Abstract
Type 1 narcolepsy is strongly (98%) associated with human leukocyte antigen (HLA) class II DQA1*01:02/DQB1*06:02 (DQ0602) and highly associated with T cell receptor (TCR) alpha locus polymorphism as well as other immune regulatory loci. Increased incidence of narcolepsy was detected following the 2009 H1N1 pandemic and linked to Pandemrix vaccination, strongly supporting that narcolepsy is an autoimmune disorder. Although recent results suggest CD4+ T cell reactivity to neuropeptide hypocretin/orexin and cross-reactive flu peptide is involved, identification of other autoantigens has remained elusive. Here we study whether autoimmunity directed against Regulatory Factor X4 (RFX4), a protein co-localized with hypocretin, is involved in some cases of narcolepsy. Studying human serum, we found that autoantibodies against RFX4 were rare. Using RFX4 peptides bound to DQ0602 tetramers, antigen RFX4-86, -95, and -60 specific human CD4+ T cells were detected in 4/10 patients and 2 unaffected siblings, but not in others. Following culture with each cognate peptide, enriched autoreactive TCRαβ clones were isolated by single-cell sorting and TCR sequenced. Homologous clones bearing TRBV4-2 and recognizing RFX4-86 in patients and one twin control of patient were identified. These results suggest the involvement of RFX4 CD4+ T cell autoreactivity in some cases of narcolepsy, but also in healthy donors.
Highlights
Type 1 Narcolepsy (T1N) is a disabling neurological disorder that affects 1/2000 individuals
To test for the presence of autoantibodies directed against regulatory factor X4 (RFX4) in T1N cases, RFX4 variant 1 (RFX4_v1), RFX4_v3 and RFX4_v4 tagged with green fluorescent protein (GFP) were expressed in human embryonic kidney (HEK) 293 T cells
HEK293TGFP+, no significant difference was observed between patients and controls for either v1, v3, or v4, anti-RFX4_v3 autoantibody levels in post-Pandemrix T1N (PP-N) cases were significantly higher compared to patients without vaccination and vaccinated controls (p-value = 0.018 and 0.006, respectively) (Fig. 1A)
Summary
Type 1 Narcolepsy (T1N) is a disabling neurological disorder that affects 1/2000 individuals. One of the most promising autoantigens first identified was T RIB231, a protein initially thought to be enriched in HCRT cells It was shown by three different studies, including one by our group, that TRIB2 autoantibody levels are elevated in recent onset narcolepsy c ases[32]. Tetramer HLA-targeted T cells facilitates identification of antigen specific TCR-groups within highly diverse TCR r epertoires[48,49], which are stochastically generated by V(D)J recombination, a process rearranging germline TCR loci in each T cell[50,51] Using this technology, there is increasing evidence that CD4 autoreactivity directed against HCRT itself may be a major autoreactive process in human narcolepsy[46,52]. Of additional interest is the fact CD8+ directed T cell activity against this intracellular antigen has been shown in a recent study of narcolepsy p atients[45]
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