Cutting Edge: Suppression of GM-CSF Expression in Murine and Human T Cells by IL-27

GM-CSF is a potent proinflammatory cytokine that plays a pathogenic role in the CNS inflammatory disease experimental autoimmune encephalomyelitis. As IL-27 alleviates experimental autoimmune encephalomyelitis, we hypothesized that IL-27 suppresses GM-CSF expression by T cells. We found that IL-27 suppressed GM-CSF expression in CD4(+) and CD8(+) T cells in splenocyte and purified T cell cultures. IL-27 suppressed GM-CSF in Th1, but not Th17, cells. IL-27 also suppressed GM-CSF expression by human T cells in nonpolarized and Th1- but not Th17-polarized PBMC cultures. In vivo, IL-27p28 deficiency resulted in increased GM-CSF expression by CNS-infiltrating T cells during Toxoplasma gondii infection. Although in vitro suppression of GM-CSF by IL-27 was independent of IL-2 suppression, IL-10 upregulation, or SOCS3 signaling, we observed that IL-27-driven suppression of GM-CSF was STAT1 dependent. Our findings demonstrate that IL-27 is a robust negative regulator of GM-CSF expression in T cells, which likely inhibits T cell pathogenicity in CNS inflammation.     

IDO upregulates regulatory T cells via tryptophan catabolite and suppresses encephalitogenic T cell responses in experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a Th1 and Th17 cell-mediated autoimmune disease of the CNS. IDO and tryptophan metabolites have inhibitory effects on Th1 cells in EAE. For Th17 cells, IDO-mediated tryptophan deprivation and small molecule halofuginone-induced amino acid starvation response were shown to activate general control nonrepressed 2 (GCN2) kinase that directly or indirectly inhibits Th17 cell differentiation. However, it remains unclear whether IDO and tryptophan metabolites impact the Th17 cell response by mechanisms other than the GCN2 kinase pathway. In this article, we show that IDO-deficient mice develop exacerbated EAE with enhanced encephalitogenic Th1 and Th17 cell responses and reduced regulatory T cell (Treg) responses. Administration of the downstream tryptophan metabolite 3-hydroxyanthranillic acid (3-HAA) enhanced the percentage of Tregs, inhibited Th1 and Th17 cells, and ameliorated EAE. We further demonstrate that Th17 cells are less sensitive to direct suppression by 3-HAA than are Th1 cells. 3-HAA treatment in vitro reduced IL-6 production by activated spleen cells and increased expression of TGF-β in dendritic cells (DCs), which correlated with enhanced levels of Tregs, suggesting that 3-HAA-induced Tregs contribute to inhibition of Th17 cells. By using a DC-T cell coculture, we found that 3-HAA-treated DCs expressed higher levels of TGF-β and had properties to induce generation of Tregs from anti-CD3/anti-CD28-stimulated naive CD4(+) T cells. Thus, our data support the hypothesis that IDO induces the generation of Tregs via tryptophan metabolites, such as 3-HAA, which enhances TGF-β expression from DCs and promotes Treg differentiation.     

Targeted overexpression of IL-18 binding protein at the central nervous system overrides flexibility in functional polarization of antigen-specific Th2 cells

The current study shows that functional polarization of Ag-specific CD4(+) Th2 cells entering the CNS during the accelerating phase of experimental autoimmune encephalomyelitis is flexible and dependent on the cytokine milieu there. Thus, targeted cell/gene therapy by Ag-specific T cells overexpressing IL-18 binding protein overrides this flexibility and induces infectious spread of T cell tolerance. Using a congenic system, we demonstrated that at this time, Ag-specific Th2 cells accumulate at the CNS but then arrest of IL-4 production. A manipulation of targeted cell/gene delivery was then used to detect whether this function is dependent on the cytokine milieu there. Targeted overexpression of IL-18 binding protein, a natural inhibitor of IL-18, restored the ability of these Ag-specific Th2 cells to produce IL-4 and subsequently induce protective spread of Th2 polarization. These findings not only suggest a novel way of therapy, but also explain why shifting the balance of Ag-specific T cells toward Th2 suppresses ongoing experimental autoimmune encephalomyelitis, whereas a direct transfer of these cells is ineffective.     

