IL-23 modulated myelin-specific T cells induce EAE via an IFNγ driven, IL-17 independent pathway

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nervous system (CNS) mediated by myelin-reactive CD4⁺ T cells. An unresolved issue that has important clinical implications concerns the cytokines produced by myelin-reactive T cells that determine their pathogenicity. Initially, IL-12 polarized, IFNγ producing Th1 cells were thought to be essential for the development of EAE. More recently, IL-23 polarized, IL-17 producing Th17 cells have been highlighted as critical encephalitogenic effectors. There is growing evidence that parallel autoimmune pathways can result in common clinical and histopathological endpoints. In the current study, we describe a form of EAE induced by the transfer of IL-23 modulated CD4⁺ T cells into IL-17 receptor (IL-17R) deficient hosts. We found that IL-23 stimulates myelin-reactive T cells to produce both IFNγ and IL-17. Surprisingly, in this model the development of EAE is IFNγ dependent. Our findings illustrate a novel mechanism by which IL-23 promotes encephalitogenicity and they further expand the spectrum of autoreactive T cells capable of mediating inflammatory demyelinating disease of the CNS.     

β-Lapachone ameliorization of experimental autoimmune encephalomyelitis

β-Lapachone is a naturally occurring quinine, originally isolated from the bark of the lapacho tree (Tabebuia avellanedae) which is currently being evaluated in clinical trials for the treatment of cancer. In addition, recent investigations suggest its potential application for treatment of inflammatory diseases. Multiple sclerosis (MS) is an autoimmune disorder characterized by CNS inflammation and demyelination. Reactive T cells including IL-17 and IFN-γ-secreting T cells are believed to initiate MS and the associated animal model system experimental autoimmune encephalomyelitis (EAE). IL-12 family cytokines secreted by peripheral dendritic cells (DCs) and CNS microglia are capable of modulating T-cell phenotypes. The present studies demonstrated that β-lapachone selectively inhibited the expression of IL-12 family cytokines including IL-12 and IL-23 by DCs and microglia, and reduced IL-17 production by CD4⁺ T-cells indirectly through suppressing IL-23 expression by microglia. Importantly, our studies also demonstrated that β-lapachone ameliorated the development on EAE. β-Lapachone suppression of EAE was associated with decreased expression of mRNAs encoding IL-12 family cytokines, IL-23R and IL-17RA, and molecules important in Toll-like receptor signaling. Collectively, these studies suggest mechanisms by which β-lapachone suppresses EAE and suggest that β-lapachone may be effective in the treatment of inflammatory diseases such as MS.     

Therapeutic gene silencing with siRNA for IL-23 but not for IL-17 suppresses the development of experimental autoimmune encephalomyelitis in rats

Gene silencing with siRNAs is important as a therapeutic tool in autoimmune diseases. In this study, we administered siRNAs specific for cytokines that may be involved in pathogenesis of experimental autoimmune encephalomyelitis (EAE). siRNA specific for IL-23p19 (siRNA-IL-23) suppressed EAE almost completely, whereas siRNA-IL-17A did not modulate the clinical course. Flow cytometric analysis revealed that siRNA-IL-23 significantly reduced the proportion of both IFN-γ⁺IL-17^? Th1 and IFN-γ^?IL-17⁺ Th17 cells in the spinal cord. Consistent with this finding, siRNA-IL-23 treatment downregulated IL-12, IL-17 and IL-23 mRNAs. These findings indicate that IL-23, but not IL-17, play an important role in the development of EAE.     

Respiratory infection with a bacterial pathogen attenuates CNS autoimmunity through IL-10 induction

Infection with viral or bacterial pathogens has been linked with the development of multiple sclerosis (MS), while infection with helminth parasites has been associated protection against MS and other autoimmune diseases. Here we have used a murine model of MS, experimental autoimmune encephalomyelitis (EAE), to examine the effect of infection with the respiratory pathogen Bordetella pertussis infection on development of CNS inflammation. The data demonstrate that infection of mice with B. pertussis significantly attenuates the clinical course of EAE induced by active immunization or cell transfer. This was reflected in a significant reduction in VLA-4 and LFA-1 expression on T cells and infiltration of IL-17⁺, IFN-γ⁺ and IFN-γ⁺IL-17⁺ CD4 T cells into the CNS. Infection with B. pertussis induced IL-10 production from dendritic cells in vitro and enhanced the frequency of IL-10-producing CD25^-Foxp3^+/− CD4⁺ T cells in vivo. Furthermore, the suppressive effects of B. pertussis infection on EAE were lost in IL-10^−/− mice. Our findings demonstrate that a bacterial infection of the respiratory tract can attenuate EAE by promoting production of the anti-inflammatory cytokine IL-10 that may suppress licensing of autoaggressive T cells in the lungs, thereby preventing their migration into the CNS.     

