Cryopreserved PBMC Analysis in T1D

Cryopreserved peripheral blood mononuclear cells (PBMC) were provided by Type 1 Diabetes TrialNet from the clinical trial NCT00505375 (7 (link)). Samples from 40 abatacept- and 19 placebo-treated individuals were analysed at baseline, year 1 and year 2. Samples were provided blinded to treatment and time of sampling; each subject’s samples from the three collection times were stained and analysed on the same day. TrialNet Coordinating Centre provided clinical and demographic metadata following submission of raw data files. PBMCs were thawed at 37°C, washed in pre-warmed basal media (RPMI1640 Glutamax with HEPES, containing 100U/mL penicillin and 100μg/mL streptomycin (all Gibco)) containing 5% human heat-inactivated AB serum (Gibco), 20mM tumour necrosis factor (TNF)-α Protease Inhibitor-2 (TAPI-2) (Calbiochem) and 50U/mL Benzonase (Millipore). Cells were rested for 1 hour at 37°C/5% CO₂ in basal media containing 10% human AB serum and 20mM TAPI-2 before washing in 1X basal media and 1X DPBS (Gibco) and staining of 2×10⁶ PBMC each for flow and mass cytometry.