Pathogenic Potential of Eomesodermin-Expressing T-Helper Cells in Neurodegenerative Diseases.

Eomesodermin-expressing (Eomes+) T-helper (Th) cells show cytotoxic characteristics in secondary progressive multiple sclerosis. We found that Eomes+ Th cell frequency was increased in the peripheral blood of amyotrophic lateral sclerosis and Alzheimer's disease patients. Furthermore, granzyme B production by Th cells from such patients was high compared with controls. A high frequency of Eomes+ Th cells was observed in the initial (acutely progressive) stage of amyotrophic lateral sclerosis, and a positive correlation between Eomes+ Th cell frequency and cognitive decline was observed in Alzheimer's disease patients. Therefore, Eomes+ Th cells may be involved in the pathology of amyotrophic lateral sclerosis and Alzheimer's disease. ANN NEUROL 2024;95:1093-1098.

Involvement of cytotoxic Eomes-expressing CD4+ T cells in secondary progressive multiple sclerosis.

Multiple sclerosis (MS), a putative autoimmune disease of the central nervous system (CNS), commonly presents as relapsing-remitting MS (RRMS), characterized by recurrent episodes of peripheral disabling symptoms resulting from inflammatory CNS damage. Many RRMS patients transition to a chronic disease course with progressive neurological dysfunctions (secondary progressive MS, SPMS), with the progression rate varying between patients and over time. SPMS pathogenesis is now linked to immune-cell-mediated processes, although the mechanisms driving SPMS transition and progression remain elusive, and SPMS lacks biomarkers and effective treatments. We report the crucial involvement of cytotoxic CD4+ T cells expressing Eomes (Eomes+ Th cells) in SPMS pathogenesis-a Th cell subset previously identified in a mouse model of late/chronic autoimmune CNS inflammation. Few Eomes+ Th cells circulate in RRMS patient peripheral blood (n = 44), primary progressive MS (PPMS) patients (n = 25), or healthy controls (n = 42), but Eomes+ Th cells were significantly increased in SPMS (n = 105, P < 0.0001). Strikingly, lymphocytes isolated from SPMS autopsy brain samples revealed CD4+ T cells infiltrating CNS that coexpressed Eomes and the cytotoxic molecule granzyme B. In particular, the Eomes+ Th cell levels were increased in SPMS patients in progressive disease phases versus SPMS patients without current disability increases (P < 0.0001). Moreover, Eomes level acted as a biomarker to predict SPMS patients at risk of disease worsening with over 80% accuracy (ROC-AUC = 0.8276). Overall, our results indicate that granzyme B-expressing Eomes+ T helper cells are involved in the pathogenesis of SPMS, with significant implications for SPMS biomarkers and therapeutic targets.

Extrapituitary prolactin promotes generation of Eomes-positive helper T cells mediating neuroinflammation.

Induction of eomesodermin-positive CD4+ T cells (Eomes+ T helper [Th] cells) has recently been correlated with the transition from an acute stage to a later stage of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Moreover, these cells' pathogenic role has been experimentally proven in EAE. While exploring how the pathogenic Eomes+ Th cells are generated during the course of EAE, we unexpectedly found that B cells and MHC class II+ myeloid cells isolated from the late EAE lesions strikingly up-regulated the expression of prolactin (PRL). We demonstrate that such PRL-producing cells have a unique potential to induce Eomes+ Th cells from naïve T cells ex vivo, and that anti-MHC class II antibody could block this process. Furthermore, PRL levels in the cerebrospinal fluid were significantly increased in the late phase of EAE, and blocking the production of PRL by bromocriptine or Zbtb20-specific siRNA significantly reduced the numbers of Eomes+ Th cells in the central nervous system (CNS) and ameliorated clinical signs in the later phase of EAE. The PRL dependency of Eomes+ Th cells was confirmed in a series of in vitro and ex vivo experiments. Collectively, these results indicate that extrapituitary PRL plays a crucial role in the CNS inflammation mediated by pathogenic Eomes+ Th cells. Cellular interactions involving PRL-producing immune cells could be considered as a therapeutic target for the prevention of chronic neuroinflammation.

[Two Cases of Digestive Organ Cancer in Patients with Situs Inversus Treated with Laparoscopic Surgery].

