Page 49 - The autoimmune hypothesis of narcolepsy and its unexplored clinical features M.S. Schinkelshoek

T cell receptor (TCR) sequencing
TRAV, TRBV and CDR3 gene segment sequences of H1N1-HA275-287-specific TCCs were amplified using PCR and a set of specifically designed primers. PCR products rendered in this way were cloned into a Promega pGEM-T Easy vector and subsequently sequenced. The TRAV and TRBV gene usage and CDR3 sequences for all generated clones were determined using IMGT/V- QUEST (Brochet et al., 2008).
Statistical analysis
Differences at baseline in participant characteristics were calculated with Student’s t-tests and Pearson’s chi-square test. Pearson’s chi-square test was also used for comparing T cell proliferation in NT1 patients as compared to healthy controls. Differences between conditions in the HLA blocking experiments were calculated by one-way ANOVA with a Bonferroni post hoc analysis. Differences between P-values below 0.05 were deemed significant. Bonferroni corrections were executed when needed. All analyses were conducted using the IBM SPSS Statistics 23 software package.
Results
Patient characteristics
We included 81 NT1 patients and 19 healthy controls. Patient characteristics are shown in Table 3.2. Notably, all NT1 patients except one were HLA- DQA1*01:02/DQB1*06:02 (HLA-DQ6) positive. Thirty-four patients had undergone a lumbar puncture for Hcrt-1 measurement; all had Hcrt-1 values in the cerebrospinal fluid that were below the cut-off value of 110 pg/mL based on the ICSD-3 criteria for NT1. NT1 patients were younger than healthy controls, but the distribution of males and females was comparable between the two groups.
H1N1 reactivity in CD4+ T cells
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