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T-cell determinants of the autoantigen, aquaporin-4

Background and Goals: Neuromyelitis optica (NMO) is associated with aquaporin-4 (AQP4)-specific IgG1 antibodies. Human IgG1 is a T cell-dependent antibody subclass, which suggests that AQP4-specific T cells may participate in NMO pathogenesis, a possibility also supported by observations that AQP4-specific antibodies are pathogenic only in the presence of CNS inflammation. We have been studying T cell recognition of AQP4 in NMO patients and in mice in order to understand the potential role of T cells in NMO pathogenesis and to develop a model of NMO. The goal of this investigation was to identify AQP4-specific T cell determinants in mice. Methods and Results: T cell reactivity was examined in C57BL/6 and SJL/J mouse strains using intact AQP4 and overlapping 20mer peptides encompassing the entire 323 amino acid sequence of M1 AQP4. Mice were immunized with murine AQP4 peptides or intact human AQP4 in complete Freund’s adjuvant. Proliferation was measured by 3H-thymidine incorporation. Immunization of C57BL/6 mice with AQP4 peptides identified T cell determinants within peptides (p) 21-40, p91-110, p166-180, and p261-280. In SJL/J mice, T cell determinants are located in p11-30, p21-40, p101-130, p126-140, and p261-280. In both strains, T cell determinants are located within putative cytoplasmic or transmembrane AQP4 domains. MHC II-restricted CD4^+ T cells specific for these individual AQP4 determinants proliferated in response to intact AQP4, indicating that they are naturally processed AQP4 determinants. However, after immunization with intact AQP4, recall to p21-40 was strongest, indicating that this region contains the immunodominant T cell epitope(s) for both strains. Using truncated AQP4 peptides within the 21-40 sequence, these T cell determinants were mapped to p25-38 and p23-38 for C57BL/6 and SJL/J mice, respectively. T cells specific for these determinants secreted IFN-g and IL-17. Preliminary data from proliferation and tetramer analysis using AQP4 20mers indicate that T cells from NMO patients also recognize some of the same regions of AQP4. Conclusions: Immunogenic T cell determinants for C57BL/6 and SJL/J mice are found within AQP4 residues 11-30, 21-40, 91-110, 101-120, 126-140, 166-180, and 261-280, all within cytoplasmic and transmembrane regions of the molecule. Our identification of these AQP4 T cell determinants should be useful for development of AQP4-targeted NMO models.

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