Recombinant human il 15

Buffy coats and non-mobilized leukapheresis were obtained from healthy volunteer donors from the DRK Dortmund and Ulm. Human PBMCs were purified by density gradient centrifugation using Pancoll solution (Pan-Biotech, Aidenbach, Germany). Primary human T cells were isolated from PBMCs by negative bead selection according to manufacturer’s recommendations (Pan T cell isolation kit, Miltenyi Biotec, Bergisch Gladbach, Germany) and activated using MACS GMP T Cell TransAct (Miltenyi Biotec, Bergisch Gladbach, Germany). T cells were cultured in TexMACS GMP medium (Miltenyi Biotec, Bergisch Gladbach, Germany) supplemented with 12.5 ng/mL of recombinant human IL-7 and 12.5 ng/mL of recombinant human IL-15 (Miltenyi Biotec, Bergisch Gladbach, Germany). T cells were transduced using lentiviral vectors 24 h after stimulation using an MOI of 5 to maintain the vector copy number of the drug product below 5. T cells were washed 3 days after stimulation to remove MACS GMP T Cell TransAct and lentiviral particles. T cells were expanded for 13–14 days with the addition of fresh culture medium every 2–3 days, before the experiments were started. Timing of T cell generation, including activation, transduction, and addition of medium was kept consistent for experiments comparing multiple donors and CAR constructs.

Cryopreserved CBMC and PBMC samples were thawed and rested overnight (18 h) at 37°C 5% CO₂ in RPMI 1640 media supplemented with 10% FBS, or media supplemented with 10 ng/mL recombinant human IL-15 (Miltenyi Biotec), as previously described (32 (link)). Subsequently, NK cells were purified by negative selection with magnetic beads (human NK cell isolation kit, Miltenyi Biotec) and used as effector cells (NK cell to target cell ratio of 5:1) in redirected cytotoxicity assays using HIV × CD16 DART molecules, performed as described above. Positive responses were defined as specific killing >20%, and data were not fit to a dose-response function for graphing due to the presence of a prozone (37 (link)).

The generation of CAR T cells was previously reported [20 (link)]. In brief, PBMCs were isolated by density gradient centrifugation from whole blood. Thereafter, T cells were purified using the Pan T Cell Isolation Kit, human (Miltenyi Biotec, Bergisch Gladbach, Germany), and T cells were activated in TexMACS™ Medium (Miltenyi Biotec) supplemented with T Cell TransAct™ (Miltenyi Biotec), 12.5 ng/mL of recombinant human interleukin IL-7, and 12.5 ng/mL of recombinant human IL-15 (Miltenyi Biotec). After 24 h of activation, T cells were transduced with vesicular stomatitis virus glycoprotein G (VSV-G) pseudotyped lentiviral particles. Three days after activation, T cells were washed and cultured with fresh TexMACS™ Medium supplemented with 12.5 ng/mL of recombinant human interleukin IL-7 and 12.5 ng/mL of recombinant human IL-15. On day 12 after transduction, CAR T cells were used in in vitro assays, and the number of viable CAR T cells was determined by staining T cells with 7-AAD (Miltenyi Biotec) and biotinylated human FOLR1 protein (Miltenyi Biotec). To generate CAR T cells from patient-derived ascites, the ascites suspension was centrifuged for 5 min at 300 g to separate the cellular fraction. T cells were transduced within the total cellular fraction of ascites. Transduction, as well as culture, was performed as described above.

NOD/SCID/IL2Rγ^null (NSG) mice were originally purchased from Jackson Laboratories, and housed and bred in the Radboud university medical center (Radboudumc) Central Animal Laboratory. Female NSG mice were used from 6 to 20 weeks of age. All animal experiments were approved by the Animal Experimental Committee of the Radboudumc and were conducted in accordance with institutional and national guidelines, permit number 10300.
Mice were i.v. injected with 10–15 × 10⁶ (cultured) PBLs containing antigen-specific CD8⁺ T cells and weekly vaccinated with 0.25–0.5 × 10⁶ peptide-loaded autologous control or PD-L silenced DCs (i.p.). To support antigen-specific CD8⁺ T cells, 0.5 μg recombinant human IL15 (Miltenyi, i.e., 2500 units) was administered i.p. every 2–3 days when indicated. At indicated time points, mice were killed and organs were analyzed for presence of antigen-specific CD8⁺ T cells by flow cytometry.