Host T cells are the main producers of IL-17 within the central nervous system during initiation of experimental autoimmune encephalomyelitis induced by adoptive transfer of Th1 cell lines

Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, has long been thought to be mediated by Th1 CD4(+) T cells. Using adoptive transfer techniques, transfer of CNS specific Th1 T cells was sufficient to induce EAE in naive mice. However, recent studies found a vital role for IL-17 in induction of EAE. These studies suggested that a fraction of IL-17-producing T cells that contaminate Th1 polarized cell lines are largely responsible for initiation of EAE. In this study, we tracked the appearance and cytokine production capacity of adoptively transferred cells within the CNS of mice throughout EAE disease. IL-17-producing, adoptively transferred cells were not enriched over the low percentages present in vitro. Thus, there was no selective recruitment and/or preferential proliferation of adoptively transferred IL-17-producing cells during the induction of EAE. Instead a large number of CNS infiltrating host T cells in mice with EAE were capable of producing IL-17 following ex vivo stimulation. The IL-17-producing T cells contained both alphabeta and gammadelta TCR(+) T cells with a CD4(+)CD8(-) or CD4(-)CD8(-) phenotype. These cells concentrated within the CNS within 3 days of adoptive transfer, and appeared to play a role in EAE induction as adoptive transfer of Th1 lines derived from wild-type mice into IL-17-deficient mice induced reduced EAE clinical outcomes. This study demonstrates that an encephalitogenic Th1 cell line induces recruitment of host IL-17-producing T cells to the CNS during the initiation of EAE and that these cells contribute to the incidence and severity of disease.     

Cytokines and effector T cell subsets causing autoimmune CNS disease

Although experimental autoimmune encephalomyelitis (EAE) is limited in its potency to reproduce the entirety of clinical and histopathologic features of multiple sclerosis (MS), this model has been successfully used to prove that MS like autoimmunity in the CNS is orchestrated by autoantigen specific T cells. EAE was also very useful to refute the idea that IFN-γ producing T helper type 1 (Th1) cells were the sole players within the pathogenic T cell response. Rather, "new" T cell lineages such as IL-17 producing Th17 cells or IL-9 producing Th9 cells have been first discovered in the context of EAE. Here, we will summarize new concepts of early and late T cell plasticity and the cytokine network that shapes T helper cell responses and lesion development in CNS specific autoimmunity.     

JAGGED1 and delta1 differentially regulate the outcome of experimental autoimmune encephalomyelitis

Notch signaling plays an important role during T cell development in the thymus and in T cell activation but the role of Notch in autoimmunity is not clear. We investigated the role of Jagged1 and Delta1 in experimental autoimmune encephalomyelitis. During experimental autoimmune encephalomyelitis, Delta1 expression is up-regulated on dendritic cells and B cells after priming while Jagged1 is up-regulated only on dendritic cells. Administration of anti-Jagged1 Ab exacerbated clinical disease while that of anti-Delta1 Ab reduced the severity of the clinical disease. In contrast, administration of Jagged1-Fc protected from disease, that of Delta1-Fc exacerbated disease. Treatment with Jagged1-Fc was associated with increased IL-10-producing Ag-specific cells in the CNS, while anti-Jagged1 decreased the frequency of IL-10-producing cells. Treatment with Delta1-Fc increased Th1 cells in the CNS, while anti-Delta-1 decreased the frequency of Th1 cells. Manipulation of Delta1 or Jagged1 had no effect on the frequency of Th17 cells or FoxP3(+) cells. Moreover, Jagged1 may play a role in CNS homeostasis because murine astrocytes specifically express Jagged1 that is up-regulated by TGF-beta, whereas IFN-gamma, TNF-alpha, and IL-17 decrease Jagged1 expression. Our study provides novel data about differential roles of Notch ligands in regulating inflammation in the periphery as well as in the CNS.     

