A single amino acid substitution prevents recognition of a dominant human aquaporin-4 determinant in the context of HLA-DRB1-03:01 by a murine TCR

Abstract

Background Aquaporin 4 (AQP4) is considered a putative autoantigen in patients with Neuromyelitis optica (NMO), an autoinflammatory disorder of the central nervous system (CNS). HLA haplotype analyses of patients with NMO suggest a positive association with HLA-DRB1∗ 03:01.We previously showed that the human (h) AQP4 peptide 281-300 is the dominant immunogenic determinant of hAQP4 in the context of HLA-DRB1∗03:01. This immunogenic peptide stimulates a strong Th1 and Th17 immune response. AQP4281-300-specific encephalitogenic CD4+ T cells should initiate CNS inflammation that results in a clinical phenotype in HLA-DRB1∗03:01 transgenic mice. Methods Controlled study with humanized experimental animals. HLA-DRB1∗03:01 transgenic mice were immunized with hAQP4281-300, or whole-length hAQP4 protein emulsified in complete Freund's adjuvant. Humoral immune responses to both antigens were assessed longitudinally. In vivo T cell frequencies were assessed by tetramer staining. Mice were followed clinically, and the anterior visual pathway was tested by pupillometry. CNS tissue was examined histologically post-mortem. Flow cytometry was utilized for MHC binding assays and to immunophenotype T cells, and T cell frequencies were determined by ELISpot assay. Results Immunization with hAQP4281-300, resulted in an in vivo expansion of antigen-specific CD4+ T cells, and an immunoglobulin isotype switch. HLA-DRB1∗03:01 TG mice actively immunized with hAQP4281-300, or with whole-length hAQP4 protein were resistant to developing a neurological disease that resembles NMO. Experimental mice show no histological evidence of CNS inflammation, nor change in pupillary responses. Subsequent analysis reveals that a single amino acid substitution from aspartic acid in hAQP4 to glutamic acid in murine (m)AQP4 at position 290 prevents the recognition of hAQP4281-300, by the murine T cell receptor (TCR). Conclusion Induction of a CNS inflammatory autoimmune disorder by active immunization of HLADRB1∗ 03:01 TG mice with human hAQP4281-300, will be complex due to a single amino acid substitution. The pathogenic role of T cells in this disorder remains critical despite these observations.