Page 44 - The autoimmune hypothesis of narcolepsy and its unexplored clinical features M.S. Schinkelshoek

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Chapter 3
T cell responses have recently been described (Latorre et al., 2018), this does not explain the strong link with HLA-DQ6. In short, the molecular mechanism underlying the HLA-DQ6 association remains to be determined.
Based on the increased incidence of NT1 after the H1N1 influenza pandemic mentioned above, we aimed to assess whether H1N1 influenza reactive CD4+ T cells are present in NT1 patients. A secondary aim was to assess whether these H1N1 influenza specific CD4+ T cells cross-react with Hcrt. We determined the crystal structure of one earlier identified H1N1 hemagglutinin peptide and two Hcrt peptides bound to the disease-predisposing HLA-DQ6 molecules to assess structural homology. Subsequently, we stimulated peripheral blood mononuclear cells with these peptides to generate specific T cell lines and clones. We then performed proliferation tests on these clones to assess both H1N1- and Hcrt-reactivity and assess HLA-DQ6 restriction, cross-reactivity and T cell receptor sequence of these clones.
Materials and methods
Subjects
Between March 2014 and June 2016, we included all consecutive NT1 patients after informed consent, recruited from the sleep clinic of the department of Neurology, Leiden University Medical Center and the Sleep-Wake Centre of Stichting Epilepsie Instellingen Nederland (SEIN), Heemstede. All patients were diagnosed with NT1 according to the International Classification of Sleep Disorders (ICSD-3; (American Academy of Sleep Medicine, 2014)). Symptom onset in all patients was after the 2009 H1N1 pandemic. Healthy controls were included in the same time period as the NT1 patients and matched for HLA and gender.
Hcrt-1 measurements
CSF samples were drawn for Hcrt-1 measurement in 34 NT1 patients. Hypocretin-1 concentrations were measured in duplicate with an I125 hypocretin-1 radioimmunoassay (Phoenix Pharmaceuticals, Mountain View, CA, USA). This assay has an intra-assay variability of <5% and a detection limit of 50 pg/mL. To adjust for inter-assay variability, Stanford reference CSF samples were included in the assay (Ripley et al., 2001, Mignot et al., 2002).