Case 1: A 53-year-old woman had a positive fecal occult blood test during an examination performed in June 2014, and she visited our department in August. Colonoscopic examination showed a type 2 rectal cancer 4 cm from the anal verge. CT showed situs inversus totalis. We performed laparoscopic abdominoperineal resection (D2) for a diagnosis of cT1b, N0, M0, Stage Ⅰrectal cancer. Case 2: A 60-year-old man had a positive fecal occult blood test. Colonoscopic examination showed a type 2 cancer of the ascending colon. Chest radiography showed dextrocardia, but the arrangement of the organs in the abdomen was normal. We performed laparoscopic ileocecal resection (D3) for a diagnosis of cT2, N0, M0, StageⅠ colon cancer. Laparoscopic surgery can be performed safely in patients with situs inversus totalis.

[Significance of preoperative radiotherapy and lateral lymph node dissection for locally advanced rectal cancer - a comparative study involving different hospitals].

The aim of this study was to compare the results between lateral lymph node dissection (LLND) alone, and preoperative radiotherapy (preRT) followed by LLND, for the treatment of locally advanced rectal cancer. This study assessed 44 consecutive patients with lower rectal cancer (clinical Stages II and III) without lateral lymph node metastasis by preoperative imaging at two hospitals. Twenty-five patients at one hospital received preoperative short-course radiation therapy (total 25 Gy) followed by a curative LLND operation (preRT group), and 19 patients at another hospital underwent a curative operation by LLND alone (non-preRT group). The 5-year locoregional relapse-free survival, disease-free survival, and overall survival rates were not different between the preRT and non-preRT groups. Although three patients in each group had lateral lymph node metastases, none developed pelvic sidewall recurrence. This study suggests that preRT followed by curative surgery with LLND for lower rectal cancers without lateral lymph node metastasis before surgery would not improve survival and local control rates.

[Experimental animal model of multiple sclerosis--transverse investigation of MS pathogenesis for therapeutic intervention].

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) in which autoreactive T cells ignites downstream pathogenic cascades. The orphan nuclear receptor, NR4A2, is identified to be a selectively upregulated gene in peripheral blood T cells from relapsing-remitting MS patients. Furthermore, selective upregulation of NR4A2 is observed in peripheral blood T cells and CNS-infiltrating T cells upon immunization with myelin peptide in experimental autoimmune encephalomyelitis (EAE). Intriguingly, IL-17 -producing helper T cells exclusively express NR4A2, suggesting that NR4A2 expression represents a pathogenic T cells in autoimmunity. In addition, a NR4A2 blockade by RNA interference ameliorated EAE, implying the intrinsic roles of NR4A2 in MS/EAE, and could serve as a novel therapeutic target of the diseases.

Pathogenic Microglia Orchestrate Neurotoxic Properties of Eomes-Expressing Helper T Cells.

In addition to disease-associated microglia (DAM), microglia with MHC-II and/or IFN-I signatures may form additional pathogenic subsets that are relevant to neurodegeneration. However, the significance of such MHC-II and IFN-I signatures remains elusive. We demonstrate here that these microglial subsets play intrinsic roles in orchestrating neurotoxic properties of neurotoxic Eomes+ Th cells under the neurodegeneration-associated phase of experimental autoimmune encephalomyelitis (EAE) that corresponds to progressive multiple sclerosis (MS). Microglia acquire IFN-signature after sensing ectopically expressed long interspersed nuclear element-1 (L1) gene. Furthermore, ORF1, an L1-encoded protein aberrantly expressed in the diseased central nervous system (CNS), stimulated Eomes+ Th cells after Trem2-dependent ingestion and presentation in MHC-II context by microglia. Interestingly, administration of an L1 inhibitor significantly ameliorated neurodegenerative symptoms of EAE concomitant with reduced accumulation of Eomes+ Th cells in the CNS. Collectively, our data highlight a critical contribution of new microglia subsets as a neuroinflammatory hub in immune-mediated neurodegeneration.

Stochasticity of individual competition and local matchup inequality for saplings in a niche-structured forest.

