Article Repulsive Guidance Molecule-a Is Involved in Th17Cell-Induced Neurodegeneration in Autoimmune Encephalomyelitis Graphical Abstract Authors Shogo Tanabe, Toshihide Yamashita Correspondence [email protected] In Brief The mechanism of neurodegeneration under inflammation in the CNS remains largely unknown. Tanabe and Yamashita demonstrate that RGMa is highly expressed in Th17 cells and induces dephosphorylation of Akt, leading to death of neurons. A neutralizing antibody to RGMa attenuates axonal degeneration and severity of Th17-cell-mediated autoimmune encephalomyelitis. Highlights RGMa binding to neogenin induces dephosphorylation of Akt in neurons RGMa expressed in Th17 cells induces neurodegeneration Depletion of RGMa rescues axonal degeneration in EAE Neutralizing antibody to RGMa attenuates severity of Th17 cellmediated EAE Tanabe & Yamashita, 2014, Cell Reports 9, 1–12 November 20, 2014 ª2014 The Authors http://dx.doi.org/10.1016/j.celrep.2014.10.038 Please cite this article in press as: Tanabe and Yamashita, Repulsive Guidance Molecule-a Is Involved in Th17-Cell-Induced Neurodegeneration in Autoimmune Encephalomyelitis, Cell Reports (2014), http://dx.doi.org/10.1016/j.celrep.2014.10.038 Cell Reports Article Repulsive Guidance Molecule-a Is Involved in Th17-Cell-Induced Neurodegeneration in Autoimmune Encephalomyelitis Shogo Tanabe1,2 and Toshihide Yamashita1,2,* 1Department of Molecular Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita-shi, Osaka 565-0871, Japan 2Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 5 Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan *Correspondence: [email protected] http://dx.doi.org/10.1016/j.celrep.2014.10.038 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). SUMMARY Multiple sclerosis (MS) is a chronic autoimmune disease characterized by inflammation, demyelination, and neurodegeneration in the CNS. Although it is important to prevent neurodegeneration for alleviating neurological disability, the molecular mechanism of neurodegeneration remains largely unknown. Here, we report that repulsive guidance molecule-a (RGMa), known to regulate axonal growth, is associated with neurodegeneration in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. RGMa is highly expressed in interleukin-17-producing CD4+ T cells (Th17 cells). We induced EAE by adoptive transfer of myelin oligodendrocyte glycoprotein (MOG)-specific Th17 cells and then inhibited RGMa with a neutralizing antibody. Inhibition of RGMa improves EAE scores and reduces neuronal degeneration without altering immune or glial responses. Th17 cells induce cultured cortical neuron death through RGMa-neogenin and Akt dephosphorylation. Our results demonstrate that RGMa is involved in Th17-cell-mediated neurodegeneration and that RGMa-specific antibody may have a therapeutic effect in MS. INTRODUCTION In multiple sclerosis (MS), immune cells, such as T cells and monocytes, infiltrate the CNS and induce inflammation, demyelination, and neurodegeneration (Trapp et al., 1999). MS pathogenesis is attributed to acquired autoimmunity to CNS components, including myelin sheaths (Stinissen et al., 1998). Previous studies have demonstrated that CD4+ T cells play critical roles in inducing CNS inflammation (Goverman, 2009). T helper type 1 cells (Th1 cells), characterized by their secretion of interferon-gamma (IFN-g), are abundant in the cerebrospinal fluid (CSF) of MS patients and are an essential initiator of encephalomyelitis (Ando et al., 1989; Rotteveel et al., 1990). A more recent study identified T helper type 17 cells (Th17 cell), which produce interleukin-17 (IL-17), as critical drivers for autoimmune diseases, including MS (Bettelli et al., 2006). Th17 cells are differentiated by IL-6 and transforming growth factor-b1 (TGF-b1), and stimulation of Th17 cells with IL-23 enhances their pathogenic activity via proinflammatory cytokine production (Dong, 2008; Lee et al., 2012). Indeed, IL-17-, IL-17 receptor-, or IL-23 receptor-deficient mice are resistant to experimental autoimmune encephalomyelitis (EAE; Hu et al., 2010; Komiyama et al., 2006; McGeachy et al., 2009). IL-17 also exerts cytotoxic effects on NG2 glial cells (Kang et al., 2013), and an in vivo imaging study revealed that Th17 cells directly contact neurons in EAE and induce neuronal dysfunction (Siffrin et al., 2010). The existing evidence suggests that Th17 cells are important mediators of inflammation in EAE and MS. As mentioned above, the mechanism of inflammation in EAE and MS has been extensively investigated. However, it is currently accepted that neurodegeneration, which is often accompanied by demyelination, is the major cause of permanent neurological disability in MS (Trapp et al., 1999; Trapp and Nave, 2008), although this phenomenon is rather poorly understood and its mechanism remains largely unknown. Neurodegeneration is induced in the regions of inflammation and begins at disease onset of MS, and it becomes irreversibly progressive in a secondary progressive stage of MS. Indeed, neurodegeneration is a major pathological hallmark in both the acute and chronic et al., 2011; phases of MS and EAE (Franklin et al., 2012; Nikic Soulika et al., 2009). Therefore, elucidation of the molecular mechanism of neurodegeneration may provide efficient neuroprotective therapy to treat progressive MS. Repulsive guidance molecule-a (RGMa) is a glycosylphosphatidylinositol-anchored membrane protein that plays an important role in axon guidance in the visual system (Severyn et al., 2009; Stahl et al., 1990). RGMa is expressed in oligodendrocytes and contributes to axonal growth inhibition after CNS injury (Hata et al., 2006; Tao et al., 2013). RGMa is also involved in apoptosis in Xenopus and chick embryos, but the precise function of RGMa in cell death remains controversial (Fujita et al., 2008; Matsunaga et al., 2004; Shin and Wilson, 2008). We previously reported that RGMa is expressed in dendritic cells and promotes T cell activation in EAE (Muramatsu et al., 2011). We immunized mice with myelin oligodendrocyte glycoprotein Cell Reports 9, 1–12, November 20, 2014 ª2014 The Authors 1 Please cite this article in press as: Tanabe and Yamashita, Repulsive Guidance Molecule-a Is Involved in Th17-Cell-Induced Neurodegeneration in Autoimmune