Secretion assay

Meshes presenting goat-anti-mouse IgG were coated with 1:5 mol/mol ratio of FITC-tagged mouse IgG-anti-human CD3 (Biolegend, clone OKT3) to non-fluorescent mouse IgG-anti-human CD3 (Bio X Cell, clone OKT3), then incubated in RPMI media at 37 °C. Protein concentration was quantified by widefield fluorescence microscopy.
T cell culture was carried out using RPMI 1640 media (Gibco) supplemented with L-glutamine (20 µM, Gibco), fetal bovine serum (FBS) (5%, GE Healthcare) and HEPES (100 µM, Sigma). For expansions, cells were thawed then rested in complete media for 12–14 hours (37° C, 5% CO₂ / 95 % air) prior to use. Positive and negative controls consisted of stimulation with Dynabeads (CTS Dynabeads CD3/CD28, Thermo Fisher) and uncoated, non-activating culture wells. T cells were seeded at 1 × 10⁶ cells in a 1 mL volume into 15.6-mm diameter wells and incubated under standard cell culture conditions. Every second day, starting on day 3, cells were reseeded at 1 × 10⁶ cells / mL. Cells were frozen when mean volume dropped below 400 fL.
Cells frozen at the end of expansion were thawed, allowed to rest, and restimulated using Dynabeads. Cells were assayed for IFNγ (Secretion assay, Miltenyi Biotech) and CD107b mobilization (anti-CD107b clone H4B4, Biolegend) at four and twelve hours after restimulation. Cells were counterstained for CD4 and CD8, as described for phenotype analysis.