Th1 not Th17 cells drive spontaneous MS-like disease despite a functional regulatory T cell response

Multiple sclerosis is considered a disease of complex autoimmune etiology, yet there remains a lack of consensus as to specific immune effector mechanisms. Recent analyses of experimental autoimmune encephalomyelitis, the common mouse model of multiple sclerosis, have investigated the relative contribution of Th1 and Th17 CD4 T cell subsets to initial autoimmune central nervous system (CNS) damage. However, inherent in these studies are biases influenced by the adjuvant and toxin needed to break self-tolerance. We investigated spontaneous CNS disease in a clinically relevant, humanized, T cell receptor transgenic mouse model. Mice develop spontaneous, ascending paralysis, allowing unbiased characterization of T cell immunity in an HLA-DR15-restricted T cell repertoire. Analysis of naturally progressing disease shows that IFNγ⁺ cells dominate disease initiation with IL-17⁺ cells apparent in affected tissue only once disease is established. Tregs accumulate in the CNS but are ultimately ineffective at halting disease progression. However, ablation of Tregs causes profound acceleration of disease, with uncontrolled infiltration of lymphocytes into the CNS. This synchronous, severe disease allows characterization of the responses that are deregulated in exacerbated disease: the correlation is with increased CNS CD4 and CD8 IFNγ responses. Recovery of the ablated Treg population halts ongoing disease progression and Tregs extracted from the central nervous system at peak disease are functionally competent to regulate myelin specific T cell responses. Thus, in a clinically relevant mouse model of MS, initial disease is IFNγ driven and the enhanced central nervous system responses unleashed through Treg ablation comprise IFNγ cytokine production by CD4 and CD8 cells, but not IL-17 responses.     

Male rats develop more severe experimental autoimmune encephalomyelitis than female rats: Sexual dimorphism and diergism at the spinal cord level

Compared with females, male Dark Agouti (DA) rats immunized for experimental autoimmune encephalomyelitis (EAE) with rat spinal cord homogenate in complete Freund’s adjuvant (CFA) exhibited lower incidence of the disease, but the maximal neurological deficit was greater in the animals that developed the disease. Consistently, at the peak of the disease greater number of reactivated CD4+CD134+CD45RC− T lymphocytes was retrieved from male rat spinal cord. Their microglia/macrophages were more activated and produced greater amount of prototypic proinflammatory cytokines in vitro. Additionally, oppositely to the expression of mRNAs for IL-12/p35, IL-10 and IL-27/p28, the expression of mRNA for IL-23/p19 was upregulated in male rat spinal cord mononuclear cells. Consequently, the IL-17+:IFN-γ+ cell ratio within T lymphocytes from their spinal cord was skewed towards IL-17+ cells. Within this subpopulation, the IL-17+IFN-γ+:IL-17+IL-10+ cell ratio was shifted towards IL-17+IFN-γ+ cells, which have prominent tissue damaging capacity. This was associated with an upregulated expression of mRNAs for IL-1β and IL-6, but downregulated TGF-β mRNA expression in male rat spinal cord mononuclear cells. The enhanced GM-CSF mRNA expression in these cells supported the greater pathogenicity of IL-17+ T lymphocytes infiltrating male spinal cord. In the inductive phase of the disease, contrary to the draining lymph node, in the spinal cord the frequency of CD134+ cells among CD4+ T lymphocytes and the frequency of IL-17+ cells among T lymphocytes were greater in male than in female rats. This most likely reflected an enhanced transmigration of mononuclear cells into the spinal cord (judging by the lesser spinal cord CXCL12 mRNA expression), the greater frequency of activated microglia/macrophages and the increased expression of mRNAs for Th17 polarizing cytokines in male rat spinal cord mononuclear cells. Collectively, the results showed cellular and molecular mechanisms underlying the target organ specific sexual dimorphism in the T lymphocyte-dependent immune/inflammatory response, and suggested a substantial role for the target organ in shaping the sexually dimorphic clinical outcome of EAE.     

