Page 48 - The autoimmune hypothesis of narcolepsy and its unexplored clinical features M.S. Schinkelshoek

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Chapter 3
Flow cytometry
Peptide-specific T cell lines generated from one patient were incubated for 30 minutes with 11 antibodies for surface staining and subsequently acquired on a LSRII instrument (BD Biosciences). Fluorochrome-labelled antibodies directed against CD3 (clone UCHT1), CD4 (clone SK3), CD5 (clone L17F12), CD7 (clone M-T701), CD14 (clone MφP9), CD27 (clone M-T271), CD28 (clone CD28.2), CD45RA (clone L48) and IgG1 (clone MOPC-21) were from BD Biosciences (San Jose, California, USA), anti-CD8 (clone 3B5) was from Invitrogen (Bleiswijk, the Netherlands) and anti-CD45 (clone HI30) from eBioscience (San Diego, California, USA). Results were analysed using FlowJo V.10 software (Schmitz et al., 2016).
T cell proliferation assay and assays for assessing HLA-DQ6 restriction
Proliferation assays were performed on T cell lines and TCCs in triplicate in 150 μl IMDM supplemented with 10% human serum in 96-well, flat-bottom plates (Corning Life Sciences, Tewksbury, USA) using 1x104 T cells stimulated with 1x105 irradiated HLA-DQA1*01:02/DQB1*06:02 (HLA-DQ6)-matched allogeneic PBMCs (3,000 RAD) in the presence or absence of either H1N1- HA275-287 or Hcrt56-68 and Hcrt87-99 (10 μg/ml). In HLA-restriction experiments, the HLA-DQA1*01:02/DQB1*06:02-matched allogeneic PBMCs were replaced by PBMCs expressing either HLA-DQA1*03:01/DQB1*03:02 (HLA-DQ8; no association with narcolepsy), HLA-DQA1*01:02/DQB1*06:03 (less frequently found in narcolepsy compared to healthy controls (Tafti et al., 2014)) or a mix of HLA-DQA1*01:02/DQB1*06:02 and HLA-DQA1*01:02/DQB1*06:03. Triplicate wells containing 104 T cells supplemented with 20 U/mL rIL-2 and 10 ng/mL IL-15 functioned as a positive control. After 48 h at 37°C, cultures were pulsed with 10 μCi/mL of 3H-thymidine and harvested 18 h later. Proliferation was measured using a MicroBeta Microplate Counter (PerkinElmer, Waltham, MA, USA). A positive response was defined as a stimulation index (SI) of 3 (defined as the mean count in the wells with peptides divided by the mean count in the wells without peptide) (Kooy-Winkelaar and Koning, 2015). Additionally, to confirm HLA restriction of the T cell lines, blocking experiments were performed in which either anti-HLA-class I (W6/32), anti-HLA-DP (B7/21), anti-HLA-DQ (SPV-L3) or anti-HLA-DR (B8.12.2) monoclonal antibodies (mAbs; locally produced) were added to the initial assay.