Kinetics and organ distribution of IL-17-producing CD4 cells in proteolipid protein 139-151 peptide-induced experimental autoimmune encephalomyelitis of SJL mice

In experimental autoimmune encephalomyelitis (EAE), the production of proinflammatory cytokines by neuroantigen-specific T cells is thought to initiate and maintain the inflammatory autoimmune pathology. Because gene knockout strategies have shown that IFN-gamma and TNF are not essential for EAE development, there is increasing interest in establishing the role of other proinflammatory cytokines, primarily IL-17 in EAE. We used an IL-17 ELISPOT assay to track the neuroantigen-specific IL-17-producing T cells at single-cell resolution in various organs of SJL mice undergoing PLP 139-151-induced EAE. Overall, the migration patterns and population kinetics of the PLP 139-151-specific IL-17-producing CD4 cells were reminiscent of the IFN-gamma-producing cells, with the exception of IL-17 producers far outnumbering the IFN-gamma and IL-2 producers in the inflamed CNS. The selective enrichment of IL-17-producing CD4 cells in the CNS is suggestive of the pathogenic role of an independent (non-Th1) IL-17-producing proinflammatory effector T cell class in EAE.     

Loss of STAT3 in CD4+ T cells prevents development of experimental autoimmune diseases

Th17 cells are implicated in CNS autoimmune diseases. We show that mice with targeted-deletion of Stat3 in CD4(+) T cells (CD4(Stat3)(-/-)) do not develop experimental autoimmune uveoretinitis (EAU) or experimental autoimmune encephalomyelitis. Defective Th17 differentiation noted in CD4(Stat3)(-/-) mice is compensated by exaggerated increases in Foxp3-, IL-10-, IL-4-, and IFN-gamma-expressing T cells, suggesting critical roles of STAT3 in shaping Ag-specific CD4(+) T cell repertoire. In mice with EAU, a high percentage of IL-17-expressing T cells in their peripheral lymphoid organs also secrete IFN-gamma while these double-expressors are absent in CD4(Stat3)(-/-) and wild-type mice without EAU, raising the intriguing possibility that uveitis maybe mediated by Th17 and IL-17-expressing Th1 cells. Resistance of Stat3-deficient mice to EAU derives in part from an inability of uveitogenic Th17 and Th1 cells to enter eyes or brain of the CD4(Stat3)(-/-) mouse because of the reduction in the expression of activated alpha4/beta1 integrins on CD4(Stat3)(-/-) T cells. Adoptive transfer of activated interphotoreceptor retinoid-binding protein-specific uveitogenic T cells induced in CD4(Stat3)(-/-) mice a severe EAU characterized by development of retinal folds, infiltration of inflammatory cells into the retina, and destruction of retinal architecture, underscoring our contention that the loss of STAT3 in CD4(+) T cells results in an intrinsic developmental defect that renders CD4(Stat3)(-/-) resistant to CNS inflammatory diseases. STAT3 requirement for IL-17 production by Th17, generation of double positive T cells expressing IL-17 and IFN-gamma, and for T cell trafficking into CNS tissues suggests that STAT3 may be a therapeutic target for modulating uveitis, sceritis, or multiple sclerosis.     

IL-22 is expressed by Th17 cells in an IL-23-dependent fashion, but not required for the development of autoimmune encephalomyelitis

Lately, IL-17-secreting Th cells have received an overwhelming amount of attention and are now widely held to be the major pathogenic population in autoimmune diseases. In particular, IL-22-secreting Th17 cells were shown to specifically mark the highly pathogenic population of self-reactive T cells in experimental autoimmune encephalomyelitis (EAE). As IL-17A itself was found to only play a minor role during the development of EAE, IL-22 is now postulated to contribute to the pathogenic function of Th17 cells. The goal of this study was to determine the role and function of IL-22 during the development of CNS autoimmunity in vivo. We found that CNS-invading encephalitogenic Th17 cells coexpress IL-22 and that IL-22 is specifically induced by IL-23 in autoimmune-pathogenic CD4+ T cells in a time- and dose-dependent manner. We next generated IL-22-/- mice, which--in contrast to the prediction that expression of inflammatory cytokines by CNS-invading T cells inevitably confers pathogenic function--turned out to be fully susceptible to EAE. Taken together, we show that self-reactive Th cells coexpress IL-17 and IL-22, but that the latter also does not appear to be directly involved in autoimmune pathogenesis of the CNS.     