Gintonin mitigates experimental autoimmune encephalomyelitis by stabilization of Nrf2 signaling via stimulation of lysophosphatidic acid receptors

Gintonin (GT), a glycolipoprotein fraction isolated from ginseng, exerts neuroprotective effects in models of neurodegenerative diseases such as Alzheimer’s disease. However, the in vivo role of GT in multiple sclerosis (MS) has not been clearly resolved. We investigated the effect of GT in myelin oligodendrocyte glycoprotein (MOG_35-55)-induced experimental autoimmune encephalomyelitis (EAE), an animal model of MS. GT alleviated behavioral symptoms of EAE associated with reduced demyelination, diminished infiltration and activation of immune cells (microglia and macrophage), and decreased expression of inflammatory mediators in the spinal cord of the EAE group compared to that of the sham group. GT reduced the percentages of CD4⁺/IFN-γ⁺ (Th1) and CD4⁺/IL-17⁺ (Th17) cells but increased the population of CD4⁺/CD25⁺/Foxp3⁺ (Treg) cells in the spinal cord, in agreement with altered mRNA expression of IFN-γ, IL-17, and TGF-ß in the spinal cord in concordance with mitigated blood–brain barrier disruption. The underlying mechanism is related to inhibition of the ERK and p38 mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) pathways and the stabilization of nuclear factor erythroid 2-related factor 2 (Nrf2) via increased expression of lysophosphatidic acid receptor (LPAR) 1–3. Impressively, these beneficial effects of GT were completely neutralized by inhibiting LPARs with Ki16425, a LPAR1/3 antagonist. Our results strongly suggest that GT may be able to alleviate EAE due to its anti-inflammatory and antioxidant activities through LPARs. Therefore, GT is a potential therapeutic option for treating autoimmune disorders including MS.     

IL-27 mediates the response to IFN-β therapy in multiple sclerosis patients by inhibiting Th17 cells

Interferon (IFN)-β is a commonly used therapy for relapsing remitting multiple sclerosis (RRMS). However its protective mechanism is still unclear and the failure of many patients to respond has not been explained. We have found that IFN-β suppressed IL-23 and IL-1β production and increased IL-10 production by human dendritic cells (DC) activated with the TLR2 and dectin-1 agonist zymosan. Furthermore, IFN-β impaired the ability of DC to promote IL-17 production by CD4⁺ T cells, but did not affect IFN-γ production. IFN-β induced IL-27 expression by DC, and neutralisation of IL-27 abrogated the suppressive effects of IFN-β on zymosan-induced IL-1 and IL-23 production and the generation of Th17 cells in vitro. Complementary in vivo studies in a mouse model showed that treatment with IFN-β enhanced expression of IL-27, and reduced IL-17 in the CNS and periphery and attenuated the clinical signs of experimental autoimmune encephalomyelitis (EAE). In addition, the significant suppressive effect of IFN-β on the ability of DC to promote Th17 cells was lost in cells from IL-27 receptor deficient mice. Finally, we showed that PBMC from non-responder RRMS patients produced significantly less IL-27 in response to IFN-β than patients who responded to IFN-β therapy. Our findings suggest that IFN-β mediates its therapeutic effects in MS at least in part via the induction of IL-27, and that IL-27 may represent an alternative therapy for MS patients that do not respond to IFN-β.     

Ingested (oral) ACTH inhibits EAE

Ingested type I IFN and SIRS peptide inhibit EAE. We examined whether another immunoactive protein, ACTH, would have similar anti-inflammatory effects in EAE after oral administration. B6 mice were immunized and gavaged with control saline or ACTH starting on the onset of disease. ACTH decreased clinical score and decreased inflammatory foci. CNS lymphocytes showed decreases in IL-17 (T(eff)) and Th1-like encephalitogenic cytokines IL-2 and IFN-γ in the ACTH fed group compared to the mock fed group. Adoptive transfer of ACTH fed splenocytes into MOG immunized recipient mice with early clinical disease suppressed disease severity compared to splenocytes from mock fed donors. The protected recipients showed decreased splenic IL-17 (T(eff)) and Th1-like cytokine IFN-γ and increased CNS secretion of immunoregulatory IL-4 and chemokine M-CSF. Splenic CD4+CD25+ FoxP3+ frequency doubled in ACTH fed compared to control fed mice. Increased immuno-regulatory IL-4 and M-CSF secreting cell populations is the mechanism of protection in adoptively protected recipients and reflects the direct action of ACTH on the immune system.     

