T cell lines and clones
In order to generate GAD-specific T cell lines, cryopreserved PBMCs from participants (3 months post immunisation with GAD-alum) were thawed and stimulated with 10 μg/ml T cell GAD for 48 h; IL-13-secreting cells were captured using an IL-13 secretion/detection kit for 1 h at 37°C (Miltenyi Biotec, Bisley, Surrey, UK). Cells were subsequently labelled with mouse anti-human IL-13-biotin followed by anti-biotin PE in a cocktail containing monoclonal mouse anti-human CD3 APC-H7 (BD Biosciences Cat. No. 560275, RRID:AB_1645476), CD14 V500 (Cat. No. 541391), CD19 V500 (Cat. No. 561121), CD8 FITC (BD Biosciences Cat. No. 555634, RRID:AB_395996) (Becton Dickinson, Oxford, UK) and CD4 allophycocyanin (APC) (BioLegend Cat. No. 317415, RRID:AB_571944) (BioLegend, London, UK) all made up according to manufacturer’s instructions for 10 min on ice. Following a washing step, the cells were magnetically labelled with anti-PE microbeads, and a viability marker, 7-amino-actinomycin D (BioLegend, London, UK), was added. The cells were washed, and magnetic separation was performed using MS MACS columns (Miltenyi Biotec, Bisley, Surry, UK) to enrich for IL-13+ cells. CD4+IL-13+ T cells were then bulk sorted (typically 100–3000 cells were sorted per condition) using a FACSARIA II SORP cell sorter (Becton Dickinson) into polypropylene tubes containing 105 irradiated mixed allogeneic PBMCs with 4 μg/ml phytohaemagglutinin (PHA) (Alere, Zug, Switzerland) in X-vivo-15 (Lonza, Slough, UK) containing 5% (wt/vol.) human AB serum (Sigma Aldrich, Gillingham, Dorset, UK). The next day, fresh medium supplemented with T cell growth factor (TCGF; final concentration 5% (wt/vol.); Cellkine, ZeptoMetrix, Buffalo, NY, USA) was added and cells were expanded in culture medium containing decreasing concentrations of TCGF for 2 weeks before testing for specificity by IL-13 ELISpot. In brief, GAD specificity was examined by direct IL-13 ELISpot (U-Cytech, Utrecht, the Netherlands) using 104 T cells in the presence of 2×105 HLA-matched PBMCs for antigen presentation and 10 μg/ml T cell GAD. Epitope specificity of lines was examined using the same approach and individual peptides from a library of 115 overlapping peptides of GAD65 (20-mers overlapping by five amino acids; 10 μg/ml; Thermo Hybaid, Ulm, Germany;) were used as stimulation. Peptides eliciting a positive IL-13 response (spot number ≥ the inter-assay mean of the diluent +3 SD) were re-screened twice to confirm the result. In addition, as controls, Th1 and Th2 cell lines were generated using specific differentiating protocols and human cellXvivo Th1 and Th2 cell differentiation kits as instructed by the manufacturer (R & D systems, Abingdon, UK).
For the isolation of GAD-specific T cell clones, GAD-reactive T cell lines were stimulated with 10 μg/ml T cell GAD, labelled as above and CD4+IL-13+ cells were single-cell sorted into 96 well U-bottom plates containing 105 irradiated mixed allogeneic PBMCs and PHA (4 μg/ml) in 200 μl complete X-VIVO media containing 5% (wt/vol.) human AB serum. The following day, the clones were supplemented with 10% (wt/vol.) TCGF and expanded in culture media containing decreasing concentrations of TCGF for 2 weeks. Clones and lines were expanded and maintained by alternate rounds of restimulation using PHA or T cell GAD and irradiated mixed allogeneic PBMCs.
In some experiments, intracellular cytokine staining was performed on GAD-specific clones and for comparison a haemagglutinin (HA)-peptide-specific flu clone following stimulation with phorbol 12-myristate 13-acetate (PMA)/ionomycin (Sigma Aldrich, Gillingham, UK) at 50 ng/ml and 1 μg/ml, respectively, or T cell GAD (10 μg/ml; Diamyd, Sweden) or GAD peptide (residues 555–567) or HA peptide (residues 306–318), accordingly (Thermo Hybaid, Ulm, Germany) at 10 μg/ml, followed by monensin and brefeldin A (Becton Dickinson) for 3 h at 37°C. The cells were then harvested and washed in PBS (Life Technologies, Paisley, UK) and stained with a live/dead marker (Aqua dead cell stain; ThermoFisher Scientific, Hemel Hempstead, UK) and following another wash in PBS, membrane staining was performed using mouse anti-human CD14 Pacific Blue (PB) (Thermo Fisher Scientific Cat. No. MHCD1428, RRID:AB_10373537), CD19 PB (Thermo Fisher Scientific Cat. No. MHCD1928, RRID:AB_10373689) (ThermoFisher Scientific), CD3 APC-H7 (Cat. No. 560275, RRID:AB_1645476, Becton Dickinson), CD4 PE-Cy5.5 (Cat. No. 560650, ThermoFisher Scientific) and CD8 PE-Cy7 (Cat. No. 344712 BioLegend) all made up according to manufacturer’s instructions for 20 min at 4°C according to manufacturers’ instructions. Cells were washed and fixed in CytoFix (Becton Dickinson) for 20 min at room temperature in the dark and subsequently permeabilised with permeabilisation solution (BioLegend) before intracellular staining using anti-human IL-13 APC (Cat. No. 501908), IL-4 FITC (Cat. No. 500807) (BioLegend) and anti-IFN-γ PE (Cat. No. 502508) (Becton Dickinson) at room temperature for 20 min. The cells were washed and resuspended for acquisition using a FACSCanto II flow cytometer (Becton Dickinson) and analysed using FlowJo v.10 (FlowJo, Ashland, OR, USA).