highly expressed in Th17 cells and was induced by cytokine IL-6-STAT3 signaling. miR-183C expression enhanced pathogenic cytokine production from Th17 cells during their development and promoted autoimmunity. Mechanistically, miR-183C in Th17 cells directly repressed expression of the transcription factor Foxo1. Foxo1 negatively regulated the pathogenicity of Th17 cells in part by inhibiting expression of cytokine receptor IL-1R1. These findings indicate that the miR-183C drives Th17 pathogenicity in autoimmune diseases via inhibition of Foxo1 and present promising therapeutic targets.

Genome-wide Analysis Identifies Bcl6-Controlled Regulatory Networks during T Follicular Helper Cell Differentiation.

T follicular helper (Tfh) cell is a unique T cell subset specialized in promoting humoral immunity. B-cell lymphoma 6 protein (Bcl6) has been identified as an obligatory transcription factor in Tfh cells; however, the molecular mechanism underlying Bcl6 function remains largely unknown. Here, we defined Bcl6 target genes in Tfh cells by analyzing genome-wide Bcl6 occupancy together with transcriptome profiling. With consensus sequences being different from those in Th9, B cells, and macrophages, Bcl6 binding in Tfh cell was closely associated with a decrease in 5-hydroxymethylcytosine (5hmC). Importantly, Bcl6 promoted Tfh cell differentiation through antagonizing IL-7R (CD127)/signal transducer and activator of transcription (STAT) 5 axis; deletion of the Bcl6 gene in T cells resulted in enhanced IL-7R-STAT5 signaling and substantial expansion of CD127(hi) non-Tfh cells. Thus, our study systemically examines Bcl6-controlled regulatory networks and provides important insights into Bcl6's biological functions in Tfh cells.

The methylcytosine dioxygenase Tet2 promotes DNA demethylation and activation of cytokine gene expression in T cells.

Epigenetic regulation of lineage-specific genes is important for the differentiation and function of T cells. Ten-eleven translocation (Tet) proteins catalyze 5-methylcytosine (5 mC) conversion to 5-hydroxymethylcytosine (5 hmC) to mediate DNA demethylation. However, the roles of Tet proteins in the immune response are unknown. Here, we characterized the genome-wide distribution of 5 hmC in CD4(+) T cells and found that 5 hmC marks putative regulatory elements in signature genes associated with effector cell differentiation. Moreover, Tet2 protein was recruited to 5 hmC-containing regions, dependent on lineage-specific transcription factors. Deletion of Tet2 in T cells decreased their cytokine expression, associated with reduced p300 recruitment. In vivo, Tet2 plays a critical role in the control of cytokine gene expression in autoimmune disease. Collectively, our findings suggest that Tet2 promotes DNA demethylation and activation of cytokine gene expression in T cells.

Smad2/3 and IRF4 play a cooperative role in IL-9-producing T cell induction.

IL-9 is a pleiotropic cytokine that can regulate autoimmune and allergic responses. Th9 cells can develop from naive T cells or Th2 cells through stimulation by TGF-ß in vitro. In this study, we demonstrated that Smad2 and Smad3 are necessary for IL-9 production from T cells in an OVA-induced asthma model using T cell-specific Smad2- and Smad3-deficient mice. Smad2 and Smad3 were also redundantly essential for TGF-ß signaling to induce histone modifications for Il9 transcription. Although Smad2/3 was recruited to the Il9 promoter by TGF-ß stimulation, they are not sufficient to activate the Il9 promoter. By the screening the transcription factors, we found that IFN regulatory factor 4 (IRF4) was essential for the Smad2/3-mediated Il9 promoter activation. In addition, Smad2/3 physically interacted with IRF4, and Smad2/3 did not bind to the Il9 promoter and could not induce Th9 in IRF4-deficient T cells. Similarly, IRF4 could not stimulate Il9 transcription in the absence of Smad2/3, and TGF-ß enhanced IRF4 recruitment to the Il9 promoter in a Smad2/3-dependent manner. We propose that Smad2/3 and IRF4 cooperatively transactivate the Il9 promoter and play an important role in regulating allergic immune responses by inducing Th9 cells.

