T cell cultures

To generate mature DCs, monocytes were cultured for 2–3 days in manufactory pre-tested X-VIVO™ 15 medium (cat#BE02-061Q or cat#BE02-060Q, Lonza) supplemented with 2% human serum (HS, Sanquin Blood Supply Foundation, not pre-tested for assay performance), 500 IU/ml IL4 and 800 IU/ml GM-CSF (cat#11340045 and cat#11343125, ImmunoTools). Subsequently, cells were re-plated at 0.5 × 10⁶/ml in fresh X-VIVO™ 15 containing 2% HS, 500 IU/ml IL4 and 800 IU/ml GM-CSF for another 3–4 days. Next, immature DCs were maturated for 2 days by adding X-VIVO™ 15 containing 2% HS and cytokines at a final concentration of 5 ng/ml IL1β, 15 ng/ml IL6, 20 ng/ml TNFα (cat#11340015, cat#11340064, cat#11343015, all ImmunoTools) and 1 µg/ml PGE2 (Prostin E2®, Pfizer).

CD3⁺ T cells, CD4⁺ T cells, CD4⁺ T_N or CD8⁺ T cells were stimulated with allogeneic DCs at a 1:10 DC:T cell ratio or with CD3/CD28 Dynabeads (cat#11131D, ThermoFisher) at a 1:1 bead: T cell ratio for 10 days in IMDM (cat#12440053, Thermofisher), supplemented with 10% HS, 50 IU/ml IL2 (Proleukin®, Chiron), 5 ng/ml IL7 and 5 ng/ml IL15 (cat#11340075 and cat#11340155, both ImmunoTools). When indicated, Akt-inhibitor VIII (cat#A6730, Sigma) or GDC-0068 (cat#HY-15186, MedChemExpress), dissolved in DMSO, was added. Control conditions were supplemented with corresponding concentrations of DMSO (≤ 0.1% in all experiments). Half of the culture volume was refreshed with medium plus cytokines and AKT-inhibitor or DMSO every 2–3 days. Cells were rechallenged with DCs or CD3/CD28 beads on day 10 and cultured for another 7 days without AKT-inhibitor or DMSO. To evaluate cytokine production profiles on day 10 or 17, T cells were restimulated overnight with DCs (1:10 DC:T cell ratio) or 1 µg/ml PMA plus 40 µg/ml ionomycin (cat# P1585 and cat#I0634, both Sigma), in the presence of Brefeldin A (Golgiplug, cat#555029, BD Biosciences) or Monensin (Golgistop, cat#554724 BD Biosciences), in absence of AKT-inhibitor or DMSO.