Identifying Dominant T cell Determinants of Aquaporin-4 in the Context of HLA-DR3

Background:  Neuromyelitis optica (NMO) is a demyelinating inflammatory disorder of the central nervous system (CNS), which is defined as the co-occurrence of optic neuritis and myelitis.  Thus far, all HLA haplotype analyses of NMO patients suggest an association with HLA-DR3.  In the majority of patients, IgG against aquaporin-4 (AQP-4), a water channel found on astrocytes, can be detected.  The role of cellular immune responses against aquaporin-4 in NMO has not been studied.  Our laboratory has initiated experiments that will characterize cellular immune responses against full-length human (h)AQP-4 in HLA-DR3 transgenic mice to identify dominant T cell determinants.

Methods:  HLA-DR3 mice are actively immunized with recombinant whole-length hAQP4. Recall assays are performed with 15-amino acid length, over-lapping peptides of hAQP4 to determine the dominant T cells epitope.  Dominant epitopes are identified by ELISPOT assay and a CFSE-based flow cytometry assay.  Individual linear peptides are being used to actively induce CNS autoimmune disease.  Clinical disease assessed prospectively longitudinally using a 5 point scale.  Involvement of the anterior visual pathway is being assessed with pupillometry.

Results:  Three peptides have been discovered to stimulate the proliferation of CD4⁺ T cells.  When actively immunized into HLA-DR3 mice, these peptides caused atypical EAE symptoms as well as lateral and bi-lateral optic neuritis.

Conclusions:  Our studies in HLA-DR3 transgenic mice will allow us to study antigen-specific T cell responses against the CNS autoantigen hAQP4.  Also, determining dominant linear AQP-4 determinants in HLA-DR3 will lead to the generation of a T cell-mediated CNS autoimmune disease of NMO.