PI3K-Akt-mTORC1-S6K1/2 axis controls Th17 differentiation by regulating Gfi1 expression and nuclear translocation of RORγ.

The PI3K-Akt-mTORC1 axis contributes to the activation, survival, and proliferation of CD4(+) T cells upon stimulation through TCR and CD28. Here, we demonstrate that the suppression of this axis by deletion of p85α or PI3K/mTORC1 inhibitors as well as T cell-specific deletion of raptor, an essential component of mTORC1, impairs Th17 differentiation in vitro and in vivo in a S6K1/2-dependent fashion. Inhibition of PI3K-Akt-mTORC1-S6K1 axis impairs the downregulation of Gfi1, a negative regulator of Th17 differentiation. Furthermore, we demonstrate that S6K2, a nuclear counterpart of S6K1, is induced by the PI3K-Akt-mTORC1 axis, binds RORγ, and carries RORγ to the nucleus. These results point toward a pivotal role of PI3K-Akt-mTORC1-S6K1/2 axis in Th17 differentiation.

1,25-dihydroxyvitamin D(3) ameliorates Th17 autoimmunity via transcriptional modulation of interleukin-17A.

A new class of inflammatory CD4(+) T cells that produce interleukin-17 (IL-17) (termed Th17) has been identified, which plays a critical role in numerous inflammatory conditions and autoimmune diseases. The active form of vitamin D, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], has a direct repressive effect on the expression of IL-17A in both human and mouse T cells. In vivo treatment of mice with ongoing experimental autoimmune encephalomyelitis (EAE; a mouse model of multiple sclerosis) diminishes paralysis and progression of the disease and reduces IL-17A-secreting CD4(+) T cells in the periphery and central nervous system (CNS). The mechanism of 1,25(OH)(2)D(3) repression of IL-17A expression was found to be transcriptional repression, mediated by the vitamin D receptor (VDR). Transcription assays, gel shifting, and chromatin immunoprecipitation (ChIP) assays indicate that the negative effect of 1,25(OH)(2)D(3) on IL-17A involves blocking of nuclear factor for activated T cells (NFAT), recruitment of histone deacetylase (HDAC), sequestration of Runt-related transcription factor 1 (Runx1) by 1,25(OH)(2)D(3)/VDR, and a direct effect of 1,25(OH)(2)D(3) on induction of Foxp3. Our results describe novel mechanisms and new concepts with regard to vitamin D and the immune system and suggest therapeutic targets for the control of autoimmune diseases.

Transcription factor Smad-independent T helper 17 cell induction by transforming-growth factor-ß is mediated by suppression of eomesodermin.

Transforming growth factor-ß (TGF-ß) has been shown to be required for Th17 cell differentiation via Smad-independent mechanisms. The molecular mechanism underlying this pathway remains to be clarified, however. We searched for genes regulated by TGF-ß through the Smad-independent pathway by using Smad2 and Smad3 double-deficient T cells and identified the transcription factor Eomesodermin (Eomes), whose expression was suppressed by TGF-ß via the c-Jun N-terminal kinase (JNK)-c-Jun signaling pathway. Inhibition of JNK strongly suppressed disease in an in vivo EAE model as well as in vitro Th17 cell induction. Overexpression of Eomes substantially suppressed Th17 cell differentiation, whereas ablation of Eomes expression could substitute for TGF-ß in Th17 cell induction in primary T cells. Eomes suppressed Rorc and Il17a promoters by directly binding to the proximal region of these promoters. In conclusion, the suppression of Eomes by TGF-ß via the JNK pathway is an important mechanism for Smad-independent Th17 cell differentiation.

SOCS1 regulates type I/type II NKT cell balance by regulating IFNgamma signaling.

