X vivo 15 medium

Bone marrow or peripheral blood samples from untreated children initially diagnosed with T-ALL were collected from the Czech Pediatric Hematology Centers. To be able to perform the here described experiments, only patients with the blast percentage higher than 80% and with a high cellularity were included. Within 24 h after aspiration, the mononuclear cells were isolated by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare, UK). All the samples were obtained with the informed consent of the children’s parents or guardians as well as the approval of the Ethical Committee of the University Hospital Motol, Prague, Czech Republic, study no. 201528848A. All experiments were performed in accordance with relevant guidelines and regulations. The isolated blasts were frozen in 90% fetal calf serum and 10% DMSO. After thawing, the blasts were maintained in X-VIVO™ 15 Medium with l-glutamine and gentamicin supplemented with 10% fetal calf serum, penicillin (100 U/mL), streptomycin (100 μg/mL), IL-7 (50 ng/mL) and insulin-transferrin-sodium selenite supplement (Sigma-Aldrich, St Louis, MO, USA).

PBMCs from healthy donor buffy coats were isolated by gradient centrifugation and used immediately or cryopreserved for later use. Cryopreserved PBMCs were thawed and rested overnight in X-VIVO 15 medium (Lonza) and 100 U/mL IL-2 (Proleukin) prior to use. CD8⁺ T cells were isolated by negative selection (EasySep, Stemcell). Isolated CD8⁺ T cells or PBMCs were activated with anti-CD3/CD28 coated Dynabeads (Gibco, 1:1 bead to cell ratio) in X-VIVO 15 medium supplemented with 50 U/mL IL-2 and 5% heat-inactivated human AB serum (Sigma-Aldrich). Small molecule inhibitors UNC2025 (Sigma-Aldrich), MRX-2843 (Medchem Express), and AC220 (quizartinib) (Medchem Express) were added to culture day 0 at 50 nM, 250 nM, 500 nM, or 750 nM. In long term cell culture assays, fresh cell culture medium with cytokines IL-2 (100 U/mL) and IL-15 (10 ng/mL, Peprotech) were replenished every 3 days, and T cells or PBMCs were split every 7 days.

PBMCs from healthy donors were isolated by Ficoll density centrifugation. Afterwards, immunomagnetic separations of CD14⁺ and CD8⁺ cells were performed with corresponding microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany). CD14⁺ cells were differentiated to immature DCs in X-VIVO15 Medium (Lonza Group, Basel, Switzerland) with 100 ng/mL IL-4 and 50 ng/mL GM-CSF (both PeproTech, Hamburg, Germany) for five days. Then, immature DCs were treated with a maturation cocktail consisting of X-VIVO15 Medium with 20 µg/mL poly I:C (Sigma-Aldrich Co., St. Louis, MO, USA), 3000 U/mL IFN-α (R&D Systems, Minneapolis, MN, USA), 1000 U/mL IFN-γ (PeproTech, Hamburg, Germany), 25 ng/mL IL-1β (PeproTech, Hamburg, Germany) and 50 ng/mL TNF-α (PeproTech, Hamburg, Germany) for 48 h. Mature DCs were incubated with peptides at a concentration of 10 µg/mL at 37 °C and 5% CO₂ for four hours. A total of 10 µg/mL of CEF pool (JPT Peptide Technologies, Berlin, Germany) were used as positive control. Meanwhile, CD8⁺ T cells were cultured in RPMI complete medium with 20 U/mL IL-2 and 20 ng/mL IL-7 (both PeproTech, Hamburg, Germany) for seven days until co-cultivation with peptide loaded mature DCs. Co-cultivation was performed with a ratio of 10:1 (T cells:DCs). On day 20, T cells were restimulated with peptide loaded mature DCs. Cells were harvested for further analysis on day 27.