Encephalomyelitis, Cell Reports (2014), http://dx.doi.org/10.1016/j.celrep.2014.10.038 Figure 1. RGMa Is Expressed in Th17 Cells (A) Intracellular labeling and subsequent flow cytometry analysis of IFN-g, IL-17, and Foxp3 in CD4+ T cells differentiated to Th0, Th1, Th17, or Treg cells. (B) Quantitative RT-PCR analysis of RGMa in CD4+ T cells differentiated to Th0, Th1, Th17, or Treg cells. The graph shows the relative expression level to that of GAPDH mRNA (n = 3). (C) Representative western blots showing the expression levels of RGMa (32 kDa) and b-actin (45 kDa) in CD4+ T cells polarized to Th0, Th1, Th17, or Treg cells. The level of RGMa was normalized to that of b-actin. (D) Quantitative RT-PCR analysis of RGMa in CD4+ T cells differentiated with cytokine combinations of TGF-b1, IL-6 and IL-23, or no cytokines (n = 3). Statistical analysis was performed by one-way ANOVA, followed by Tukey-Kramer tests for (B) and (D) (***p < 0.001, *p < 0.05). NS, not significant. Error bars represent mean ± SEM. (MOG)35–55 or proteolipid protein (PLP)139–151 and treated these mice with an RGMa-neutralizing antibody before neurological symptoms of EAE appeared to determine if interfering with RGMa-signaling could ameliorate EAE. The observed preventive effect fits well with the observation that RGMa facilitates T cell activation by dendritic cells. Thus, RGMa plays an important role in initiation of inflammation in EAE. However, the therapeutic effect of this antibody on progressive EAE remained to be determined. This issue is important from a clinical point of view, but information regarding the roles of RGMa in EAE or MS progression is scarce. The present study sought to elucidate the function of RGMa in progression of EAE. Our results demonstrate that RGMa is involved in Th17-cell-induced neurodegeneration in EAE. We found high RGMa expression in Th17 cells, and RGMa depletion with the neutralizing antibody attenuated the severity of EAE induced by the adoptive transfer of Th17 cells. Moreover, RGMa depletion reduced acute axonal degeneration in inflammatory lesions without altering immune or glial responses. Indeed, coculture of Th17 cells and cortical neurons revealed that Th17 cells induced neuronal cell death in an RGMa-dependent manner. Collectively, we identified the molecular mechanism of Th17-induced neurodegeneration, and our findings demonstrate that RGMa in Th17 cells is a major inducer of neurodegeneration in EAE. 2 Cell Reports 9, 1–12, November 20, 2014 ª2014 The Authors RESULTS RGMa Is Expressed in Th17 Cells We investigated RGMa expression in each type of CD4+ T cell that plays critical roles in the pathologies of EAE and MS. Naive CD4+ T cells were isolated from wild-type (WT) mouse spleen and differentiated in vitro into Th0 (IL-2), Th1 (IL-2 + IL-12), Th17 (IL-6 + TGF-b1 + IL-23), and regulatory T cells (Treg; IL2 + TGF-b1). The differentiation status was confirmed by intracellular staining and flow cytometry analysis (Figure 1A). RGMa expression levels were determined in T cells by quantitative RT-PCR and western blot analysis. While Th0, Th1, and Treg cells expressed low levels of RGMa, abundant mRNA and protein levels of RGMa were detected in Th17 cells (Figures 1B and 1C). Th17 cells have crucial roles in promoting inflammation and tissue injury to facilitate the development of autoimmune diseases. It has been reported that Th17 cells require IL-23 stimulation to maintain and enhance these activities (Dong, 2008; Lee et al., 2012). To examine whether IL-23 was required to express RGMa, Th17 cells were differentiated by IL-6 and TGF-b1 with or without IL-23. Although Th17 cells differentiated by only TGF-b1 + IL-6 express low levels of RGMa, IL-23 stimulation enhanced RGMa expression in Th17 cells (Figure 1D). These observations suggest that RGMa may be associated with Th17-cell-induced disease progression. Please cite this article in press as: Tanabe and Yamashita, Repulsive Guidance Molecule-a Is Involved in Th17-Cell-Induced Neurodegeneration in Autoimmune Encephalomyelitis, Cell Reports (2014), http://dx.doi.org/10.1016/j.celrep.2014.10.038 Th17-EAE as assessed by EAE scores in the late stage of disease progression (Figure 2A). Although the day of onset was not altered by RGMa antibody treatment, the accumulated EAE scores and maximum scores were significantly decreased by RGMa antibody treatment (Figures 2B–2D). In this model, 58.8% of Th17-EAE mice died within 30 days after transfer. However, RGMa antibody treatment reduced the mortality rate to 21.4% (Figure 2E). These data demonstrate that RGMa inhibition reduced the severity and mortality of Th17induced EAE. Figure 2. RGMa Inhibition Ameliorates EAE Severity Induced by 2D2-Th17 Cells (A) Th17 cells from 2D2 MOG35–55 TCR transgenic mice were adoptively transferred to wild-type irradiated recipient mice (2.0 3 106). The graph shows EAE scores in mice treated with control IgG (n = 17) or anti-RGMa antibody (n = 14) after 2D2-Th17 cell transfer. (B–D) The average data of onset day (B), maximum scores (C), and accumulated scores (D) after 2D2-Th17 cell transfer. (E) The rate of disease mortality within 30 days after adoptive transfer. Statistical analysis was performed by two-way ANOVA followed by Bonferroni tests for (A), Student’s t test for (B)(D), *p < 0.05, **p < 0.01. Error bars represent mean ± SEM. RGMa Inhibition Attenuates Th17-Induced EAE Severity To examine the role of RGMa in Th17 cells, we employed a mouse model of Th17-induced EAE. We adoptively transferred in vitro differentiated Th17 cells from 2D2 MOG35-55-specific T cell receptor (TCR) transgenic mice to irradiated WT recipient mice (Th17-EAE; Bettelli et al., 2003; Ja¨ger et al., 2009; Rothhammer et al., 2011). This model system allowed us to estimate the effect of RGMa in the EAE progression phase apart from the function of RGMa in dendritic-cell-induced T cell responses. An RGMa-specific neutralizing antibody was administered intraperitoneally to Th17-EAE mice every 7 days. RGMa antibody treatment attenuated the severity of RGMa-Specific Antibody Does Not Affect Immune Responses in Th17-EAE As RGMa modulates T cell responses and promotes inflammation, we examined whether treatment with an RGMa antibody suppressed immune cell activation or infiltration. In EAE, inflammatory