Suppressor of cytokine signaling-1 (SOCS1) has been shown to be an essential negative regulator of cytokine responses, including those of IFNγ, IL-2, IL-4 and IL-7. SOCS1 deficiency resulted in hyperactivation not only of T cells in general but also of NKT cells specifically. Consistent with previous reports, T- and NKT-cell-specific deletion of Socs1 in mice resulted in enhanced sensitivity to ConA-induced hepatitis. Compared with wild-type (WT) NKT cells, SOCS1-deficient NKT cells produced larger quantities of IFNγ in response to ConA and proliferated faster in response to IL-2 and IL-15. To our surprise, however, SOCS1-deficient NKT cells did not respond to the synthetic glycolipid ligand alpha-galactosylceramide (α-GalCer), though they did respond to sulfatide. α-GalCer-CD1d-tetramer-positive type I NKT [invariant NKT (iNKT)] cells were marginally detected in the periphery of SOCS1-conditional knockout (cKO) mice, suggesting that most of the SOCS1-deficient NKT cells at the periphery were type II NKT cells. Consistently, invariant Vα14 expression was much lower in SOCS1-deficient NKT cells than in WT NKT cells, indicating that iNKT cell homeostasis was abnormal in SOCS1-cKO mice. This reduction in iNKT cells was not observed in mice of an IFNγ-deficient background. These results suggest that SOCS1 is an important regulator of the balance between type I and type II NKT cells at the periphery.

Smad2 and Smad3 are redundantly essential for the TGF-beta-mediated regulation of regulatory T plasticity and Th1 development.

Although it has been well established that TGF-beta plays a pivotal role in immune regulation, the roles of its downstream transcription factors, Smad2 and Smad3, have not been fully clarified. Specifically, the function of Smad2 in the immune system has not been investigated because of the embryonic lethality of Smad2-deficient mice. In this study, we generated T cell-specific Smad2 conditional knockout (KO) mice and unexpectedly found that Smad2 and Smad3 were redundantly essential for TGF-beta-mediated induction of Foxp3-expressing regulatory T cells and suppression of IFN-gamma production in CD4(+) T cells. Consistent with these observations, Smad2/Smad3-double KO mice, but not single KO mice, developed fatal inflammatory diseases with higher IFN-gamma production and reduced Foxp3 expression in CD4(+) T cells at the periphery. Although it has been suggested that Foxp3 induction might underlie TGF-beta-mediated immunosuppression, TGF-beta still can suppress Th1 cell development in Foxp3-deficient T cells, suggesting that the Smad2/3 pathway inhibits Th1 cell development with Foxp3-independent mechanisms. We also found that Th17 cell development was reduced in Smad-deficient CD4(+) T cells because of higher production of Th17-inhibitory cytokines from these T cells. However, TGF-beta-mediated induction of RORgamma t, a master regulator of Th17 cell, was independent of both Smad2 and Smad3, suggesting that TGF-beta regulates Th17 development through Smad2/3-dependent and -independent mechanisms.

Gfi1 negatively regulates T(h)17 differentiation by inhibiting RORgammat activity.

T(h) cells have long been divided into two subsets, T(h)1 and T(h)2; however, recently, T(h)17 and inducible regulatory T (iTreg) cells were identified as new T(h) cell subsets. Although T(h)1- and T(h)2-polarizing cytokines have been shown to suppress T(h)17 and iTreg development, transcriptional regulation of T(h)17 and iTreg differentiation by cytokines remains to be clarified. In this study, we found that expression of the growth factor independent 1 (Gfi1) gene, which has been implicated in T(h)2 development, was repressed in T(h)17 and iTreg cells compared with T(h)1 and T(h)2 lineages. Gfi1 expression was enhanced by the IFN-gamma/STAT1 and IL-4/STAT6 pathways, whereas it was repressed by the transforming growth factor-beta1 stimulation at the promoter level. Over-expression of Gfi1 strongly reduced IL-17A transcription in the EL4 T cell line, as well as in primary T cells. This was due to the blockade of recruitment of retinoid-related orphan receptor gammat to the IL-17A promoter. In contrast, IL-17A expression was significantly enhanced in Gfi1-deficient T cells under T(h)17-promoting differentiation conditions as compared with wild-type T cells. In contrast, the impacts of Gfi1 in iTregs were not as strong as in T(h)17 cells. Taken together, these data strongly suggest that Gfi1 is a negative regulator of T(h)17 differentiation, which represents a novel mechanism for the regulation of T(h)17 development by cytokines.