Cutting edge: central nervous system plasmacytoid dendritic cells regulate the severity of relapsing experimental autoimmune encephalomyelitis

Plasmacytoid dendritic cells (pDCs) have both stimulatory and regulatory effects on T cells. pDCs are a major CNS-infiltrating dendritic cell population during experimental autoimmune encephalomyelitis but, unlike myeloid dendritic cells, have a minor role in T cell activation and epitope spreading. We show that depletion of pDCs during either the acute or relapse phases of experimental autoimmune encephalomyelitis resulted in exacerbation of disease severity. pDC depletion significantly enhanced CNS but not peripheral CD4(+) T cell activation, as well as IL-17 and IFN-gamma production. Moreover, CNS pDCs suppressed CNS myeloid dendritic cell-driven production of IL-17, IFN-gamma, and IL-10 in an IDO-independent manner. The data demonstrate that pDCs play a critical regulatory role in negatively regulating pathogenic CNS CD4(+) T cell responses, highlighting a new role for pDCs in inflammatory autoimmune disease.     

Cutting edge: Alternative signaling of Th17 cell development by sphingosine 1-phosphate

Sphingosine 1-phosphate (S1P) in blood and lymph controls T cell traffic and proliferation through type 1 S1P receptor (S1P(1)) signals, but suppression of IFN-gamma generation has been the only consistently observed effect on T cell cytokines. The fact that S1P enhances the development of Th17 cells from Ag-challenged transgenic S1P(1)-overexpressing CD4 T cells suggested that the S1P-S1P(1) axis may promote the expansion of Th17 cells in wild-type mice. In a model of Th17 cell development from CD4 T cells stimulated by anti-CD3 plus anti-CD28 Abs and a mixture of TGF-beta1, IL-1, and IL-6, S1P enhanced their number and IL-17-generating activity the same as IL-23. As for IL-23 enhancement of Th17 cell development, that by S1P was prevented by IL-4 plus IFN-gamma and by IL-27. The prevention of S1P augmentation of Th17 cell development by the S1P receptor agonist and down-regulator FTY720 implies that FTY720 immunosuppression is attributable partially to inhibition of Th17-mediated inflammation.     

Anti-MS4a4B treatment abrogates MS4a4B-mediated protection in T cells and ameliorates experimental autoimmune encephalomyelitis

Recent data show that anti-CD20 therapy is effective for some autoimmune diseases, including multiple sclerosis (MS). However, the efficacy of anti-CD20 therapy for MS is largely limited because anti-CD20 antibodies target only B cells. In previous studies, we have investigated the function of MS4a4B, a novel CD20 homologue, in T cell proliferation. Here, we found that MS4a4B regulates not only T cell proliferation but also T cell apoptosis. Knockdown of MS4a4B by MS4a4B-siRNA or MS4a4B-shRNA-expressing vector promoted apoptosis in primary T cells and T32 cell line. In contrast, vector-driven over-expression of MS4a4B reduced apoptosis in EL-4 cells. Machinery analysis showed that MS4a4B-mediated T cell survival was associated with decreased activity of caspases 3, 8 and 9. Interestingly, binding of anti-MS4a4B antibodies to T cells induced activated T cells to undergo apoptosis. To test whether anti-MS4a4B antibody interferes with MS4a4B-mediated protection of T cells, we injected anti-MS4a4B antibodies into mice with experimental autoimmune encephalomyelitis (EAE). The results show that anti-MS4a4B treatment ameliorated the severity of EAE, accompanied by decreased Th1 and Th17 cell responses and reduced levels of pro-inflammatory cytokines in the central nervous system, suggesting that MS4a4B may serve as a target of antibody-based therapy for T cell-mediated diseases.     