lesions are formed by accumulated immune cells; therefore, we quantified the number of inflammatory lesions by assessing hematoxylin and eosin (H&E)-stained spinal cord of Th17-EAE mice 30 days after transfer. We did not observe any alteration in the number of inflammatory lesions in mice treated with an RGMa-specific antibody compared with mice treated with control immunoglobulin G (IgG) (Figure 3A). Moreover, we assessed the extent of demyelination with fluoromyelin staining. The demyelinated area was not altered by RGMa-specific antibody treatment (Figure 3B). These histological analyses suggest that RGMa antibody treatment did not affect inflammatory lesion formation or demyelination processes. We next examined whether RGMa-specific antibody affects infiltration of Th17 cells to the CNS. T cells from 2D2 mice could be distinguished by their expression of TCR Va 3.2, which is only expressed in 2D2 T cells (Bettelli et al., 2003; Ja¨ger et al., 2009). Thirty days after transfer, mononuclear cells were isolated from the brain and spinal cord, and the number of exogenous T cells (CD4+, TCR Va 3.2+ cells) was quantified by flow cytometry analysis. The number of infiltrated exogenous T cells was not different in either the brain or spinal cord between the control IgG and RGMa-specific antibody treatment groups (Figure 3C). We confirmed that these exogenous T cells were Th17 cells by performing intracellular cytokine staining (Figure 3D). Furthermore, to determine whether the RGMa antibody suppressed T cell activation, we stimulated T cells isolated from Th17-EAE mouse spleen with MOG35–55 peptide and measured proliferation activity with bromodeoxyuridine (BrdU) incorporation. We did not observe any differences in proliferation activity between control IgG and RGMa-specific antibody-treated mice (Figure 3E). In EAE, astrocyte and microglia activation contributes to disease exacerbation (Colombo et al., 2012; Ding et al., 2014). Thus, we examined whether RGMa-specific antibody treatment reduced astrocyte and microglia activation by performing immunohistochemistry with glial fibrillary acidic protein (GFAP) and Iba1 antibodies. We determined that RGMa-specific antibody treatment did not alter the activation state of astrocyte or microglia (Figure 3F). Collectively, these results indicate that RGMa-specific antibody treatment did not affect immune responses, demyelination, or gliosis in Th17-EAE mice. Cell Reports 9, 1–12, November 20, 2014 ª2014 The Authors 3 Please cite this article in press as: Tanabe and Yamashita, Repulsive Guidance Molecule-a Is Involved in Th17-Cell-Induced Neurodegeneration in Autoimmune Encephalomyelitis, Cell Reports (2014), http://dx.doi.org/10.1016/j.celrep.2014.10.038 Figure 3. RGMa Is Not Involved in Immune Responses in Th17-EAE Mice (A) H&E staining of cervical spinal cord sections of control IgG- and anti-RGMa-treated Th17-EAE mice 30 days after transfer. Arrowheads indicate EAE lesions. The graph shows the number of EAE lesions per section (control IgG, n = 3; anti-RGMa, n = 4). Scale bar represents 100 mm. (legend continued on next page) 4 Cell Reports 9, 1–12, November 20, 2014 ª2014 The Authors Please cite this article in press as: Tanabe and Yamashita, Repulsive Guidance Molecule-a Is Involved in Th17-Cell-Induced Neurodegeneration in Autoimmune Encephalomyelitis, Cell Reports (2014), http://dx.doi.org/10.1016/j.celrep.2014.10.038 RGMa-Specific Antibody Reduces Neuronal Damage in Th17-EAE To address the role of RGMa in Th17-EAE, we performed immunohistochemistry to analyze the expression of neogenin, which is a receptor of RGMa, in the spinal cord of Th17-EAE mice. Although neogenin was not expressed in noninflammatory lesions, neogenin signals were detected in inflammatory lesions and colocalized with axons immunostained with an axonal neurofilament (SMI-312) antibody (Figure 4A). Based on this result, we hypothesized that RGMa contributes to neurodegeneration in Th17-EAE. To assess the extent of neuronal damage, we stained the sections for amyloid precursor protein (APP), which is a marker of axonal damage, and found that the number of APP signals in lesions was significantly decreased in Th17-EAE mice treated with the RGMa-specific antibody (Figure 4B). In addition, we stained the sections with SMI-312 antibody to assess whether RGMa-specific antibody preserves axons in inflammatory lesions. SMI-312 staining revealed that RGMa antibody treatment significantly preserved axons in the lesions of dorsal column (Figure 4C). These results suggest that RGMa is involved in acute axonal degeneration in Th17EAE mice. RGMa in Th17 Cells Induces Neuronal Cell Death A recent study demonstrated that Th17 cells induce neurodegeneration via cell-cell contacts both in vitro and in vivo (Siffrin et al., 2010). Furthermore, RGMa is a membrane protein and is involved in apoptosis (Shin and Wilson, 2008). These lines of evidence and our in vivo results prompted us to investigate whether RGMa expressed in Th17 cells can directly induce neurodegeneration. Cortical neurons and Th17 cells were cocultured with or without RGMa-specific antibody, and neuronal cell death was detected by TUNEL staining. Th17 cells, but not Th0 cells or conditioned medium from Th17 cells, induced neuronal apoptosis. Moreover, this effect was inhibited by the RGMa-specific antibody treatment (Figures 5A and 5B). As we demonstrated that IL-23 stimulation was required to express RGMa in Th17 cells, cortical neurons were cocultured with Th17 cells differentiated by only TGF-b1 and IL-6. Coculturing with unstimulated Th17 cells resulted in fewer TUNELpositive cells than incubation with IL-23-stimulated Th17 cells (Figure 5C). These results demonstrate that Th17 cells directly induce neuronal cell death through RGMa and that IL-23 exposure is required to exert this effect. Neogenin is an RGMa receptor and has various physiological functions, including roles in neurite outgrowth and cell death (De Vries and Cooper, 2008). We next examined whether neogenin inhibition reduced Th17-cell-induced neuronal death. Knock- down of neogenin expression in cortical neurons was carried out by small interfering RNA (siRNA) transfection, and the knockdown efficiency was confirmed by assessing mRNA and protein levels (Figures 5D and 5E). Although