Promotion of IL-4- and IL-5-dependent differentiation of anti-mu-primed B cells by ascorbic acid 2-glucoside.

The stable ascorbic acid derivative 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) was used to investigate the role of ascorbic acid (AA) in B cell differentiation in vitro. AA-2G is stable in a solution unlike AA but is hydrolyzed by cellular alpha-glucosidase to release AA. Mouse spleen B cells were primed for 2 days with an anti-mu antibody in the presence of interleukin (IL)-4 and IL-5 and then washed and recultured with AA-2G in the presence of IL-4 and IL-5. AA-2G, but not AA, dose-dependently increased IgM production, the greatest enhancement being 150% at concentrations of more than 0.5mM. In the absence of IL-4 and IL-5, primed B cells produced a negligible amount of IgM, and AA-2G had no effect. AA-2G-induced IgM production in the presence of IL-4 and IL-5 was inhibited by the alpha-glucosidase inhibitor castanospermine. Intracellular AA content, depleted during the priming period, increased by adding AA-2G at the start of reculture. Treatment of B cells with AA-2G resulted in an increase in the number of IgM-secreting cells, CD138-positive cells and CD45R/B220-negative cells. The number of viable cells in untreated cultures decreased gradually, but the decrease was significantly attenuated by AA-2G, resulting in about 70% more viable cells in AA-2G-treated cultures. AA-2G caused a slight but reproducible enhancement of DNA synthesis and a slight decrease in the number of cells with a sub-G1 DNA content. These results demonstrated that AA released from AA-2G enhanced cytokine-dependent IgM production in anti-mu-primed B cells and suggest that its effect is caused through promoting the differentiation of B cells to plasma cells and attenuating the gradual decrease in the number of viable cells.

STAT6 Inhibits TGF-beta1-mediated Foxp3 induction through direct binding to the Foxp3 promoter, which is reverted by retinoic acid receptor.

It has been shown that transforming growth factor beta1 (TGF-beta1) is critical in the generation of CD4(+)CD25(+)Foxp3(+)-inducible regulatory T cells (iTregs) from naïve CD4(+)T cells. However, in contrast to natural Tregs, TGF-beta1-induced iTregs rapidly lose both Foxp3 expression and suppression activity. We found that TGF-beta1-induced Foxp3 levels were maintained by the addition of the anti-interleukin 4 (IL-4) antibody or by STAT6 gene deletion. Thus, IL-4 is an important suppressor of Foxp3 induction, and T helper 2 development is a major cause for the disappearance of iTreg during long culture. Using promoter analysis in EL4 cells and primary T cells, we identified a silencer region containing a STAT6 binding site. STAT6 binding to this site reduced TGF-beta1-mediated Foxp3 promoter activation and chromatin modification. Retinoic acid has also been shown to suppress loss of Foxp3 induced by TGF-beta1. Retinoic acid in the presence of TGF-beta1 reduced STAT6 binding to the Foxp3 promoter and enhanced histone acetylation, thereby reverting the effect of IL-4. We propose that antagonistic agents for neutralizing IL-4 could be a novel strategy to facilitate inducible Treg cell generation and the promotion of tolerance in Th2-dominated diseases such as allergy.

Foxp3 inhibits RORgammat-mediated IL-17A mRNA transcription through direct interaction with RORgammat.

The cytokine, transforming growth factor-beta1 (TGF-beta1), converts naive T cells into regulatory T cells that prevent autoimmunity. However, in the presence of interleukin (IL)-6, TGF-beta1 has also been found to promote differentiation into IL-17-producing helper T (Th17) cells that are deeply involved in autoimmunity and inflammation. However, it has not been clarified how TGF-beta1 and IL-6 determine such a distinct fate. Here we found that a master regulator for Th17, retinoic acid-related orphan receptor gammat (RORgammat), was rapidly induced by TGF-beta1 regardless of the presence of IL-6. IL-6 reduced Foxp3 expression, and overexpression of Foxp3 in a T cell line resulted in a strong reduction of IL-17A expression. We have characterized the IL-17A promoter and found that RORgammat binding is sufficient for activation of the minimum promoter in the HEK 293T cells. RORgammat-mediated IL-17A promoter activation was suppressed by forced expression of Foxp3. Foxp3 directly interacted with RORgammat through exon 2 region of Foxp3. The exon 2 region and forkhead (FKH) domain of Foxp3 were necessary for the suppression of RORgammat-mediated IL-17A promoter activation. We propose that induction of Foxp3 is the mechanism for the suppression of Th17 and polarization into inducible Treg.

