0065 Immunity to Hypocretin and Influenza A in Patients with Narcolepsy Type 1 and Type 2

Abstract Introduction Narcolepsy type 1 (NT1) is caused by the loss of hypocretin/orexin (HCRT) neurons and is strongly associated with human leukocyte antigen (HLA)-DQA1*01:02/DQB1*06:02 (DQ0602, ~98%). Antigen-specific T cell responses suggest adaptive immunity against HCRT and pH1N1/Pandemrix in NT1. However, the mechanisms behind HCRT neuron loss and the molecular mimicry between influenza A and HCRT remain unclear, and immune responses in NT2 are largely unexplored. Methods Using DQ0602 DNA-barcoded (dCODE) dextramers loaded with pHA273-287, NP17-31, and HCRTNH2 peptides, we characterized peptide-DQ0602 (pDQ0602)-restricted CD4+ T cells in peripheral blood mononuclear cells from DQ0602-positive NT1, NT2, and healthy controls (HC), followed by single-cell RNA sequencing (scRNA-seq). Additionally, cerebrospinal fluid (CSF) cells were incubated with dextramers with a panel of 16 peptides and subsequently analyzed by scRNA-seq. Results We found a significantly higher frequency of pDQ0602-restricted CD4+ T cells reactive to HCRTNH2, pHA273-287, and NP17-31 in the PBMCs of DQ0602-positive NT2 patients compared with NT1 patients and HC. Close-to-onset NT2 patients (≤12 months) showed higher frequencies of pDQ0602-restricted CD4+ T cells than late-onset patients (>12 months), particularly significant for HCRT54-66-NH2 and NP17-31, whereas NT1 showed no such trend. In those patients (mainly NT1) under follow-up, we observed an initial increase followed by a subsequent decrease in pDQ0602-restricted CD4+ T cells among narcoleptic patients with at least two clinical visits, with mean intervals of 18.0 and 41.6 months, respectively. Furthermore, we detected a tendency toward higher frequencies of B cells, CD4+ T cells, and pDCs in the CSF of narcoleptic patients, and significantly higher frequencies of CD69+CD44+ Tem, CD69-CD44+Tcm, and CD69-CXCR4- Teff cells among CSF CD4+ T cells in NT1 patients. Lastly, we found that an N-terminal HCRT peptide, HCRT25-39, was recognized by a large number of NT1 TCR clones, suggesting a potential immune target in NT1. Conclusion This work provides evidence for the antigen-specific immune response targeting HCRT and influenza A antigens in NT2, suggesting disease progression from NT2 to NT1, particularly in DQ0602-positive individuals. Support (if any) The authors declare no potential conflicts of interest. This work was supported by the National Natural Science Foundation of China (32441101, 82341243) and the National Key Research and Development Program of China (2023YFE0118500).