Rh il 2

PBMCs/T cells were cultured at 37°C with 5% CO₂. Upon thawing, PBMCs were washed with PBS (300xg, 5min) and resuspended at a density of 2 × 10⁶ cells/ml in X-VIVO™ 15 (Lonza) medium supplemented with 200U/ml rhIL-2 (Immunotools) and seeded into 24-well plates (1 ml/well). Adherent cells were allowed to attach for 4 h. After 4 h, non-adherent cells were collected, counted, adjusted to a cell density of 1 × 10⁶ cells/ml with X-VIVO™ 15 (Lonza) medium supplemented with 200U/ml rhIL-2 (Immunotools) and re-seeded into 24-well plates (1ml/well). To each well, 5 µl of ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (StemCell Technologies) was added and the T cells activated for 72–96 h prior to gene editing.

Genetically modified DCs stimulated with tumor antigens were seeded in a 12-well plate (Nunc) at a density of 1.8 × 10⁵ cell/0.5 ml/well. Splenocytes obtained from C57BL/6 healthy female mice and stored in liquid nitrogen were thawed, centrifuged (7 min, 192 × g), resuspended in RPMI (Gibco) supplemented with 10% FBS(Sigma-Aldrich), and applied at a density of 1.8 × 10⁶ cells/0.5 ml/well to 12-well plates with DCs. The wells were supplemented with rh IL-2 (200 U/ml, ImmunoTools). Mixed culture of transduced DC and splenocytes was carried out for 5 days under standard conditions (37°C, 5% CO₂). After that, the spleen cell phenotype and their cytotoxic activity as well as the concentration of cytokines in the supernatants collected from the 5-day mixed culture were evaluated.

First HA-1 transgenic T cell receptor (TCR) transduced CD8⁺ T cells were used as a model system. Here, 1 × 10⁴ ex vivo cDCs, generated from HLA-A2⁺HA-1⁻ HPC donors, were pre-incubated with/without 2.5 µg/mL cytochalasin D (CytD, Sigma Aldrich) for 1 h at 37 °C in 75 µL plain IMDM in round-bottom 96-wells plates. Then, 25 µL IMDM with/without HA-1 short-peptide or HA-1 long-peptide (VARFAEGLEKLKECVLHDDLLEARRPRAHEZL) was added (final peptide concentration 5 µM) and cells were incubated for 2 h at 37 °C. Whereupon overnight maturation was induced as described before. Next, 100µL supernatant was harvested and 100µL IMDM/10% FCS containing overnight rested 1 × 10⁴ HA-1 transgenic TCR T cells were added [23 (link)] (cDC:T cell ratio 1:1), CD107a antibody (Biolegend) and Brefeldin A (BD) was added to each well. The following day, T cell activation and intracellular cytokine production was analyzed using flow cytometry. Next, alloSCT patient PBMC containing low frequencies of HA-1 specific CD8⁺ T cells were used. Here, 1 × 10⁵ ex vivo cDC1s, generated from HLA-A2⁺HA-1⁻ HPC donors, were co-cultured with 1 × 10⁶ patient-derived PBMCs in IMDM/10% HS. At day 4, 1 mL fresh IMDM/10% HS containing 50 U/mL rh IL-2 (Immunotools) and 5 ng/mL rh IL-15 (Immunotools) was added. At day 7, cells were harvested and HA-1 specific T cell expansion was assessed using flow cytometry.