重症肌无力患者血清Th1/Th2/Th17细胞因子的变化及意义

目的：分析重症肌无力（MG）患者血清CD4^＋ T细胞主要细胞因子的水平,探讨不同亚型CD4^＋ T细胞分泌的细胞因子在MG发病机制中的作用。方法：用ELISA测定93例MG患者和34名健康对照者血清中各项细胞因子（IL-2、IL-12、IFN-γ、TNF-α、IL-4、IL-10、IL-13和IL-17）的水平,分组行统计学分析。结果：与健康对照组相比,MG患者Th1细胞相关各细胞因子（IL-2、IL-12、IFN-γ及TNF-α）均明显升高,差异有统计学意义（P〈0.05）;Th2细胞相关的细胞因子IL-4、IL-10差异无统计学意义（P〉0.05）,仅IL-13水平升高;Th17细胞的细胞因子IL-17水平差异无统计学意义（P〉0.05）。MG眼肌型与全身型患者血清中各细胞因子水平的差异无统计学意义（P〉0.05）,在不同病程的MG患者中差异也无统计学意义（P〉0.05）。结论：Th1细胞因子在MG发病机制中发挥重要作用,而Th2细胞及其细胞因子在MG机制中的角色各异。     

Encephalitogenic T cells are present in Lewis rats protected from autoimmune encephalomyelitis by coimmunization with MBP73-84 and its analog

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) that is mediated by T helper 1 (Th1) CD4⁺ T cells. Lewis rats can be protected from actively induced EAE by coimmunization with the encephalitogenic myelin basic protein (MBP) epitope 73–84 and its single alanine-substituted analog 1028. Although analog 1028 cannot induce either active or passive EAE, it does elicit a Th1-like response that is cross-reactive with MBP73–84. Analog 1028 can effectively inhibit clinical EAE in a dose-dependent manner when rats are coimmunized with the encephalitogenic peptide MBP73–84 and 1028 in complete Freund adjuvant (CFA). Stimulation of cells from MBP73–84:1028—coimmunized protected rats proliferate and secrete interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in vitro in response to MBP73–84. Furthermore, coimmunized protected rats harbor a population of MBP73–84—reactive potentially encephalitogenic T cells, because splenocytes from these rats can be stimulated to transfer passive EAE to naive recipients. Thus, the protection of coimmunized rats by analog 1028 is not due to the inhibition of priming of MBP73–84—reactive T cells or alteration of the cytokine secretion profile of the MBP73–84—reactive cell population. Rather, MBP73–84—reactive potentially encephalitogenic T cells are primed in these protected animals. © 1996 Wiley-Liss, Inc.     

Cytokine production in the central nervous system of Lewis rats with experimental autoimmune encephalomyelitis: dynamics of mRNA expression for interleukin-10, interleukin-12, cytolysin,tumor necrosis factor α and tumor necrosis factor β

The kinetics of mRNA expression in the central nervous system (CNS) for a series of putatively disease-promoting and disease-limiting cytokines during the course of experimental autoimmune encephalomyelitis (EAE) in Lewis rats were studied. Cytokine mRNA-expressing cells were detected in cryosections of spinal cords using in situ hybridization technique with synthetic oligonucleotide probes. Three stages of cytokine mRNA expression could be distinguished: (i) interleukin (IL)-12, tumor necrosis factor (TNF)-β (=lymphotoxin-α) and cytolysin appeared early and before onset of clinical signs of EAE; (ii) TNF-α peaked at height of clinical signs of EAE; (iii) IL-10 appeared increasingly at and after clinical recovery. The early expression of IL-12 prior to the expression of interferon-γ (IFN-γ) mRNA shown previously is consistent with a role of IL-12 in promoting proliferation and activation of T helper 1 (Th1) type cells producing IFN-γ. The TNF-β mRNA expression prior to onset of clinical signs favours a role for this cytokine in disease initiation. A pathogenic effector role of TNF-α was suggested from these observations that TNF-α mRNA expression roughly paralleled the clinical signs of EAE. This may be the case also for cytolysin. IL-10-expressing cells gradually increased to high levels in the recovery phase of EAE, consistent with a function in down-regulating the CNS inflammation. From these data we conclude that there is an ordered appearance of putative disease-promoting and -limiting cytokines in the CNS during acute monophasic EAE.     

