Fig. 1

A. T cell proliferation assay reflecting a robust reaction to loop C of AQP4. T cells from two C57Bl6 mice (wt-MOG) immunized with myelin-oligodendrocyte glycoprotein (MOG) peptide 35–55 showed proliferative activity as measured by incorporation of tritiated-thymidine only in the presence of the MOG antigen (red bars). C57Bl6 mice immunized with AQP4 peptides (wt-AQP4) did not react against any AQP4 antigen. T cells from AQP4 null mice (KO naïve) do not inherently react to AQP4 peptides unless the mice are immunized with the peptides (KO1-AQP4 and KO2-AQP4). Among those tested, a peptide corresponding to the extracellular loop C generated a robust reaction only in T cells from AQP4 null mice immunized against a mix of AQP4 peptides 56–69 (loop A), 135–53 (loop C), and 212–30 (loop E), exposed to cells in culture alone (brown bars) or as a mixture of all three peptide (yellow bars). Results shown are counts per minute means +/− SEM of triplicate reactions. B. ELISPOT assay was used to determine the number of IL-17 and interferon-gamma (IFN-γ) cytokine-producing cells in Th17 polarized (Th17-pol) and unpolarized (unpol) cell cultures exposed to AQP4 loop C peptide (AQP4_135–53) versus no stimulation (NS). Unpolarized AQP4-reactive T cells expressed significant levels of both IL-17 and IFN-γ compared to unstimulated controls. After polarization to a Th17 phenotype, the number of IL-17 producing cells almost doubles while the number of IFN-γ producing cells is nearly undetectable, but the frequency of IL-17-producing cells also increases in the unstimulated culture (while IFN-γ-producing cells remains low)