Th17 cells increased the number of TUNEL-positive neurons that were transfected with control siRNA, this effect was attenuated by neogenin knockdown in neurons (Figure 5F). Neogenin knockdown by itself or coculturing with Th0 cells did not affect the number of TUNELpositive neurons. Therefore, Th17-cell-induced neuronal death is dependent on RGMa expressed in Th17 cells and neogenin in neurons. RGMa Is Not Involved in T Cell-Neuron Adhesion As we demonstrated that contact between Th17 cells and neurons was required to induce neuronal death, we next examined whether RGMa is involved in T cell-neuron adhesion. Th0 or Th17 cells were labeled with calcein-AM and plated on cortical neurons for 6 hr. Nonadherent cells were removed by several washes and the fluorescent intensity was measured. After a 6 hr incubation, although Th17 cells adhered to neurons significantly more than Th0 cells, RGMa antibody treatment had no effect on the adherent activity of either Th0 or Th17 cells (Figures 6A and 6B). This result excludes the possibility that RGMa in Th17 cells is involved in adhesion to neurons. RGMa in Th17 Cells Mediates Akt Dephosphorylation in Neurons To determine which signaling pathways Th17 cells affect to induce neuronal death, we investigated the expression of cellsurvival-related molecules in neurons cocultured with Th17 cells. We examined the phosphorylation of Akt and extracellular signal-related kinase (ERK), which are antiapoptotic factors that are dephosphorylated by RGMa-neogenin signaling (Endo and Yamashita, 2009 and Manning and Cantley, 2007). We isolated neurons from the T cell-neuron coculture system with anti-CD4 magnetic beads and confirmed that CD4+ T cells were not contaminated with neurons by flow cytometry (Figure 7A). Akt in neurons cocultured with Th17 cells was dephosphorylated compared to neurons cocultured with Th0 cells, and RGMa-specific antibody significantly inhibited Th17 cellinduced Akt dephosphorylation (Figures 7B and 7C). In contrast, while ERK1 phosphorylation was not altered in neurons cocultured with Th17 cells, ERK2 was dephosphorylated in neurons cocultured with either Th0 or Th17 cells (Figures 7D–7F). As both Th0 and Th17 cells dephosphorylated ERK2 to a similar extent in neurons and RGMa-specific antibody did not modulate ERK2 phosphorylation, we concluded that ERK dephosphorylation is not involved in Th17-induced neuronal death. These (B) Fluoromyelin staining of cervical spinal cord sections in both groups. The graph shows the percentages of demyelinated area in sections (control IgG, n = 3; anti-RGMa, n = 3). Scale bar represents 50 mm. (C) The number of infiltrated CD4+, TCR Va 3.2+ cells in the brain and spinal cord of control IgG- and anti-RGMa-treated Th17-EAE mice were identified by flow cytometry analyses (control IgG, n = 4; anti-RGMa, n = 4). (D) The percentages of IL-17+ cells in infiltrated CD4+, TCR Va 3.2+ cells in the brain and spinal cord. (E) Splenocytes were isolated from control IgG- and anti-RGMa-treated Th17-EAE mice 30 days after transfer and restimulated with MOG35–55 peptide for 3 days. Proliferation was measured by BrdU incorporation (control IgG, n = 3; anti-RGMa, n = 4). (F) Immunohistochemical staining for Iba1 (green) and GFAP (red). Scale bar represents 60 mm. Statistical analysis was performed by Student’s t test for (A)–(C) and one-way ANOVA for (E) (*p < 0.05). Error bars represent mean ± SEM. Cell Reports 9, 1–12, November 20, 2014 ª2014 The Authors 5 Please cite this article in press as: Tanabe and Yamashita, Repulsive Guidance Molecule-a Is Involved in Th17-Cell-Induced Neurodegeneration in Autoimmune Encephalomyelitis, Cell Reports (2014), http://dx.doi.org/10.1016/j.celrep.2014.10.038 Figure 4. RGMa Antibody Suppresses Axonal Damage in EAE Lesions (A) Immunohistochemical staining for axons (SMI312, green) and neogenin (red) in EAE lesions and normal sites of spinal cord. Scale bar represents 30 mm. (B) Immunohistochemical staining for APP to detect axonal damage in EAE lesions of control IgG- and RGMa antibody-treated Th17-EAE mice. The graph shows the number of APP signals in EAE lesions per mm2 (control IgG, n = 3; antiRGMa, n = 3). Scale bar represents 30 mm. (C) Axonal neurofilament (SMI-312) staining in the spinal cord of intact and Th17-EAE mice treated with control IgG or anti-RGMa. Representative images show the EAE lesion sites (dorsal column) of Th17-EAE mice and the same regions in intact mice. The graph shows the percentage of preserved axons in EAE lesions or the corresponding regions in intact mice (intact, n = 3; control IgG, n = 4; anti-RGMa, n = 4). Scale bar represents 20 mm. Statistical analysis was performed by Student’s t test for (B), and one-way ANOVA followed by Tukey Kramer test for (C). *p < 0.05. Error bars represent mean ± SEM. DISCUSSION results strongly suggest that dephosphorylation of Akt in neurons by RGMa expressed in Th17 cells plays a critical role in neuronal death. 6 Cell Reports 9, 1–12, November 20, 2014 ª2014 The Authors In the present study, we elucidated a mechanism of neurodegeneration in Th17-cell-induced EAE. In MS and EAE, activated immune cells infiltrate the CNS and induce demyelination and neurodegeneration. These phenomena aggravate the disease condition and limit spontaneous functional recovery. Because neurodegeneration is a major pathological hallmark in both the acute and chronic phases of MS and EAE (Franklin et al., et al., 2011; Soulika et al., 2012; Nikic 2009), identifying ways to inhibit neurodegeneration is an important therapeutic strategy for the effective treatment of MS. RGMa was first identified as an axon repulsive guidance molecule in the retina (Severyn et al., 2009; Monnier et al., 2002). Subsequent studies revealed that RGMa has other functions, such as growth cone collapse and neural tube closure (Conrad et al., 2007; Niederkofler et al., 2004). RGMa is expressed in glial cells, including oligodendrocytes, and potently inhibits axonal growth in the CNS (Hata et al., 2006). Indeed, RGMa inhibition promotes drastic axonal growth and recovery after spinal cord injury (Hata et al., 2006; Kitayama et al., 2011). On the other hand, RGMa regulates inflammation through T cell activation, and an RGMa polymorphism is associated with the expression of Please cite this