Transduction of multiple effects of sphingosine 1-phosphate (S1P) on T cell functions by the S1P1 G protein-coupled receptor

Sphingosine 1-phosphate (S1P) in blood, lymph, and immune tissues stimulates and regulates T cell migration through their S1P(1) (endothelial differentiation gene encoded receptor-1) G protein-coupled receptors. We show now that S1P(1)Rs also mediate suppression of T cell proliferation and cytokine production. Uptake of [(3)H]thymidine by mouse CD4 T cells stimulated with anti-CD3 mAbs plus either anti-CD28 or IL-7 was inhibited up to 50% by 10(-9)-10(-6) M S1P. Suppression by S1P required Ca(2+) signaling and was reduced by intracellular cAMP. S1P decreased CD4 T cell generation of IFN-gamma and IL-4, without affecting IL-2. A Th1 line from D011.10 TCR transgenic mice without detectable S1P(1) was refractory to S1P until introduction of S1P(1) by retroviral transduction. S1P then evoked chemotaxis, inhibited chemotaxis to CCL-5 and CCL-21, and suppressed Ag-stimulated proliferation and IFN-gamma production. Thus, S1P(1) signals multiple immune functions of T cells as well as migration and tissue distribution.     

Cutting edge: loss of α4 integrin expression differentially affects the homing of Th1 and Th17 cells

The neutralization of α4 integrin is currently used as treatment in several autoimmune diseases and is thought to prevent the entry of most immune cells in target tissues. In this study, we showed that selective deletion of α4 integrin in T cells did not prevent but delayed the development of experimental autoimmune encephalomyelitis. Whereas both Th1 and Th17 cells infiltrate the CNS of wild-type mice, T cells present in the CNS of mice lacking α4 integrin were mainly enriched in Th17 cells, suggesting that this T cell subset uses other integrins to access the CNS. In contrast, α4 integrin expression is important for Th1 cells to enter the CNS and for the stability of their Th1-associated genetic program. Therefore, our data suggest that anti-α4 integrin Ab treatment may be more efficient in the treatment of Th1- rather than Th17-mediated disease.     

Retinal self-antigen induces a predominantly Th1 effector response in Axl and Mertk double-knockout mice

The TAM family of receptors (Tyro3, Axl, and Mertk) plays an important role in the negative regulation of response of dendritic cells (DCs) and macrophages to pathogenic stimuli, and mice lacking this receptor family develop spontaneous lupus-like systemic autoimmunity against a variety of tissues, including retina. To study the molecular mechanism underlying the TAM regulation of APC functions and subsequent effects on the induction of an autoimmune response against the eye, we examined CD4 T cell differentiation following retinal self-antigen immunization. CD4 T cells prepared from naive or interphotoreceptor retinoid-binding protein (IRBP)1-20-immunized Axl and Mertk double-knockout (dko) mice reacted to activation using anti-CD3 and anti-CD28 Abs or to bolster by self-antigen in vitro with a predominantly Th1 effector response, as characterized by increased IFN-γ production and higher frequency of IFN-γ-positive CD4 T cells. The Th17 effector response to IRBP immunization was similar in dko mice to that in wild-type controls, as shown by ELISA measurement of IL-17A in the culture medium and flow cytometric analysis of IL-17A-secreting CD4 T cells. Interestingly, APCs or DCs isolated from IRBP-immunized dko mice exhibited a greater ability to drive the Th1 response. The production of two driving cytokines for Th1 differentiation, IL-12 and IL-18, was dramatically increased in dko DCs and macrophages, and LPS stimulation bolstered their production. The preferential development into the Th1 subset in dko mice suggests that the cytokine milieu produced by the mutant mice in vivo or by mutant APCs in vitro selectively creates a differentiation environment favoring the Th1 effector response.     