Loss of suppressor of cytokine signaling 1 in helper T cells leads to defective Th17 differentiation by enhancing antagonistic effects of IFN-gamma on STAT3 and Smads.

Suppressor of cytokine signaling 1 (SOCS1) is an important negative regulator for cytokines; however, the role of SOCS1 in Th17 differentiation has not been clarified. We generated T cell-specific SOCS1-deficient mice and found that these mice were extremely resistant to a Th17-dependent autoimmune disease model, experimental autoimmune encephalomyelitis. SOCS1-deficient naive CD4(+) T cells were predominantly differentiated into Th1 and poorly into Th17 in vitro. These phenotypes were canceled in IFN-gamma(-/-) background, suggesting that a large amount of IFN-gamma in SOCS1-deficient T cells suppressed Th17 differentiation. IL-6 plus TGF-beta enhanced retinoic acid receptor-related orphan receptor (ROR)-gammat expression and suppressed IFN-gamma production in wild-type T cells, whereas these effects were severely impaired in SOCS1-deficient T cells. These phenotypes can be partly explained by STAT3 suppression by enhanced SOCS3 induction through hyper-STAT1 activation in SOCS1-deficient T cells. In addition, SOCS1-deficient T cells were much less sensitive to TGF-beta. Suppression of Th1 differentiation by TGF-beta was impaired in SOCS1-deficient T cells. TGF-beta-mediated Smad transcriptional activity was severely inhibited in SOCS1-deficient cells in the presence of IFN-gamma. Such impairment of TGF-beta functions were not observed in SOCS3-overexpressed cells, indicating that suppression of Smads was independent of SOCS3. Therefore, SOCS1 is necessary for Th17 differentiation by suppressing antagonistic effect of IFN-gamma on both STAT3 and Smads. Induction of SOCS3 can partly explain IFN-gamma-mediated STAT3 suppression, while other mechanism(s) will be involved in IFN-gamma-mediated Smad suppression. SOCS1-deficient T cells will be very useful to investigate the molecular mechanism for the STAT1-mediated suppression of Th17 development.

Inhibition of free radical-induced erythrocyte hemolysis by 2-O-substituted ascorbic acid derivatives.

Inhibitory effects of 2-O-substituted ascorbic acid derivatives, ascorbic acid 2-glucoside (AA-2G), ascorbic acid 2-phosphate (AA-2P), and ascorbic acid 2-sulfate (AA-2S), on 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative hemolysis of sheep erythrocytes were studied and were compared with those of ascorbic acid (AA) and other antioxidants. The order of the inhibition efficiency was AA-2S> or =Trolox=uric acid> or =AA-2P> or =AA-2G=AA>glutathione. Although the reactivity of the AA derivatives against AAPH-derived peroxyl radical (ROO(*)) was much lower than that of AA, the derivatives exerted equal or more potent protective effects on AAPH-induced hemolysis and membrane protein oxidation. In addition, the AA derivatives were found to react per se with ROO(*), not via AA as an intermediate. These findings suggest that secondary reactions between the AA derivative radical and ROO(*) play a part in hemolysis inhibition. Delayed addition of the AA derivatives after AAPH-induced oxidation of erythrocytes had already proceeded showed weaker inhibition of hemolysis compared to that of AA. These results suggest that the AA derivatives per se act as biologically effective antioxidants under moderate oxidative stress and that AA-2G and AA-2P may be able to act under severe oxidative stress after enzymatic conversion to AA in vivo.