article in press as: Tanabe and Yamashita, Repulsive Guidance Molecule-a Is Involved in Th17-Cell-Induced Neurodegeneration in Autoimmune Encephalomyelitis, Cell Reports (2014), http://dx.doi.org/10.1016/j.celrep.2014.10.038 Figure 5. RGMa in Th17 Cells Induces Neuronal Cell Death In Vitro (A) Representative figures show dual staining with TUNEL (red) and b-tubulin III (green) in cortical neurons. Cortical neurons were cocultured with CD4+ T cells polarized to Th0 or Th17 cells with or without anti-RGMa antibody (10 mg/ml). Scale bar represents 50 mm. (B) The rate of TUNEL+ apoptotic neurons in the coculture system (n = 3; CM, conditioned medium). (C) The rate of TUNEL+ apoptotic neurons cocultured with IL-23-stimulated or unstimulated Th17 cells (n = 3). (D) The graph shows the relative expression level of neogenin mRNA quantified by quantitative RT-PCR (n = 3). (E) Neogenin expression was detected by western blot analysis and normalized to a-tubulin. (F) The rate of TUNEL+ neurons transfected with control or neogenin siRNA and cocultured with Th0 or Th17 cells (n = 3). Statistical analysis was performed by one-way ANOVA followed by Tukey Kramer tests for (B), Student’s t test for (C) and (D), and two-way ANOVA followed by Bonferroni test for (F). Error bars represent mean ± SEM. IFN-g and tumor necrosis factor-a (TNF-a) in the CSF of MS patients (Muramatsu et al., 2011; Nohra et al., 2010). The present results demonstrate a function of RGMa wherein its expression by Th17 cells induces neuronal damage. Several reports have suggested the involvement of RGMa in cell death. Neogenin overexpression induced cell death in both chick embryos and human embryonic kidney 293 (HEK293) cells, but this effect was inhibited by RGMa (Matsunaga et al., 2004; Fujita et al., 2008). In contrast, RGMa overexpression induced cell death via neogenin signaling in Xenopus (Shin and Wilson, 2008). Thus, the bidirectional effect of RGMa in cell death may be cell context dependent. Our in vitro results indicated that RGMa in Th17 cells induced neuronal death. However, recombinant RGMa by itself did not induce neuronal death (data not shown), suggesting that additional molecules or a specific inflammatory environment may be required for RGMa to facilitate neuronal death. In fact, the proinflammatory cytokine TNF-a induces neogenin upregulation in a nuclear factor-kappa B (NF-kB)-dependent manner (Mirakaj et al., 2012). Indeed, our histological analysis of Th17-EAE mice indicated that axons expressed neogenin only in inflammatory lesions. Accordingly, in EAE lesions, inflammation leads to neogenin upregulation in neurons; therefore RGMa in Th17 cells may be able to drive degenerative signaling in neurons. We found that inhibition of RGMa attenuated the severity of EAE induced by 2D2-Th17 cells. To elucidate the underlying mechanism, we examined the possibility that the RGMa antibody could suppress inflammation. However, treatment with the RGMa-neutralizing antibody did not alter the number of lesions, Th17 cell infiltration activity, T cell activation, or gliosis. These results suggest that RGMa is not involved in inflammation and contributes to alternative functions of Th17 cells in EAE. While Th17 cells induce inflammation by secreting Cell Reports 9, 1–12, November 20, 2014 ª2014 The Authors 7 Please cite this article in press as: Tanabe and Yamashita, Repulsive Guidance Molecule-a Is Involved in Th17-Cell-Induced Neurodegeneration in Autoimmune Encephalomyelitis, Cell Reports (2014), http://dx.doi.org/10.1016/j.celrep.2014.10.038 Figure 6. RGMa Is Not Involved in T CellNeuron Adhesion (A) Representative figures demonstrate adherent calcein AM-labeled Th0 or Th17 cells to cortical neurons. T cells and neurons were cultured for 6 hr. (B) Quantitative analysis of (A). Fluorescent intensity was measured after 6 hr of coculture (n = 5). Statistical analysis was performed by a Student’s t test (*p < 0.05). Error bars represent mean ± SEM. proinflammatory cytokines, such as IL-17, IL-21, and IL-22, it has been reported that Th17 cells are involved in processes other than inflammation (Dong, 2008). For example, Th17 cells express receptor activator of NF-kB ligand (RANKL) on their surface, which allows them to stimulate osteoclasts and promote bone destruction (Sato et al., 2006). It is well known that CNS neurodegeneration is a pathological hallmark of MS, and a previous in vivo imaging study revealed that Th17 cells adhere to axons and induce axonal degeneration in brain stem lesions (Siffrin et al., 2010). However, the mechanisms underlying Th17 cellmediated neurodegeneration in MS were largely unknown. Our in vivo findings demonstrate that RGMa inhibition is neuroprotective in Th17-EAE, and our in vitro results indicate that RGMa expressed by Th17 cells is required for Th17-cell-mediated neuronal death. In addition, RGMa in Th17 cells induced dephosphorylation of Akt, which is an antiapoptotic factor (Dudek et al., 1997). Notably, dephosphorylation of Akt is involved in both apoptosis and axonal degeneration (Wakatsuki et al., 2011). Hence, it is possible to speculate that Akt dephosphorylation mediates axonal degeneration in Th17-EAE lesions. We performed intraperitoneal administration of the RGMa antibody to Th17-EAE mice. This administration route is considered to allow the antibody to contact majority of the circulating Th17 cells. Histological analysis showed that the RGMa antibody treatment did not affect immune responses or demyelination. Therefore, RGMa is not involved in immune responses, except T cell activation induced by dendritic cells, or demyelination process. Considering the mechanism of action of the RGMa antibody in the model, intrathecal administration of the RGMa antibody is expected to show more effective neuroprotective activity than systemic administration. However, it should be noted that systemic administration of the RGMa antibody may be feasible and useful in treating MS. We also showed that IL-23 stimulation led to RGMa upregulation in Th17 cells. IL-23 is known to promote inflammation and enhance Th17 cell pathogenic activities (Dong, 2008). In fact, our in vitro experiments indicated that IL-23-unstimulated Th17 cells exert lower neurotoxicity than IL-23-stimulated Th17 cells. There are two possible reasons