Activation of mixed glia by Aβ-specific Th1 and Th17 cells and its regulation by Th2 cells

Microglia are innate immune cells of the CNS, that act as antigen-presenting cells (APC) for antigen-specific T cells and respond to inflammatory stimuli, such as amyloid-beta (Aβ), resulting in the release of neurotoxic factors and pro-inflammatory cytokines. Astrocytes can also act as APC and modulate the function of microglia. However, the role of distinct T cell subtypes, in particular Th17 cells, in glial activation and subsequent modulatory effects of Th2 cells are poorly understood. Here, we generated Aβ-specific Th1, Th2, and Th17 cells and examined their role in modulating Aβ-induced activation of microglia in a mixed glial culture, a preparation which mimics the complex APC types in the brain. We demonstrated that mixed glia acted as an effective APC for Aβ-specific Th1 and Th17 cells. Addition of Aβ-specific Th2 cells suppressed the Aβ-induced IFN-γ production by Th1 cells and IL-17 production by Th17 cells with glia as the APC. Co-culture of Aβ-specific Th1 or Th17 cells with glia markedly enhanced Aβ-induced pro-inflammatory cytokine production and expression of MHC class II and co-stimulatory molecules on the microglia. Addition of Aβ-specific Th2 cells inhibited Th17 cell-induced IL-1β and IL-6 production by mixed glia and attenuated Th1 cell-induced CD86 and CD40 expression on microglia. The modest enhancement of MHC class II and CD86 expression on astrocytes by Aβ-specific Th1 and Th17 was not attenuated by Th2 cells. These data indicate that Aβ-specific Th1 and Th17 cells induce inflammatory activation of glia, and that this is in part regulated by Th2 cells.     

Priming of microglia with IFN-γ impairs adult hippocampal neurogenesis and leads to depression-like behaviors and cognitive defects

Neuroinflammation driven by interferon-gamma (IFN-γ) and microglial activation has been linked to neurological disease. However, the effects of IFN-γ-activated microglia on hippocampal neurogenesis and behavior are unclear. In the present study, IFN-γ was administered to mice via intracerebroventricular injection. Mice received intraperitoneal injection of ruxolitinib to inhibit the JAK/STAT1 pathway or injection of minocycline to inhibit microglial activation. During a 7-day period, mice were assessed for depressive-like behaviors and cognitive impairment based on a series of behavioral analyses. Effects of the activated microglia on neural stem/precursor cells (NSPCs) were examined, as was pro-inflammatory cytokine expression by activated microglia. We showed that IFN-γ-injected animals showed long-term adult hippocampal neurogenesis reduction, behavior despair, anhedonia, and cognitive impairment. Chronic activation with IFN-γ induces reactive phenotypes in microglia associated with morphological changes, population expansion, MHC II and CD68 up-regulation, and pro-inflammatory cytokine (IL-1β, TNF-α, IL-6) and nitric oxide (NO) release. Microglia isolated from the hippocampus of IFN-γ-injected mice suppressed NSPCs proliferation and stimulated apoptosis of immature neurons. Inhibiting of the JAK/STAT1 pathway in IFN-γ-injected animals to block microglial activation suppressed microglia-mediated neuroinflammation and neurogenic injury, and alleviated depressive-like behaviors and cognitive impairment. Collectively, these findings suggested that priming of microglia with IFN-γ impairs adult hippocampal neurogenesis and leads to depression-like behaviors and cognitive defects. Targeting microglia by modulating levels of IFN-γ the brain may be a therapeutic strategy for neurodegenerative diseases and psychiatric disorders.     