Raloxifene Suppresses Experimental Autoimmune Encephalomyelitis and NF-κB-Dependent CCL20 Expression in Reactive Astrocytes

Recent clinical data have led to the consideration of sexual steroids as new potential therapeutic tools for multiple sclerosis. Selective estrogen receptor modulators can exhibit neuroprotective effects like estrogen, with fewer systemic estrogen side effects than estrogen, offering a more promising therapeutic modality for multiple sclerosis. The important role of astrocytes in a proinflammatory effect mediated by CCL20 signaling on inflammatory cells has been documented. Their potential contribution to selective estrogen receptor modulator-mediated protection is still unknown. Using a mouse model of chronic neuroinflammation, we report that raloxifene, a selective estrogen receptor modulator, alleviated experimental autoimmune encephalomyelitis–an animal model of multiple sclerosis–and decreased astrocytic production of CCL20. Enzyme-linked immunosorbent assay, immunohistochemistry imaging and transwell migration assays revealed that reactive astrocytes express CCL20, which promotes Th17 cell migration. In cultured rodent astrocytes, raloxifene inhibited IL-1β-induced CCL20 expression and chemotaxis ability for Th17 migration, whereas the estrogen receptor antagonist ICI 182,780 blocked this effect. Western blotting further indicated that raloxifene suppresses IL-1β-induced NF-κB activation (phosphorylation of p65) and translocation but does not affect phosphorylation of IκB. In conclusion, these data demonstrate that raloxifene provides robust neuroprotection against experimental autoimmune encephalomyelitis, partially via an inhibitory action on CCL20 expression and NF-κB pathways in reactive astrocytes. Our results contribute to a better understanding of the critical roles of raloxifene in treating experimental autoimmune encephalomyelitis and uncover reactive astrocytes as a new target for the inhibitory action of estrogen receptors on chemokine CCL20 expression.     

Ability of a nondepleting anti-CD4 antibody to inhibit Th2 responses and allergic lung inflammation is independent of coreceptor function

Nondepleting anti-CD4 Abs have been used in vivo to induce Ag-specific immunological tolerance in Th1 responses, including tissue allograft rejection and autoimmune diabetes. To examine whether this Ab (YTS177.9) acts by provoking a Th2 shift, we tested the effect in a mouse model of allergic lung inflammation. Interestingly, nondepleting anti-CD4 treatment induces tolerance to allergens as well, especially when given during initial priming. In vitro studies indicate that the effect of the Ab is independent of CD4 coreceptor function, as Ab treatment also inhibits proliferation and induces a persistent anergy in naive CD4 T cells stimulated by anti-CD3/CD28. Moreover, the Ab stimulated a distinct pattern of tyrosine phosphorylation in T cells even in the absence of TCR triggering, suggesting that signaling through CD4 alone induces significant physiological changes in T cell function. These results show that tolerance induced by anti-CD4 triggering is not a simple shift in Th1/Th2 effector function or depletion of Ag-specific cells, but may instead induce a persistent clonal anergy capable of blocking subsequent immunity.     

Increased expression of SLAM receptors SLAMF3 and SLAMF6 in systemic lupus erythematosus T lymphocytes promotes Th17 differentiation

Altered T cell function in systemic lupus erythematosus (SLE) is determined by various molecular and cellular abnormalities, including increased IL-17 production. Recent evidence suggests a crucial role for signaling lymphocyte activation molecules (SLAMs) in the expression of autoimmunity. In this study, we demonstrate that SLAMF3 and SLAMF6 expression is increased on the surface of SLE T cells compared with normal cells. SLAM coengagement with CD3 under Th17 polarizing conditions results in increased IL-17 production. SLAMF3 and SLAMF6 T cell surface expression and IL-17 levels significantly correlate with disease activity in SLE patients. Both naive and memory CD4(+) T cells produce more IL-17 in response to SLAM costimulation as compared with CD28 costimulation. In naive CD4(+) cells, IL-17 production after CD28 costimulation peaks on day 3, whereas costimulation with anti-SLAMF3 and anti-SLAMF6 Abs results in a prolonged and yet increasing production during 6 d. Unlike costimulation with anti-CD28, SLAM costimulation requires the presence of the adaptor molecule SLAM-associated protein. Thus, engagement of SLAMF3 and SLAMF6 along with Ag-mediated CD3/TCR stimulation represents an important source of IL-17 production, and disruption of this interaction with decoy receptors or blocking Abs should mitigate disease expression in SLE and other autoimmune conditions.