for this result. First, RGMa expression in Th17 cells was not sufficient to induce neuronal death. Second, neogenin expression in neurons was inadequate because of low levels of proinflammatory cytokines from Th17 cells. It was reported that IL-23 is secreted from macrophages 8 Cell Reports 9, 1–12, November 20, 2014 ª2014 The Authors and contributes to inflammation following brain ischemia (Shichita et al., 2009). IL23 is also an essential factor for the development of EAE (McGeachy et al., 2009 and Langrish et al., 2005). The present study revealed that IL-23 contributes to both inflammation and neuronal damage via Th17 cells. As RGMa has also an adherent function (Lah and Key, 2012), it was suggested that the RGMa antibody could reduce neuronal death by inhibiting T cell-neuron adhesion. However, the RGMa antibody did not affect adhesion between T cells and neurons in the present study. Interestingly, Th17 cells adhered to neurons more efficiently than Th0 cells. T cells express various types of adhesion molecules, such as very late antigen 4 (VLA-4) and lymphocyte function-associated antigen 1 (LFA-1; Rothhammer et al., 2011; Tian et al., 2000). Th17 cells may express high levels of adhesion molecules that facilitate interactions with neurons. Indeed, treatment with the RGMa antibody did not completely inhibit Th17-cell-induced neuronal death. Thus, the neurotoxic effects of T cells may depend partially on the strength of adhesion between T cells and neurons. The proposed mechanisms that contribute to neuronal damage in MS are antibodies, complements, mitochondrial dysfunction caused by aberrant ion channels, and proinflammatory mediators from macrophages, microglia, and astrocytes (Davalos et al., 2012; Friese et al., 2007; Mathey et al., 2007; Mead et al., 2002, 2004; Schattling et al., 2012; Storch et al., 2002; Wang et al., 2005). However, effective treatments for neurodegeneration in patients with MS have not been developed until now. Here, we report a molecule involved in both inflammation and neurodegeneration in MS. It should also be noted that degeneration of the optic nerve is also induced in Th17-EAE mice (Herges et al., 2012). Therefore, the RGMa antibody may be useful for protecting neurons from the attack of Th17 cells in the optic nerve as well as the spinal cord of MS patients. Our present study builds on our previous results (Muramatsu et al., 2011) and reveals a mechanism of Th17 cellinduced neurodegeneration in EAE. Collectively, our findings indicate that RGMa inhibition could be an effective treatment to ameliorate both inflammation and subsequent neurodegeneration in MS. EXPERIMENTAL PROCEDURES Animals C57BL/6J WT mice were purchased from SLC Japan. 2D2 MOG35–55 TCR transgenic mice were a kind gift from Dr. Masaaki Murakami (Osaka University, Please cite this article in press as: Tanabe and Yamashita, Repulsive Guidance Molecule-a Is Involved in Th17-Cell-Induced Neurodegeneration in Autoimmune Encephalomyelitis, Cell Reports (2014), http://dx.doi.org/10.1016/j.celrep.2014.10.038 Figure 7. RGMa in Th17 Cells Induces Akt Dephosphorylation in Neurons (A) Schema (left) for T cell removal from the T cell-neuron coculture system. T cell removal was confirmed by flow cytometry analysis of CD4 labeling (right). (B) Phospho-Akt levels in neurons were detected by western blot. (C) The quantification of phospho-Akt obtained from (B) (n = 4), normalized to total Akt levels. (D) Phospho-ERK expression in neurons was detected. (E) The quantification of ERK1 phosphorylation obtained from (D) (n = 4), normalized to total ERK levels. (F) The quantification of ERK2 phosphorylation levels obtained from (D) (n = 4). Statistical analysis was performed by oneway ANOVA followed by Tukey-Kramer tests (*p < 0.05, **p < 0.01). Error bars represent mean ± SEM. 145-2C11, eBioscience) and anti-CD28 (2.5 mg/ml, 37.51, eBioscience) antibodies for 4 days in the presence of appropriate cytokines and antibodies. For the differentiation of each type of T cell, the following cytokines and antibodies were used. Th0: IL-2 (10 ng/ml, R&D Systems); Th1: IL-2 (10 ng/ml), IL-12 (10 ng/ml, R&D Systems) and anti-IL-4 antibody (25% culture supernatant of hybridoma, HB-188); Th17: IL-6 (30 ng/ml, R&D Systems), TGF-b1 (3 ng/ml, R&D Systems), IFN-g antibody (2% culture supernatant of hybridoma, HB-170), and presence or absence of IL-23 (20 ng/ml, R&D Systems); and Treg: IL-2 (10 ng/ml) and TGF-b1 (5 ng/ml). After 4 days, T cell differentiation efficiency was examined by flow cytometry analyses. Intracellular Cytokine Staining Differentiated T cells were collected and stimulated with phorbol 12-myristate 13-acetate (PMA: 100 ng/ml, Sigma-Aldrich), ionomycin (750 ng/ml, Calbiochem), and brefeldin A (1 mg/ml, SigmaAldrich) for 4 hr at 37 C. Cells were fixed in fixation buffer (eBioscience), permeablized in permeabilization buffer (eBioscience), and stained with fluorescent-labeled IFN-g (1:250, BioLegend), IL-17A (1:100, BioLegend), CD4 (1:100, BioLegend), and Foxp3 (1:100, BioLegend) antibodies diluted in permeabilization buffer for 30 min at 4 C. Data were collected with a FACS Canto II (BD Biosciences) and analyzed with FlowJo software (Tree Star). Osaka, Japan). All experiments adhered to the guidelines for the care and use of laboratory animals of Osaka University. In Vitro T Cell Differentiation CD4+ T cells were purified from spleen with a CD4+ T cell isolation kit II (Miltenyi Biotech). Isolated T cells were stimulated with plate-bound anti-CD3ε (5 mg/ml, Reverse Transcription and qRT-PCR Total RNA was extracted from neurons and each type of T cell with an RNeasy Mini Kit (QIAGEN), and reverse transcription was performed using a PrimeScript RT Master Mix (Takara), both according to the manufacturers’ protocols. Quantitative RT-PCR was performed with SYBR green real-time PCR master mix (Applied Biosystems) with oligonucleotide primer sets corresponding to the cDNA sequences of glyceraldehyde phosphate dehydrogenase (GAPDH), RGMa, and neogenin as follows: mouse GAPDH, 50 -TGTGTCCGTCGTGGA Cell Reports 9, 1–12, November 20, 2014 ª2014 The Authors 9 Please cite this article in press as: Tanabe and Yamashita, Repulsive Guidance Molecule-a Is Involved in Th17-Cell-Induced Neurodegeneration in Autoimmune Encephalomyelitis, Cell Reports (2014), http://dx.doi.org/10.1016/j.celrep.2014.10.038 TCTGA-30 , 50 - TTGCTGTTGAAGTCGCAGGAG-30 ; mouse RGMa, 50 -TTGGAG GATGGCAAGATGCT-30 , 50 -CCCGAGTCCTTTCAAACAGATG-30 ; and mouse neogenin, 50 -TCCAAACACAATAAGCCTGACG-30 , 50 - ATGGGACCAAATCTG CATTAACT-30 (sense and antisense, respectively). PCR reactions and analyses were carried out using a 7300 real-time PCR system (Applied Biosystems). The relative intensity versus GAPDH and the fold change relative to control were calculated. Induction of EAE and Disease Analysis Induction of Th17-EAE using 2D2 mice was performed as previously described (Ja¨ger et al., 2009). CD4+ T cells were isolated from spleen of 8- to 12-weeks-old 2D2 MOG35-55 TCR-specific transgenic mice by using a CD4+ T cell isolation kit II. CD4+ T cells were stimulated with anti-CD3ε antibody (2.5 mg/ml) in the presence of irradiated splenocytes (3,000 rad) under Th17-polarizing conditions, including IL-23. After 5 days, cells were collected and re-stimulated with plate-bound anti-CD3ε (5 mg/ml) and antiCD28 (2.5 mg/ml) antibodies for 2 days. The differentiation status was checked on day 5 by intracellular cytokine staining, and 2.0 3 106 cytokine-producing cells were intravenously injected into 8-weeks-old female mice. The recipient mice were irradiated with X-ray before injection (500 rad). Three hundred micrograms of rabbit isotype control IgG (I-5006, Sigma-Aldrich) or rabbit anti-RGMa antibody (28045, Immuno-Biological Laboratories [IBL]) was administered intraperitoneally every 7 days. We monitored and assessed the EAE scores daily according to the following criteria: 0, no abnormalities noted; 1, loss of tail reflex; 2, partial hind limb paralysis; 3, complete hind limb paralysis; 4, front and hind limb paralysis; and 5, moribund state. EAE scores were assessed by investigator who was blinded to the treatments. Histological Analysis Thirty days after the transfer of 2D2 Th17 cells, mice were anesthetized with a cocktail of domitor (0.3 mg/kg), dormicum (4 mg/kg), and butorphanol (5 mg/ kg) and transcardially perfused ice-cold PBS followed by 4% PFA (Merck). The cervical spinal cords were dissected and postfixed with 4% PFA overnight at 4 C, then immersed in phosphate-buffered 20% sucrose (pH 7.2) overnight at 4 C. The spinal cords were then embedded in optimal cutting temperature compound (Tissue-Tek), and cross-sections were cut at 40 mm thickness on a cryostat. For immunohistochemistry, sections were permeabilized in PBS containing 0.1% Triton X-100 and blocked with 3% normal goat serum (Sigma-Aldrich) for 1 hr at room temperature. Sections were incubated with primary antibodies overnight at 4 C and then incubated with fluorescent-labeled secondary antibodies for 1 hr at room temperature. The primary antibodies were used as follows: rabbit anti-Iba1 (1:3,000, 01919741, Wako), mouse anti-GFAP (1:1,000, G-A-5, Sigma-Aldrich), rabbit antiAPP (1:200, Sigma-Aldrich), rabbit anti-neogenin (1:500, NBP1-89651, Novus Biologicals), and mouse anti-axonal neurofilament (1:1,000, SMI-312, Covance). For secondary antibodies, Alexa 488- or Alexa Fluor 568-conjugated goat anti-rabbit IgG or mouse IgG (1:500, Molecular Probes) were used. To assess demyelination in the spinal cord, we stained myelin with green fluorescent lipophilic dye (FluoroMyelin, 1:500, Invitrogen) for 30 min at room temperature. The labeled sections were imaged under confocal microscopy (FV1200, Olympus). To assess the numbers of inflammatory lesions, we performed H&E staining (Muto Kagaku). Inflammatory lesions were defined as accumulation of more than ten cells. The numbers of inflammatory lesions were counted from five to eight sections per mouse. For APP signal quantification, ten inflammatory lesions per mouse were imaged under confocal microscopy, and the number of APP signals was quantified by ImageJ software (US NIH). Counted APP signals were normalized to the area of lesions per mm2. For quantification of the axon preservation, the spinal cord sections were stained with SMI-312 antibody, and the number of SMI-312+ axons in inflammatory lesions of dorsal column was quantified by ImageJ software. The average of axon number in intact mice was defined as 100%, and we calculated the percentage of preserved axons from EAE mice treated with control IgG or anti-RGMa. For quantification of demyelinated areas, fluoromyelin-positive areas in white matter were quantified by ImageJ, and the percentages of demyelinated areas in the total white matter areas were calculated. 10 Cell Reports 9, 1–12, November 20, 2014 ª2014 The Authors Flow Cytometry Analysis of CNS-Infiltrating Immune Cells At 30 days after 2D2 T cell transfer, mice were anesthetized and transcardially perfused with ice-cold PBS. The brain and spinal cord were dissected out and minced with a scalpel. The minced tissues were digested with 0.1% collagenase D (Roche Applied Science) containing 2.5 mM calcium chloride at 37 C for 30 min. We prepared single-cell suspensions by trituration, and the cells were resuspended in 30% Percoll (GE Healthcare). Next, 70% Percoll was layered underneath and centrifugation was performed at 2,000 rpm for 20 min at room temperature. We isolated infiltrated immune cells from the interfaces of 30%/70% Percoll gradients. For flow cytometry analysis, cells were treated with anti-CD16/32 antibody (1:100, eBioscience) for 10 min on ice to block Fc receptors and then stained with fluorescent-labeled antibodies for TCR Va 3.2 TCR Va 3.2 (1:250, eBioscience), CD11b (1:100, BioLegend), CD4 (1:100, BioLegend), and/or CD45 (1:100, BioLegend). For intracellular cytokine staining, cells were stimulated with PMA (100 ng/ml), ionomycin (750 ng/ml), and brefeldin A (1 mg/ml) at 37 C for 4 hr. After fixation and permeabilization, cells were stained with antibodies for TCR Va 3.2 (1:250, eBioscience), IL-17A (1:100, BioLegend), and/or CD4 (1:100, BioLegend). Proliferation of T Cells T cell proliferation was measured with Cell Proliferation ELISA, BrdU (colorimetric) (Roche Diagnostics). Splenocytes were isolated from Th17-EAE mice at 30 days after transfer and stimulated with MOG35–55 peptide (0, 1, 5, or 10 mg/ml, CS Bio) in 96-well plates (12.5 3 105/ml). After 48 hr, BrdU was added, and the cells were cultured for an additional 24 hr. BrdU incorporation