Overlapping AQP4 peptides.

<p>Overlapping AQP4 peptides.</p

Functional characterization of AQP4_22–36 peptide specific TCL8.2.

<p>Representative T cell line (TCL) reactive to AQP4_22–36. (A) TCL8.2 cells were restimulated with irradiated syngeneic splenocytes in the presence of increasing concentrations of AQP4_22–36. The proliferative response was determined by ³[H] thymidine incorporation. Mean c.p.m. of triplicate cultures + SD. (B) Cytokine production of TCL8.2 cells in response to increasing concentrations of AQP4_22–36 as measured by cytometric bead array in the cell culture supernatant collected at 48 h after initiation of restimulation. Mean cytokine concentrations + SD of triplicate cultures. (C) Intracellular cytokine staining of TCL8.2 after 6 restimulation cycles.</p

Screening of T cell responses with pools of AQP4 peptides.

<p>Mice were immunized with full length AQP4 protein/CFA. Draining lymph node cells and splenocytes were restimulated <i>in vitro</i> with pools of overlapping AQP4 peptides (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016083#pone-0016083-t001" target="_blank">Tables 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016083#pone-0016083-t002" target="_blank">2</a>). (A) Proliferative responses of AQP4-sensitized lymphocytes to 21 pools of overlapping AQP4 peptides as measured by ³[H] thymidine incorporation (c.p.m.). Mean of triplicate cultures + SD or stimulation indices as calculated by deviding the c.p.m. values of each peptide pool by the background c.p.m. This experiment was performed twice with similar results. (B) In a second round of restimulation, T cells out of each pool were stimulated with the individual peptides of the parental pool in the presence of irradiated syngeneic splenocytes as APCs. Proliferative responses of T cell pools that showed antigen specific responses to at least one individual splenocytes with a stimulation index of at least 3.0 are depicted. Mean of ³[H] thymidine incorporation (c.p.m.) of triplicate cultures + SD are shown.</p

Antibody response to AQP4.

<p>Wild type C57BL/6 mice were immunized with full length AQP4/CFA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016083#s4" target="_blank">Materials and Methods</a>. Unimmunized mice or mice immunized with MOG_35–55/CFA were used as controls. Sera of mice from each group were collected and tested for AQP4 specific antibodies in a cell based flow cytometry analysis. Subclass specification was performed by using fluorochrome labeled anti-mouse Ig antibodies specific for IgM, IgA, IgE, IgG1, IgG2a, IgG2b, and IgG3 (FITC labeled) or total IgG H+L (AlexaFluor488 labeled). (A) Representative histogram plots illustrating the MFIs for AQP4 specific IgG H+L in the various test groups. (B) Representative histogram plots of AQP4 specific Ig classes and IgG subclasses in AQP4 immunized animals. (C) ΔMFIs + SEM (n = 4) for AQP4 specific IgG H+L in naive control mice, MOG_35–55 immunized mice, or full length AQP4 immunized mice. (D) ΔMFIs + SEM (n = 4) for individual anti-AQP4 Ig classes and IgG subclasses in full length AQP4 immunized mice.</p

Immunization of C57BL/6 mice with AQP4_22–36 or AQP4_289–303 does not induce signs of spinal cord disease or optic neuritis.

<p>Groups of wild type C57BL/6 mice (n = 4 per group) were immunized with PBS/CFA, MOG_35–55/CFA, AQP4_22–36 (peptide 8)/CFA, or AQP4_289–303 (peptide 97)/CFA. (A) EAE scores. Note that AQP4 peptide immunized mice did not develop signs of disease while MOG_35–55/CFA immunized animals showed classical paraparesis. (B, C) Mice that had been immunized with MOG_35–55/CFA, AQP4_22–36 (peptide 8)/CFA, or AQP4_289–303 (peptide 97)/CFA were analysed by histology. Representative sections of spinal cords (B) and optic nerves (C) are shown. Paraffin sections were stained with luxol fast blue (LFB)-PAS to illustrate demyelination or with antibodies to CD3 or Mac-3 to visualize T cell or macrophage infiltrates, respectively. Spinal cord areas that are shown at higher magnification are marked by frames. Scale bars are 500 µm and 200 µm for the spinal cord sections and 100 µm for the optic nerve sections as indicated.</p

Peptide pools.

<p>Peptide pools.</p

Immunodominant I-A^b restricted epitopes of AQP4 and sequence homology of human vs mouse AQP4.

<p>(A) Topological model of AQP4 (M1 translational isoform) according to Crane and coworkers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016083#pone.0016083-Crane1" target="_blank">[8]</a>. The core immunogenic (I-A^b restricted) determinant of AQP4 in the N-terminus is highlighted in dark blue. The remainder of the immunogenic T cell epitopes is highlighted in cyan. The numbers indicate the amino acid residue position in the AQP4 protein sequence. (B) Amino acid sequence alignment of human and mouse AQP4 M1 isoforms. I-A^b restricted T cell determinants are highlighted (cyan). The dominant immunogenic epitope is represented by peptide 8 as indicated by a box (peptide sequence in dark blue). T cell epitopes common to M1 and M23 isoforms of AQP4 are underlined. Asterisks indicate sequence identity at the corresponding sequence position.</p

Additional file 4: of Effectors of Th1 and Th17 cells act on astrocytes and augment their neuroinflammatory properties

ELISA of astrocyte culture supernatants. Concentration of CCL2, CCL20, and IL-6 was measured in the culture supernatants of astrocytes cultured in medium, Th1 and Th17 supernatants. Since we did not remove T cells factors, to exclude any background resulting from T cells, supernatants used in these experiments to stimulate astrocytes were also tested for these factors. CCL2 and IL-6 was not detected in T cell supernatants whereas little amount of CCL20 was detected in Th17 supernatants. Actual concentration of CCL20 released by astrocytes was calculated by subtracting the concentrations obtained for Th17 supernatants. The results are mean ± SEM (n = 4) with *p ≤ 0.05 and **p ≤ 0.01. (TIFF 216 kb

Additional file 2: of Effectors of Th1 and Th17 cells act on astrocytes and augment their neuroinflammatory properties

Clinical course of WT and α4−/− mice. EAE severity was monitored in WT and α4−/− mice following immunization with MOG35–55 in CFA. WT mice showed the signs of classical EAE whereas α4−/− mice had an ataxic EAE syndrome. Data is represented as mean clinical score + SEM (n = 6). (TIFF 419 kb

Additional file 5: of Effectors of Th1 and Th17 cells act on astrocytes and augment their neuroinflammatory properties

Inhibition of reactive astrocytosis affects Th17 infiltration into the CNS. Expression of Rorc mRNA was measured in the spinal cord of wild-type (WT) or GFAP TK (Tg) mice with established EAE where at day 7 following onset of clinical symptoms the mice were either challenge with PBS (controls) or ganciclovir (GCV) to deplete astrocytes. The basal expression was adjusted to WT-PBS controls and fold changes in Rorc mRNA expression was determined for the other groups. Each point represents data from one mouse. Here we used Mann-Whitney test. (TIFF 276 kb