Epigenetic regulation of Th1 and Th2 cell development

All cells of the body, regardless of the tissue type, contain the same genetic material, but express this genetic material differently. Epigenetics is one process by which differential gene expression within a cell is regulated. Epigenetic mechanisms involve postsynthetic modifications to DNA and/or DNA-associated histones that do not change the DNA sequence itself, but which remodel chromatin, are passed along at each cell division, and occur during and after early development. The CD4⁺ T cell best represents a cell in which epigenetic mechanisms are used to affect mature cell physiology. As a naïve CD4⁺ T cell develops into either a Th1 or Th2 cell that secretes predominantly IFN-γ or IL-4, respectively, the expression of one cytokine gene and the permanent silencing of the other is orchestrated using epigenetic mechanisms. Because there appears to be an association between Th1/Th2 cell immunity, behavior, and/or disease, it is possible that an environmentally induced epigenetic change that occurs during Th1/Th2 cell development could explain how certain Th1/Th2-associated conditions develop. This article will review basic epigenetic mechanisms and what is known about how these mechanisms influence cytokine gene expression in a naïve CD4⁺ T cell as it develops into a Th1 or Th2 cell.     

Cytokine phenotype of human autoreactive T cell clones specific for the immunodominant myelin basic protein peptide (83–99)

Experimental allergic encephalomyelitis (EAE), an animal model resembling multiple sclerosis (MS), is mediated by myelin antigen-specific CD4+ T cells secreting cytokines such as interferon-γ (IFN-γ), tumor necrosis factor-β (TNF-β), and the proinflammatory cytokine TNF-α—all associated with the T-helper-1 (Th1) T cell subset. Based on numerous similarities between MS and EAE, it has been postulated that Th1-like T cells are involved in the pathogenesis of MS. Production of proinflammatory cytokines such as IFN-γ and, in particular, TNF-α/β by autoreactive T cells is considered crucial for the initiation and amplification of inflammatory brain lesions and possibly also for direct myelin damage. In contrast, regulatory cytokines such as interleukin-4 (IL-4), IL-10, and IL-13, which are associated with the Th2-like phenotype, may play a role in the resolution of relapses. Although the human T cell response to myelin basic protein (MBP) is well characterized in terms of antigen specificity, HLA restriction, and T cell-receptor (TCR) usage, little is known about the cytokine pattern of these autoreactive T cells. To gain such information, conditions for studying cytokine secretion by human autoreactive T cell clones (TCC) were established. The cytokine secretion profile of human autoreactive CD4+ TCC, specific for myelin basic protein peptide (83–89) [MBP(83–99)], a candidate autoantigen in MS, was investigated. Our results show that TCC cytokine production in long-term culture was stable. In addition, the correlation of various cytokines within specific TCC revealed differences compared to murine T cells. The comparison of 30 human MBP(83–99)-specific TCC demonstrated heterogeneity in cytokine secretion, with a continuum between Th1- and Th2-like cells rather than distinct Th1 or Th2 subsets. These data are important for further investigation of the potential role of cytokines in the inflammatory process of MS, and provide a powerful tool to investigate therapeutic interventions with respect to their influence on cytokine secretion of autoreactive T cells. © 1996 Wiley-Liss, Inc.     

Programmed Death Ligand-1 on Microglia Regulates Th1 Differentiation <Emphasis Type="Italic">via</Emphasis> Nitric Oxide in Experimental Autoimmune Encephalomyelitis

Microglia are considered to be potential antigen-presenting cells and have the ability to present antigen under pathological conditions. Nevertheless, whether and how microglia are involved in immune regulation are largely unknown. Here, we investigated the suppressive activity of microglia during experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein, with the goal of understanding their role in regulating the T cell reaction. Using flow cytometric analysis, we found that microglia were characterized by increased cell number and up-regulated programmed death ligand-1 (PD-L1) at the peak phase of EAE. Meanwhile, both the CD4⁺ T cells and microglia that infiltrated the central nervous system expressed higher levels of PD1, the receptor for PD-L1, accompanied by a decline of Th1 cells. In an ex vivo co-culture system, microglia from EAE mice inhibited the proliferation of antigen-specific CD4⁺ T cells and the differentiation of Th1 cells, and this was significantly inhibited by PD-L1 blockade. Further, microglia suppressed Th1 cells via nitric oxide (NO), the production of which was dependent on PD-L1. Thus, these data suggest a scenario in which microglia are involved in the regulation of EAE by suppressing Th1-cell differentiation via the PD-L1-NO pathway.