Type 1 narcolepsy is strongly (98%) associated with human leukocyte antigen (HLA) class II DQA101:02/DQB106:02 (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. 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.