Laquinimod reverses RR-EAE and inhibits inflammatory T cell responses via a direct effect on myeloid APC.

<p>(A) Daily oral laquinimod treatment reverses relapsing remitting EAE. SJL/J mice were immunized with PLP p139-151 and treated with laquinimod or vehicle (n = 9) at the remission phase (arrow points to the start of the treatment). (B) Lesion quantification showed reduced total number of meningeal and parenchymal inflammatory foci in SJL/J mice treated with laquinimod after first exacerbation of the disease. Representative Luxol fast blue-H&E staining of the cerebellum is shown. (C, D) Laquinimod-treated myeloid APC inhibit differentiation of naive T cells into Th1 and Th17 cells. Whole splenic in vivo laquinimod-treated or untreated CD11b⁺ cells were used as APC in co-culture with untreated naive (CD4⁺CD44⁻CD62L⁺) T cells from MOG p35-55 TCR-transgenic mice (2D2). Conversely, naive T cells were isolated from laquinimod- or vehicle-treated 2D2 mice and cultured with purified vehicle-treated CD11b⁺ cells and Ag (MOG p35-55). Polarization of naïve T cells into Th1 lineage was induced by IL-12 (C) and polarization into Th17 lineage was induced by IL-23, IL-6 and TGF-β (D). Intracellular cytokine staining for IFN-γ and IL-17 after three days in culture is shown. For all experiments, data shown are representative of two independent experiments. For EAE disease course and other experiments, mean disease score and mean ± s.e.m. are displayed; *P<0.05, Mann-Whitney U test.</p

Identification of naturally processed AQP4 determinants.

<p>AQP4 peptides were tested for their ability to induce T cell proliferative responses in intact hAQP4-primed (A) C57BL/6 and (B) SJL/J mice. Each 20-mer AQP4 peptide is indicated by first residue. Mice were immunized subcutaneously with 100 µg recombinant intact hAQP4 in CFA. 10–12 days later, lymph node cells were cultured in vitro for recall responses to the indicated human or mouse overlapping 20-mer peptides. Data are shown as stimulation indices (SI's) of mean proliferative responses in the presence of peptide (25 µg/ml) compared to the absence of antigen (background). Standard errors (+/− SEM) are shown for proliferative responses tested in triplicate. Recall to intact hAQP4 is shown for comparison.</p

Characterization of the minimal core T cell epitope within AQP4 p21-40.

<p>AQP4 p21-40-specific T cells were restimulated with truncated peptides to determine the core of the p21-40 determinant in (A, B) p21-40 specific C57BL/6 cell lines and (C, D) p21-40 specific SJL/J primary lymph node cells. Cell lines were restimulated with irradiated syngeneic splenic APC and various concentrations of p21-40 or truncated peptides. After 48 hours (cell lines) or 72 hours (LN), cultures were pulsed with ³H-thymidine and harvested 16 hours later. Data shown represent means of triplicates +/− SEM.</p

AQP4 p21-40 is a naturally processed immunodominant determinant of intact AQP4.

<p>(A) C57BL/6 (above) and SJL/J (below) mice were immunized subcutaneously with 100 µg recombinant intact hAQP4 in CFA. Lymph nodes were harvested at day 10–12 and cultured in the presence of various concentrations of either intact hAQP4 (left), or individual murine AQP4 peptides (right) for 4 days. Proliferation was measured by ³H-thymidine incorporation, and are presented as mean cpm +/− SEM for triplicates. (B) C57BL/6 (above) and SJL/J (below) m21-40 primed lymph node cells were assayed for cytokine production and proliferation in response to m21-40 peptide. Supernatants were collected after 72 hr for IFN-γ and IL-17A for ELISA. Data are presented as mean +/− SEM for triplicates. (C) Proliferation of C57BL/6 T cell lines specific to p21-40, p91-110, p166-180, and p261-280 were assayed following re-stimulation with various concentrations of intact hAQP4 (left) and self-peptides (right). Data are shown as stimulation indices (SI's) of triplicates of antigen proliferation over no antigen conditions (background).</p

Localization of identified murine AQP4 T cell epitopes.

<p>AQP4 peptides that elicited proliferative responses in (A) C57BL/6 and (B) SJL/J mice are located within putative transmembrane and cytoplasmic domains. (C) Sequences of human (hAQP4) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015050#pone.0015050-Yang1" target="_blank">[34]</a> and murine <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015050#pone.0015050-Turtzo1" target="_blank">[17]</a> AQP4 (mAQP4). Dashes represent homologous regions between the two species.</p

Effect of switching on the relapse rate measured as incidence rate ratio (IRR), using a Poisson regression model, after adjusting for baseline EDSS, disease duration and time on first-line disease modifying therapy.

<p>Within group comparison compares the relapse rate before and after the switch for each group. The between group comparison compares treatment effect on immunosuppressant and natalizumab groups with non-switchers or between natalizumab and immunosuppressants.</p