Ecologists have recently accepted the notion that species coexistence involves both niche and neutral processes, but few studies have explained how both of these opposite views can explain coexistence in the same community. Here we focus on competition among sessile organisms and explored first the extent to which species-based niche reflects local "matchups" between nearby individuals, using 726 saplings of 10 temperate tree species, and second the members engaging in the matchups, which have rarely been quantified despite the importance in mixed-species forests. Growth responses to light showed considerable species-level differences, suggesting commonly seen regeneration niches. Outcomes of the individual matchups were basically predictable from the species mean response, but also with substantial contribution of within-species variation. We found strong imbalance in matchup frequencies, such that some individuals meet more individuals of differing species but others meet fewer, as well as many isolated, competition-free ones. The niche and neutral processes appear to reflect, respectively, between- and within-species differences, and our findings suggest that even when niche segregation is discernible, the role of stochasticity for the frequency of local competition, as well as its outcomes, cannot be discounted in species coexistence.

Immune-mediated neurodegenerative trait provoked by multimodal derepression of long-interspersed nuclear element-1.

Neurodegeneration is a process involving both cell autonomous and non-cell autonomous neuron loss, followed by a collapse of neural networks, but its pathogenesis is poorly understood. We have previously demonstrated that Eomes-positive helper T (Eomes + Th) cells recognizing LINE-1(L1)-derived prototypic antigen ORF1 mediate neurotoxicity associated with the neurodegenerative pathology of experimental autoimmune encephalomyelitis (EAE). Here, we show that Eomes + Th cells accumulate in the CNS of mouse models of authentic neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD), and secrete the neurotoxic granzyme B after encounter with ORF1 antigen. Multimodal derepression of neuronal L1 transcription is observed in EAE and ALS/AD models during neurodegeneration in active and cell cycle-mediated manner, respectively. These data suggest that the adventitious concurrence of immune-mediated neurodegenerative traits by Eomes + Th cells and ectopic expression of L1-derived antigen(s) in the inflamed CNS may materialize a communal and previously unappreciated pathogenesis of neurodegeneration.

Neuropilin-1 (NRP1) expression distinguishes self-reactive helper T cells in systemic autoimmune disease.

Pathogenic T helper cells (Th cells) that respond to self-antigen cannot be easily distinguished from beneficial Th cells. These cells can generate systemic autoimmune disease in response to widely expressed self-antigens. In this study, we have identified neuropilin-1 (NRP1) as a cell surface marker of self-reactive Th cells. NRP1+ Th cells, absent in non-regulatory T cell subsets in normal mice, appeared in models of systemic autoimmune disease and strongly correlated with disease symptoms. NRP1+ Th cells were greatly reduced in Nr4a2 cKO mice, which have reduced self-reactive responses but showed normal responses against exogenous antigens. Transfer of NRP1+ Th cells was sufficient to initiate or accelerate systemic autoimmune disease, and targeting NRP1-expressing Th cells therapeutically ameliorated SLE-like autoimmune symptoms in BXSB-Yaa mice. Peripheral NRP1+ Th cells were significantly increased in human SLE patients. Our data suggest that self-reactive Th cells can be phenotypically distinguished within the Th cell pool. These findings offer a novel approach to identify self-reactive Th cells and target them to treat systemic autoimmune disease.

Suppression of experimental autoimmune encephalomyelitis by ghrelin.

Ghrelin is a recently identified gastric hormone that displays strong growth hormone-releasing activity mediated by the growth hormone secretagogue receptor. While this unique endogenous peptide participates in the regulation of energy homeostasis, increases food intake, and decreases energy expenditure, its ability to inhibit the production of proinflammatory cytokines in vitro indicates its role in the regulation of inflammatory process in vivo. Here we examine the effect of exogenous ghrelin on the development of experimental autoimmune encephalomyelitis (EAE), a representative model of multiple sclerosis. In the C57BL/6 mouse model of EAE induced by sensitization to myelin oligodendrocyte glycoprotein 35-55 peptide, we found that alternate-day s.c. injections of ghrelin (5 mug/kg/day) from day 1 to 35 significantly reduced the clinical severity of EAE. The suppression of EAE was accompanied by reduced mRNA levels of proinflammatory cytokines such as TNF-alpha, IL-1beta, and IL-6 in the spinal cord cellular infiltrates and microglia from ghrelin-treated mice at the peak of disease, suggesting the role of ghrelin as an antiinflammatory hormone. Consistently, ghrelin significantly suppressed the production of proinflammatory cytokines in LPS-stimulated microglia in vitro. These results shed light on the new role of ghrelin in the regulation of inflammation with possible implications for management of human diseases.

