Page 47 - The autoimmune hypothesis of narcolepsy and its unexplored clinical features M.S. Schinkelshoek

Peripheral blood mononuclear cell (PBMC) isolation
Blood was drawn from all patients and healthy controls. PBMCs were extracted using Ficoll-Paque (GE Healthcare, Chicago, USA) gradient reagent. The first experiments were performed on fresh PBMCs, but in the remainder the isolated PBMCs were subsequently frozen in 10% dimethyl sulfoxide (DMSO; Sigma Aldrich, Saint Louis, USA) in fetal calf serum (FCS; Sigma Aldrich, Saint Louis, USA). These samples were stored until use in liquid nitrogen vessels.
Antigen-specific T cell lines
After isolating or thawing of PBMCs of NT1 patients and healthy controls, 1x106 cells were put into culture in Iscove’s Modified Dulbecco’s Medium (IMDM; Lonza, Basel, Switzerland) supplemented with L-glutamine (Thermo Fisher, Waltham, USA), 10% (pooled) human serum (NHS) and a mixture of the H1N1-HA275-287, Hcrt56-68 and Hcrt87-99 peptides. After every 5 days r-interleukin 2 (20 U/mL final concentration; rIL-2; Novartis, Basel, Switzerland) and interleukin 15 (10 ng/mL final concentration, IL-15; R&D, Minneapolis, USA) were added to each culture (Kooy-Winkelaar and Koning, 2015).
Peptide-specific T cell clone generation
Peptide-specific T cell clones (TCCs) were generated from T cell lines specific for H1N1-HA275-287 from 3 NT1 patients by limiting dilution in culture medium containing 106 irradiated feeder cells/mL, 20 U/mL rIL-2, 10 ng/mL IL-15 and 1 μg/mL PHA (“feeder mix”; Remel, Lenexa, USA). The cells were stimulated with 20 U/mL rIL-2 and 10 ng/mL in 10% human serum/IMDM after 5 days. After 10 days, growing wells were transferred to 24-well plates and cultured in feeder mix until a confluent layer of cells was formed. For the T cell lines of 9 NT1 patients, streptavidin-PE-HLA-DQ6- H1N1-HA275-287 and streptavidin-PE-HLA-DQ6-Hcrt56-68 and -Hcrt87-99 tetramers were used to directly stain HLA-H1N1-HA275-287-, -Hcrt56-68- and -Hcrt87-99 -specific T cells. Tetramers were produced essentially as described (Ooi et al., 2017). These tetramer-positive CD4+ T cells were sorted by flow cytometry on a FACS-Aria III instrument (BD Biosciences) and expanded as described above. Clones were subsequently generated from the identified H1N1-HA275-287-specific and/or Hcrt56-68- or Hcrt87-99-specific T cells, as described previously (Kooy-Winkelaar and Koning, 2015). All T cell lines used for TCC generation were derived from frozen NT1 patient samples.
H1N1 reactivity in CD4+ T cells
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