Immunodominant T Cell Determinants of Aquaporin-4, the Autoantigen Associated with Neuromyelitis Optica

Abstract

Autoantibodies that target the water channel aquaporin-4 (AQP4) in neuromyelitis optica (NMO) are IgG1, a T cell-dependent Ig subclass. However, a role for AQP4-specific T cells in this CNS inflammatory disease is not known. To evaluate their potential role in CNS autoimmunity, we have identified and characterized T cells that respond to AQP4 in C57BL/6 and SJL/J mice, two strains that are commonly studied in models of CNS inflammatory diseases. Mice were immunized with either overlapping peptides or intact hAQP4 protein encompassing the entire 323 amino acid sequence. T cell determinants identified from examination of the AQP4 peptide (p) library were located within AQP4 p21-40, p91-110, p101-120, p166-180, p231-250 and p261-280 in C57BL/6 mice, and within p11-30, p21-40, p101-120, p126-140 and p261-280 in SJL/J mice. AQP4-specific T cells were CD4+ and MHC II-restricted. In recall responses to immunization with intact AQP4, T cells responded primarily to p21-40, indicating this region contains the immunodominant T cell epitope(s) for both strains. AQP4 p21-40-primed T cells secreted both IFN-γ and IL-17. The core immunodominant AQP4 21-40 T cell determinant was mapped to residues 24-35 in C57BL/6 mice and 23-35 in SJL/J mice. Our identification of the AQP4 T cell determinants and characterization of its immunodominant determinant should permit investigators to evaluate the role of AQP4-specific T cells in vivo and to develop AQP4-targeted murine NMO models.

Identification of a Point Mutation Impairing the Binding between Aquaporin-4 and Neuromyelitis Optica Autoantibodies

“…On the discovery of AQP4 as the target for NMO-IgG (34), we have later added a key step forward by demonstrating that AQP4 tetramers per se do not contain the NMO-IgG epitope, which is in fact generated by AQP4 aggregation into OAPs (2). After several studies aimed at identifying the key determinant for such an epitope assembly (10,35,36), we demonstrate that AQP4 aggregation into OAPs is essential but alone not sufficient to generate such an epitope. We show that the epitope is generated within the OAPs by key inter-tetrameric interactions between extracellular loops A, C, and E.…”

NADPH Oxidase Modifies Patterns of MHC Class II–Restricted Epitopic Repertoires through Redox Control of Antigen Processing

“…The relative abundance of the peptides was quantified using the area under corresponding peaks. Abundance of the cleavage product MOG [42][43][44][45][46][47][48][49][50][51][52][53][54][55] relative to MOG (corresponding destruction of the I-A b binding region) was calculated using the following formula: RPA = (P x /S x )/(P 0 /S 0 ), where RPA indicates relative peptide abundance, P x indicates product (MOG [42][43][44][45][46][47][48][49][50][51][52][53][54][55] ) of sample, S x indicates substrate (MOG ) of sample, P 0 indicates product (MOG [42][43][44][45][46][47][48][49][50][51][52][53][54][55] ) without hydrogen peroxide, and S 0 indicates substrate (MOG ) without hydrogen peroxide.…”

“…The immune epitope database and analysis resource MetaSVMp (a quantitative binding affinity program that employs the Immune Epitope Database was used to compute relative affinities between I-A b and regions of MOG 1-125 as previously described (48). The inverse of the IC 50 by cysteine cathepsins using input sites from the MEROPS database.…”

“…To further interrogate the ability of NOX2 to alter processing patterns of a single Ag, we measured the efficiency of WT and Cybb 2/2 BMMfs to process four distinct HEL epitopes (HEL [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] , HEL [31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47] , HEL [48][49][50][51][52][53][54][55][56][57][58][59][60][61][62] , and HEL 74-90 ) from whole HEL protein and present them to epitope-specific CD4 + T cell hybridomas (Hb1.9, H30.44, H46.13, and B04, respectively) (26). To achieve this, T cell hybridomas...…”

“…To achieve this, T cell hybridomas were first stably transfected with an NFA-T-enhanced IL-2 promoter-driven eGFP construct, followed by multiple rounds of selection by FACS to enrich those hybridoma clones that expressed eGFP only following presentation of their cognate peptide epitope by APCs (25). When added to BMMfs that were previously pulsed with HEL protein, we found that WT and Cybb 2/2 BMMfs were equally able to induce eGFP expression in two of the hybridomas (Hb1.9 and H46.13), indicating that the processing/presentation efficiency of HEL [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] and HEL [48][49][50][51][52][53][54][55][56][57][58][59][60][61][62] peptides were unaffected by NOX2 (Fig. 2D).…”