Orphan nuclear receptor NR4A2 expressed in T cells from multiple sclerosis mediates production of inflammatory cytokines.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) mediated by Th17 and Th1 cells. DNA microarray analysis previously showed that NR4A2, an orphan nuclear receptor, is strongly up-regulated in the peripheral blood T cells of MS. Here, we report that NR4A2 plays a pivotal role for mediating cytokine production from pathogenic T cells. In experimental autoimmune encephalomyelitis (EAE), an animal model of MS, NR4A2, was selectively up-regulated in the T cells isolated from the CNS. Strikingly, a forced expression of NR4A2 augmented promoter activities of IL-17 and IFN-gamma genes, leading to an excessive production of these cytokines. Conversely, treatment with siRNA for NR4A2, resulted in a significant reduction in the production of IL-17 and IFN-gamma. Furthermore, treatment with NR4A2 siRNA reduced the ability of encephalitogenic T cells to transfer EAE in recipient mice. Thus, NR4A2 is an essential transcription factor for triggering the inflammatory cascade of MS/EAE and may serve as a therapeutic target.

An Optimized Preparation Method for Long ssDNA Donors to Facilitate Quick Knock-In Mouse Generation.

Fluorescent reporter mouse lines and Cre/Flp recombinase driver lines play essential roles in investigating various molecular functions in vivo. Now that applications of the CRISPR/Cas9 genome-editing system to mouse fertilized eggs have drastically accelerated these knock-in mouse generations, the next need is to establish easier, quicker, and cheaper methods for knock-in donor preparation. Here, we reverify and optimize the phospho-PCR method to obtain highly pure long single-stranded DNAs (ssDNAs) suitable for knock-in mouse generation via genome editing. The sophisticated sequential use of two exonucleases, in which double-stranded DNAs (dsDNAs) amplified by a pair of 5'-phosphorylated primer and normal primer are digested by Lambda exonuclease to yield ssDNA and the following Exonuclease III treatment degrades the remaining dsDNAs, enables much easier long ssDNA productions without laborious gel extraction steps. By microinjecting these donor DNAs along with CRISPR/Cas9 components into mouse zygotes, we have effectively generated fluorescent reporter lines and recombinase drivers. To further broaden the applicability, we have prepared long ssDNA donors in higher concentrations and electroporated them into mouse eggs to successfully obtain knock-in embryos. This classical yet improved method, which is regaining attention on the progress of CRISPR/Cas9 development, shall be the first choice for long donor DNA preparation, and the resulting knock-in lines could accelerate life science research.

Stromal expression of cancer-associated fibroblast-related molecules, versican and lumican, is strongly associated with worse relapse-free and overall survival times in patients with esophageal squamous cell carcinoma.

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment play an essential role in the tumor progression of esophageal squamous cell carcinoma (ESCC). The present study aimed to investigate the expression of CAF-related molecules, versican, periostin and lumican, in cancer stroma, to provide prognostic stratification for patients with ESCC after surgery. A total of 106 patients with ESCC who underwent curative esophagectomy without preoperative chemotherapy or radiotherapy were enrolled. The expression of CAF-related stromal proteins, including versican, periostin and lumican, was examined using immunohistochemistry, and the prognostic value was assessed by Kaplan-Meier survival analysis, and univariate and multivariate Cox regression models. The expression of versican, periostin and lumican was found specifically in the stromal component of ESCC. Kaplan-Meier analysis demonstrated that, compared with a low expression level, a high expression level of versican, periostin or lumican in the cancer stroma was significantly associated with worse relapse-free survival (RFS) and overall survival times in patients with ESCC. The prognostic values of stromal versican and lumican remained significant in a stratified analysis of stage I patients. Moreover, univariate and multivariate analysis revealed that high stromal versican or lumican expression was an independent prognostic factor for RFS in the patients. The present study demonstrated that CAF-related molecules, including versican, periostin and lumican, were expressed in the stroma of ESCC, and that stromal expression of versican and lumican in particular may have clinical utility as a prognostic biomarker for poor RFS in postoperative patients with ESCC.

Synthetic retinoid AM80 inhibits Th17 cells and ameliorates experimental autoimmune encephalomyelitis.

