Proleukin

293T cells were cultivated with complete DMEM medium (10% FCS, 2 mM L-glutamine, 10 U/ml penicillin-streptomycin). Ba/F3 and 32D cells were cultivated with complete RPMI 1640 medium (10% FCS, 2 mM L-glutamine, 10 U/ml penicillin-streptomycin) (all from Gibco, Thermo Fisher Scientific) supplemented with IL-3 (1 ng/ml; ImmunoTools). IL-3 stimulation was performed with 10 ng/ml IL-3 for 20 minutes.
The hSTAT5B^N642H, hSTAT5B, and B6N WT T cells were isolated from LNs and spleens from 8- to 12-week-old mice. Following T cell activation by anti-CD3 (BD), T cells were grown in complete RPMI 1640 medium containing 10 mM HEPES, 1× MEM nonessential amino acids, 50 μM β-mercaptoethanol (all from Gibco, Thermo Fisher Scientific), 1 mM sodium pyruvate (MilliporeSigma), and 100 U/ml human IL-2 (ProleukinÒ; Novartis).
Cytokine stimulation of T cells was performed with human IL-2 (100 U/ml; ProleukinÒ; Novartis), murine IL-4 (100 ng/ml; R&D Systems), or murine IL-7 (10 ng/ml; R&D Systems).
The 293T and 32D cell lines were gifts of M. Hengstschläger (Center of Pathobiochemistry and Genetics, Institute of Medical Genetics, Medical University of Vienna, Vienna, Austria) and F. Grebien (Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria), respectively. The Ba/F3 cell line was provided by A. D’Andrea (Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA).

Transduced T cells were sorted using the FACSAria II (BD Biosciences). Before sorting, cells were labeled with the Live/Dead near-IR fixable dye and resuspended in sterile PBS, supplemented with 2% BSA and 2 mM EDTA (Sigma, Gillingham, UK). The gating strategy was based on forward scatter (FSC) and side scatter (SSC), exclusion of debris and aggregates, and Live/Dead dye exclusion. ZsGreen⁺ cells were then sorted in sterile PBS, supplemented with 2% BSA and 2 mM EDTA, and cultured in X-Vivo 15 (Lonza), supplemented with 5% human serum (Seralabs) and 500 U/mL of Proleukin (Novartis) at a cell density of 1 × 10⁶ cells/mL for 16–24 h before being cryopreserved in CryoStor CS10 (Sigma) until use. Before processing the cells in the C1 Single-Cell Auto Prep system, the cells were thawed and incubated for 12–16 h at 37°C and 5% CO₂ in complete medium.

Upon thawing, CD3⁺ T cells were resuspended in X-Vivo 15 (Lonza, Huddersfield, UK), supplemented with 5% human serum (Seralabs, Hayward Heath, UK) and 500 U/mL of Proleukin (Novartis, Camberley, UK) at a cell density of 1 × 10⁶ cells/mL. T cells were activated with Transact (Miltenyi, Bisley, UK) following the manufacturer’s recommendations for 6 h before proceeding to viral transduction.
The rLV.EF1.ZsGreen1-9 lentivirus (Clontech, Saint-Germain-en-Laye, France) was thawed on ice and added to the cell suspension. The cell suspension containing the lentiviral vector was seeded into MACS guanosine monophosphate (GMP) Cell Expansion Bags (Miltenyi, Bisley, UK) and incubated for 48 h at 37°C and 5% CO₂. An equal volume of fresh medium containing 1,000 U/mL of Proleukin was added to the bags at days 3 and 5 after transduction. At day 7, the transduction efficiency was evaluated by flow cytometry; thereby, a cell aliquot was labeled with the Live/Dead near-infrared (IR) fixable dye (Life Technologies, Waltham, MA, USA) following the manufacturer’s instructions. The remaining culture was resuspended in sterile PBS and processed for FACS or cryopreservation in CryoStor CS10.