title: 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).
  
  
  CONCLUSIONS
  Induction of a CNS inflammatory autoimmune disorder by active immunization of HLA-DRB1*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.
authors.affiliation: Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center at Dallas, Dallas, TX, United States of America.
authors.forename: Benjamine
authors.initials: B
authors.lastname: Arellano
authors.affiliation: Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center at Dallas, Dallas, TX, United States of America.
authors.forename: Rehana
authors.initials: R
authors.lastname: Hussain
authors.affiliation: Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center at Dallas, Dallas, TX, United States of America.
authors.forename: William A
authors.initials: WA
authors.lastname: Miller-Little
authors.affiliation: Department of Pathology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX, United States of America.
authors.forename: Emily
authors.initials: E
authors.lastname: Herndon
authors.affiliation: Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center at Dallas, Dallas, TX, United States of America.
authors.forename: Doris
authors.initials: D
authors.lastname: Lambracht-Washington
authors.affiliation: Histocompatibility and Transplant Immunology, Department of Pathology and Genomic Medicine, The Methodist Hospital Physician Organization, Houston, TX, United States of America.
authors.forename: Todd N
authors.initials: TN
authors.lastname: Eagar
authors.affiliation: Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center at Dallas, Dallas, TX, United States of America.
authors.forename: Robert
authors.initials: R
authors.lastname: Lewis
authors.affiliation: Opexa Therapeutics, The Woodlands, TX, United States of America.
authors.forename: Don
authors.initials: D
authors.lastname: Healey
authors.affiliation: Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center at Dallas, Dallas, TX, United States of America.
authors.forename: Steven
authors.initials: S
authors.lastname: Vernino
authors.affiliation: Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center at Dallas, Dallas, TX, United States of America.
authors.forename: Benjamin M
authors.initials: BM
authors.lastname: Greenberg
authors.affiliation: Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center at Dallas, Dallas, TX, United States of America.|Neurology Section, VA North Texas Health Care System, Medical Service, Dallas, TX, United States of America.|Department of Neurology, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.
authors.forename: Olaf
authors.initials: O
authors.lastname: Stüve
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D051401:Aquaporin 4 / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D002454:Cell Differentiation; D049109:Cell Proliferation; D004195:Disease Models, Animal; D004681:Encephalomyelitis, Autoimmune, Experimental / Q000276:immunology; D005260:Female; D059811:HLA-DRB1 Chains / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009471:Neuromyelitis Optica / Q000235:genetics / Q000276:immunology*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D014611:Vaccination
chemical_list: C496030:AQP4 protein, human; C496031:Aqp4 protein, mouse; D051401:Aquaporin 4; D059811:HLA-DRB1 Chains; C553502:HLA-DRB1*03:01 antigen; D011948:Receptors, Antigen, T-Cell