Recent evidence suggests that interleukin-17-producing CD4(+) T cells (Th17 cells) are the dominant pathogenic cellular component in autoimmune inflammatory diseases, including multiple sclerosis. It has recently been demonstrated that all-trans retinoic acid can suppress Th17 differentiation and promote the generation of Foxp3(+) regulatory T cells via retinoic acid receptor signals. Here, we investigated the effects of AM80, a synthetic retinoid with enhanced biological properties to all-trans retinoic acid, on Th17 differentiation and function and evaluated its therapeutic potential in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. AM80 treatment was more effective than all-trans retinoic acid in inhibiting Th17 differentiation in vitro. Oral administration of AM80 was protective for the early development of EAE and the down-modulation of Th17 differentiation and effector functions in vivo. Moreover, AM80 inhibited interleukin-17 production by splenic memory T cells, in vitro-differentiated Th17 cells, and central nervous system-infiltrating effector T cells. Accordingly, AM80 was effective when administered therapeutically after the onset of EAE. Continuous AM80 treatment, however, was ineffective at inhibiting late EAE symptoms despite the maintained suppression of RORgammat and interleukin-17 expression levels by central nervous system-infiltrating T cells. We reveal that continuous AM80 treatment also led to the suppression of interleukin-10 production by a distinct T cell subset that expressed both Foxp3 and RORgammat. These findings suggest that retinoid signaling regulates both inflammatory Th17 cells and Th17-like regulatory cells.

NKT cell-dependent amelioration of a mouse model of multiple sclerosis by altering gut flora.

Improved hygiene has been suggested to influence certain autoimmune disorders, such as multiple sclerosis. In this study, we addressed whether altering the composition of gut flora may affect susceptibility to experimental autoimmune encephalomyelitis (EAE), an animal model of MS. We administered a mixture of non-absorbing antibiotics, kanamycin, colistin, and vancomycin (KCV), orally to mice induced to develop EAE. The antibiotic treatment, beginning 1 week prior to sensitization, altered the composition of gut flora and, intriguingly, also ameliorated the development of EAE. While this result was associated with a reduced production of pro-inflammatory cytokines from the draining lymph node cells, a reduction of mesenteric Th17 cells was found to correlate with disease suppression. In addition, we found that Valpha14 invariant NKT (iNKT) cells were necessary for maintaining the mesenteric Th17 cells. The homologous effects of KCV treatment and iNKT cell depletion led us to speculate that KCV treatment may suppress EAE by altering the function of iNKT cells. Consistent with this hypothesis, KCV treatment did not suppress EAE that was induced in iNKT cell-deficient mice, although it was efficacious in mice that lacked Valpha19 mucosal-associated invariant T cells. Thus, gut flora may influence the development of EAE in a way that is dependent on iNKT cells, which has significant implications for the prevention and treatment of autoimmune diseases.

Eomes-expressing T-helper cells as potential target of therapy in chronic neuroinflammation.

Reserch progresses in understanding the pathogenicity of multiple sclerosis (MS) in the last couple of decade has enabled us to develop new drug entities available in the clinic. However, we still have not succeeded in preventing conversion from relapsing-remitting MS (RR-MS) to secondary progressive MS (SP-MS) and curing this intractable form of MS. Furthermore, diagnosis is usually retrospective and subjective, relying on gradual worsening of neurological signs/symptoms. This is obviously due to the lack of understanding for the pathogenicity driving disease progression in MS and of reliable biomarkers reflecting the progressive or stationary disease status. Two relevant components are involved in brain pathology of SP-MS, neurodegeneration and inflammation. Neurodegeneration may occur spontaneously in a neuron-intrinsic manner under chronic inflammation, such as glutamate excitotoxicity, mitochondrial/oxidative injury with iron deposit in the brain, and loss of trophic support. Meanwhile, inflammation is usually associated with recurrent relapse and the cumulative infiltration of immune cells, including T cells, B cells, and myeloid cells of peripheral or CNS origin, could ignite the processes of neurodegeneration. Especially, the higher frequency of leptomeningeal follicle-like structures observed in SP-MS patients suggests that immune cells sheltered behind a blood-brain barrier is still active under smoldering CNS inflammation. Recent successes in Ocrelizumab for primary progressive in MS (PP-MS) and Siponimod for SP-MS reappraised the importance of immune cells for pathogenesis progressive MS. Accordingly, our recent comparative analysis between MS and its animal model, experimental autoimmune encephalomyelitis (EAE), raises a new possibility that ectopic expression of eomesodermin (Eomes) in helper T (Th) cells constitutes a previously unappreciated subset of Th cells with cytotoxic potential against neuronal cells. In this review article, I will summarize the mechanisms proposed on pathogenesis of SP-MS and propose a new pathogenic mechanism for neurodegeneration mediated by unique cytotoxic Th cells.

