Que Inhibits T Cell Proliferation Figure 4. Que treatment decreases the number of T cells in the spleen and lymph nodes and inhibites proliferation of effector T cells. A. Schematic diagram displaying the time course of immunization and Que pre-treatment. B. CD4+/CD8+ cells in the spleen and lymph nodes were quantified by FACs after Que treatment. The scatter plot (lower panel) shows the number of CD4+/CD8+ cells per million cells. In both cases, the number of cells was decreased upon Que treatment. C. Mononuclear cells isolated from the spleens of mice were immunized with MOG35–55 peptide with or without Que treatment, and were stimulated with 0, 1, 10 or 100 mg/ml of MOG35–55 peptide. The proliferation was significantly suppressed assayed by CFSE. D. Effector T cells (CD4+CD252) isolated from spleen of na¨ıve mice are labeled with CFSE. After stimulation with anti-CD3, dilution of CFSE shows that CD4+CD252 populations proliferated much less vigorously after Que treatment than vehicle. E–F. IL-2 expression in effector T cells is decreased after Que-treatment assayed by either real-time PCR (E) or ELISA (F). Data represent mean 6 SEM of five independent experiments (N = 5, *, p,0.05). doi:10.1371/journal.pone.0042746.g004 days after immunization. Moreover, CC1+ oligodendrocytes (OLs) were dramatically decreased in the EAE mice with or without Que treatment as compared to unimmunized animals, while these cells were more conserved in the Que treatment group (Fig. 2C). Ki67 was used to detect the proliferative oligodendrocyte precursors (OPCs) (NG2+) and a number of Ki67+/NG2+ cells were seen distributed throughout the white matter in the EAE model (Figure S2). Such cells were also detected after Que treatment, however likely, to a lesser extent (Figure S2), while such proliferative OPCs were rare in normal controls. Together, these results indicate that Que treatment protects the spinal cord from demyelination and loss of OLs in EAE mouse models and OPC proliferation is a prominent response to demyelination. Que Reduces Infiltration of T Cells in the EAE Spinal Cord Since myelin-specific effector T cells migrate into CNS and initiate demyelination [4], dose the treatment of Que affect the accumulation of T cells in the spinal cord of EAE model? The immunohistochemical results indicate that both CD8+ and CD4+ T cells were barely detected in unimmunized mice and control (Fig. 3A, E), while a large number of CD4+ T cells were found to accumulate in the spinal cord of the EAE model (Fig. 3B), especially distributed throughout the white matter. These cells were significantly decreased in the spinal cord of the Que-treated group (Fig. 3C–D). The CD8+ T cells were also observed infiltrated into spinal cord (Fig. 3F) and were reduced after Que treatment (Fig. 3F–H) with the exception that the number of CD8+ T cells was much fewer than that of CD4+ cells. Que Attenuates MOG35–55-specific Immune Response and Inhibits Effector T Cell Proliferation The myelin-specific effector T cells are generated in periphery lymph organs. To confirm the reduction of CD4+ and CD8+ T cells found in the spinal cord and to reveal their source, we detected the CD4+/CD8+ T cell numbers from spleen and lymph nodes using FACS 10 days after immunization with Que pre- treatment for 1 week (Fig. 4A) and found that Que treatment resulted in reduced numbers of CD4+ and CD8+ T cells as compared with untreated EAE mice (Fig. 4B). To test whether Que attenuated MOG35–55-specific T cells generation, we measured the in vitro stimulated proliferation of T cells isolated from spleens 10 days after MOG35–55 immunization, with or without Que pre-treatment. The results show that MOG35–55- stimatuating proliferation of T cells from primed mice spleen were significantly decreased in Que treated mice (Fig. 4C). To further examine whether the reduction of T cells comprise a non-specific proliferation inhibitive effect by Que, we isolated CD4+CD252 T cells (effector T cells) from spleen of na¨ıve C57BL/6 mice and stimulated the cells with anti-CD3 Ab with/ without Que treatment, dilution of CFSE results exhibit that CD4+CD252 T cells from Que treatment proliferated much less vigorously than that from untreated EAE mice (Fig. 4D). Additionally, Que decrease the expression IL-2, a potent T cell growth factor, assayed by either real-time PCR (Fig. 4E) or ELISA (Fig. 4F). Together, these results indicate Que can either attenuate MOG35–55-specific immune response or inhibit effector T cell proliferation. Que Diminishes Accumulation of CD4+ T Cells that Reduces OLs Loss and Demyelination in EAE Spinal Cords As the infiltrated T cells initiate OLs damage and local immunoresponse, dose reduced CD4+ T cells rescue demyelin- ation and protect OLs from loss? The double labeling experiments with CC1/MBP and CD4 showed CD4+ cells were abundantly distributed in the white matter upon EAE induction (Fig. 5A,C, arrowhead), and were specifically localized to demyelination lesions (Fig. 5A), while CC1+ OLs were absent from these areas (Fig. 5C). These observations suggest that the accumulation of CD4+ cells could lead to loss of OLs and demyelination. Treatment of Que, however, decreased the number of infiltrated CD4+ cells, with a subsequent increase in the number of CC1+ OLs (Fig. 5D), observed adjacent to a single appearing CD4+ T cell, accompanied with normal myelin staining (Fig. 5B). Que Inhibits Activation of Microglia and Astrocytes in EAE Spinal Cord As local immunoresponse is initially triggered by infiltrated T cells and contributed to the neuronal damage and demyelination, we observed CD68+ and CD11b+ cells which represent activated microglia/macrophages, were extensively populated throughout the spinal cord in the EAE model and were significantly diminished by Que treatment (Fig. 6A,B). Similarly, more activated GFAP+ astrocytes were found in the spinal cord of EAE mice as compared to normal controls as well as Que treated EAE mice, indicating that the activation of microglia and astroctyes in EAE had been attenuated by Que treatment (Fig. 6C). Discussion In the present study, C57BL/6 mice immunized with MOG35– 55 peptide display the disease symptoms associated with demye- lination, glial activation, and CD4+ T cell infiltration in the spinal cord. These findings indicate that our EAE models are similar with previous reports and mimic the onset of MS, the T cell mediated autoimmune demyelinating disease [18]. Our study demonstrates the beneficial effects of the administration of Que, a current antipsychotic drug, on the MOG-induced EAE model. Specifi- cally, Que treatment delays the onset, prevents deterioration of EAE symptoms, prevents demyelination, and inhibits generation of effector T cells in peripheral lymphatic organs that may results in reducing local inflammation in the spinal cord. It has been previously reported that Que is protective against myelin breakdown in the cupriozne-induced demyelination model by up-regulation of SOD activity [16]. Our present observations provide further evidence to demonstrate the beneficial effects of Que on myelin forming OLs in EAE spinal cord (Fig 2, 5). Enhanced MBP expression and the number of CC1+ mature OLs PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e42746