was measured according to the manufacturer’s protocol. Coculture of T Cells and Neurons Cortical neurons were collected from the cerebral cortices of embryonic day 17 to 18 mice embryos. Harvested cortex was minced and digested in 0.25% trypsin for 15 min at 37 C. Cells were triturated and passed through 70 mm nylon cell strainer. The resultant cell suspension was diluted with Neurobasal medium (Life Technologies) supplemented with B-27 (Life Technologies), GlutaMAX (Life Technologies), and penicillin/streptomycin (Gibco) and plated on culture slides coated with poly-D-lysine (Sigma-Aldrich) at a density 5.0 3 105 cells per ml. Three days after plating, differentiated Th0 or Th17 cells were added at 1:1 ratio to the neurons with or without anti-RGMa antibody (10 mg/ml, IBL). TUNEL Labeling and Immunocytochemistry Cell death was detected by TUNEL staining with the In Situ Cell Death Detection Kit, TMR red (Roche Applied Science) according to the manufacturer’s protocol. Briefly, cells were fixed with 4% PFA and permeabilized with 1% sodium citrate containing 0.1% Triton X-100 for 5 min. Then, cells were reacted with TMR-dUTP in the presence of terminal deoxynucleotidyl-transferase enzyme for 1 hr at 37 C. For immunocytochemistry, cells were blocked with 3% normal goat serum in PBS containing 0.1% Triton X-100 for 1 hr and incubated with anti-b Tubulin III antibody (1:2,000, TUJ1, Covance) overnight at 4 C. Cells were washed three times and incubated with Alexa 488-conjugated goat anti-mouse IgG (1:500, Molecular Probes) and DAPI (1 mg/ml, Santa Cruz Biotechnology) for 1 hr. After three washes, slides were mounted with fluorescent mounting medium (Dako). Neuronal apoptosis was quantified using fluorescence microscopy (BZ-9000, Keyence). To count the total cells, DAPI+ cells were counted and then TUNEL+ cells were counted from ten random fields. The rate of TUNEL-positive cells was calculated as the number of TUNEL/DAPI 3 100 (%). Adhesion Assay for Neurons and T Cells Differentiated T cells were labeled with 5 mg/ml 30 ,60 -Di(O-acetyl)-40 ,50 -bis [N,N-bis(carboxymethyl)aminomethyl] fluorescein, tetraacetoxymethyl ester (calcein-AM, Dojindo) for 30 min at 37 C. After three washes with RPMI1640 medium, 2.0 3 105 T cells were added to cortical neurons (2.5 3 105) that were cultured for 3 days and incubated at 37 C with rabbit IgG (10 mg/ml, Sigma-Aldrich) or RGMa antibody (10 mg/ml). After 6 hr incubation, nonadherent cells were removed by washing with RPMI medium containing 0.5% bovine serum albumin four times. The fluorescent intensity was measured with a SpectraMAX (Molecular Devices) at excitation and emission wavelengths of 485 and 538 nm, respectively. Please cite this article in press as: Tanabe and Yamashita, Repulsive Guidance Molecule-a Is Involved in Th17-Cell-Induced Neurodegeneration in Autoimmune Encephalomyelitis, Cell Reports (2014), http://dx.doi.org/10.1016/j.celrep.2014.10.038 siRNA Transfection Cortical neurons (5.0 3 106 cells) were suspended in 100 ml Nucleofector solution (Amaxa Biosystems) containing 500 pmol control (MISSION siRNA Universal negative control, Sigma-Genosys) or neogenin siRNA (Sigma-Genosys). Electroporation was performed by program O-005 as described by the manufacturer’s protocol (Amaxa Biosystems). After 3 days, neogenin expression was confirmed by quantitative RT-PCR and western blot, and Th0 or Th17 cells were added to siRNA-electroporated neurons. The neogenin siRNA sequences were 50 -CAAUUCCAUGGAUAGCAAU-30 and 50 -AUUGCUAUCCA UGGAAUUG-30 . Western Blot T cells were lysed in lysis buffer containing 150 mM NaCl, 1% Triton X-100, 20 mM HEPES (pH 7.4), 10% glycerol, 5 mM EDTA, and complete protease inhibitor cocktail (Roche Applied Science). Neurons and T cells were cocultured for 6 hr. Neurons were separated from the coculture system with antiCD4 microbeads (Miltenyi Biotech), and unlabeled fractions were regarded as neurons. Neurons were lysed in radioimmunoprecipitation assay (RIPA) buffer containing 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM Tris-HCl (pH 7.5), complete protease inhibitor cocktail, and phosSTOP phosphatase inhibitor (Roche Applied Science). After incubation on ice for 30 min, lysates were centrifuged at 15,000 rpm for 30 min at 4 C, and the supernatants were collected. Protein concentrations were determined with a bicinchoninic acid assay (BCA) kit (Pierce). Thirty micrograms of each sample were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). Membranes were blocked with 5% bovine serum albumin in PBS containing 0.05% Tween-20 for 1 hr and incubated with rat anti-RGMa (2 mg/ml, MAB2458, R&D Systems), rabbit anti-phospho Akt (1:1,000, 9271, Cell Signaling Technology), rabbit antitotal Akt (1:1,000, 9272, Cell Signaling Technology), rabbit anti-phospho ERK (1:1,000, 4370, Cell Signaling Technology), rabbit anti-total ERK (1:1,000, 9102, Cell Signaling Technology), rabbit anti-neogenin (1:1,000, H-175, Santa Cruz), rabbit anti-b-actin (1:1,000, 4970, Cell Signaling Technology), or mouse anti-a-tubulin (1:250, sc-5286, Santa Cruz) in blocking solution overnight at 4 C. After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibody to rat, rabbit, or mouse IgG (1:3,000, Cell Signaling Technology) for 1 hr. Detection was performed by Pierce Western Blotting Substrate Plus (Pierce) and RAS-3000 (Fuji Film). Statistics Student’s t tests, one-way ANOVA followed by Tukey-Kramer tests, and twoway ANOVA followed by Bonferroni tests were performed with GraphPad Prism 6 software. p < 0.05 was considered significant. AUTHOR CONTRIBUTIONS S.T. designed and performed the experiments and analyzed the data. T.Y. coordinated and directed the project. T.Y. and S.T. conceived the project and wrote the manuscript. ACKNOWLEDGMENTS We thank M. Murakami and D. Kamimura (Osaka University) for their kind gift of 2D2 TCR transgenic mice, H. Ishii (Columbia University) for technical advice, and R. Muramatsu (Osaka University) for helpful suggestions. This work was supported by a grant for Core Research for Evolutional Science and Technology from the Japan Science and Technology Agency to T.Y. and by a Grant-inAid for Scientific Research (S) from the Japan Society for the Promotion of Sciences (25221309) to T.Y. 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