Circulating exosomes suppress the induction of regulatory T cells via let-7i in multiple sclerosis.

Multiple sclerosis (MS) is a T cell-mediated autoimmune disease of the central nervous system. Foxp3+ regulatory T (Treg) cells are reduced in frequency and dysfunctional in patients with MS, but the underlying mechanisms of this deficiency are unclear. Here, we show that induction of human IFN-γ-IL-17A-Foxp3+CD4+ T cells is inhibited in the presence of circulating exosomes from patients with MS. The exosomal miRNA profile of patients with MS differs from that of healthy controls, and let-7i, which is markedly increased in patients with MS, suppresses induction of Treg cells by targeting insulin like growth factor 1 receptor (IGF1R) and transforming growth factor beta receptor 1 (TGFBR1). Consistently, the expression of IGF1R and TGFBR1 on circulating naive CD4+ T cells is reduced in patients with MS. Thus, our study shows that exosomal let-7i regulates MS pathogenesis by blocking the IGF1R/TGFBR1 pathway.

The clinical implication and molecular mechanism of preferential IL-4 production by modified glycolipid-stimulated NKT cells.

OCH, a sphingosine-truncated analog of alpha-galactosylceramide (alphaGC), is a potential therapeutic reagent for a variety of Th1-mediated autoimmune diseases through its selective induction of Th2 cytokines from natural killer T (NKT) cells. We demonstrate here that the NKT cell production of IFN-gamma is more susceptible to the sphingosine length of glycolipid ligand than that of IL-4 and that the length of the sphingosine chain determines the duration of NKT cell stimulation by CD1d-associated glycolipids. Furthermore, IFN-gamma production by NKT cells requires longer T cell receptor stimulation than is required for IL-4 production by NKT cells stimulated either with immobilized mAb to CD3 or with immobilized "alphaGC-loaded" CD1d molecules. Interestingly, transcription of IFN-gamma but not that of IL-4 was sensitive to cycloheximide treatment, indicating the intrinsic involvement of de novo protein synthesis for IFN-gamma production by NKT cells. Finally, we determined c-Rel was preferentially transcribed in alphaGC-stimulated but not in OCH-stimulated NKT cells and was essential for IFN-gamma production by activated NKT cells. Given the dominant immune regulation by the remarkable cytokine production of ligand-stimulated NKT cells in vivo, in comparison with that of (antigen-specific) T cells or NK cells, the current study confirms OCH as a likely therapeutic reagent for use against Th1-mediated autoimmune diseases and provides a novel clue for the design of drugs targeting NKT cells.

Pathological mechanism of secondary-progressive multiples sclerosis and its animal model.

Development of acute experimental autoimmune encephalomyelitis (EAE) depends on Th17 cells expressing the nuclear factor NR4A2, which we have previously reported to be upregulated in peripheral blood T cells from patients of multiple sclerosis (MS). EAE induced in mice lacking NR4A2 in T cells showed a great reduction in Th17-mediated acute symptoms, whereas a late-onset disease independent of NR4A2 was still inducible. We identified cytotoxic T-cell-like CD4+ T cells expressing the T-box transcription factor Eomesodermin (Eomes) as a pathogenic component for the development of the late-onset disease. Furthermore, T cell-specific deletion of the Eomes gene or Eomes-specific RNA interference in vivo remarkably ameliorated the late-onset EAE. Intriguingly, similar Eomes-expressing CD4+ T cells are increased in the peripheral blood and cerebrospinal fluid only from patients with secondary-progressive MS accompanied by neurodegenerative symptoms, but not in relapsing-remitting MS. Mechanistic analysis revealed that granzyme B was secreted by Eomes-expressing CD4+ T cells and the activation of protease-activated receptor-1 by granzyme B is involved in the neuroinflammation observed in the late-onset EAE.