Page 64 - The autoimmune hypothesis of narcolepsy and its unexplored clinical features M.S. Schinkelshoek

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Chapter 4
Even though indirect evidence points strongly to a T cell mediated autoimmune response, the exact key immune cell populations in this response remain elusive. We apply another approach to identifying the immune components involved in the autoimmune response that destroys the hypocretin-producing neurons. In this study, we use mass cytometry, a high-dimensional single-cell analysis technique that permits an in-depth examination of immune cell subtypes simultaneously, together with algorithm-guided analysis approaches. This technique has already been shown to be able to separate patients and controls based on their specific immune cell cluster profile (van Unen et al., 2016). We aim to primarily identify populations of immune cells in the peripheral blood that are enriched in NT1 patients with recent disease onset compared with healthy controls.
Materials and methods
Patient samples
NT1 patients were included at the outpatient clinics of the Leiden University Medical Center, Leiden, the Netherlands; Sleep-Wake Centre of Stichting Epilepsie Instellingen Nederland, Heemstede, the Netherlands; Department of Rehabilitation, University of Witten/Herdecke, Witten/Herdecke, Germany; Department of Neurology, Inselspital, Bern University Hospital and University of Bern, Bern, Switzerland. Included patients were grouped based on time from disease onset. Recent disease onset was defined as first symptom onset within 2 years of blood draw. The diagnosis of NT1 was based on the criteria described in the third edition of the International Classification of Sleep Disorders (ICSD, 2014). All patients gave their written informed consent to take part in the research program. We also included non-related healthy individuals of which HLA-DQB1*06:02 status was known. Part of these healthy individuals were participants from a panel of randomly selected Dutch individuals (van Rooijen et al., 2012); all other healthy individuals were recruited in Witten and Bern. All mass cytometry experiments and their initial analysis were performed blinded. This study was approved by the Medical Ethical Committee of the Leiden University Medical Center (protocol P17.169) and those in Witten (Ethik- Kommission Universität Witten-Herdecke; 211/2017) and Bern (Kantonale Ethikkommission Bern (KEK); ID 2018-01564).