Expansion protocol was adapted from previous protocols with minor modifications.10 Sorted cells were expanded in Roswell Park Memorial Institute 1640 media supplemented with l-glutamine, penicillin/streptomycin, sodium pyruvate, and 10% of human AB pooled serum, in the presence of recombinant human IL-2 (1000 U/mL) (Novartis) and Dynabead Human T-activator CD3/CD28 (Life Technologies) in a 1:1 cell to bead ratio over two 7-day rounds of expansion. During the second round of expansion, 8 ng/mL TAC (Sigma) dissolved in dimethyl sulfoxide (DMSO) (Sigma), based on the published trough level in patients with stable liver or kidney allografts,6,15,16 or empty DMSO control added to cell media (final concentration of DMSO in culture was 1.92 × 10−4% volume).