X vivo 20 medium

H9 cells were cultured on gelatin-coated plates with a feeder layer of MEF for 5 days, then the cells were subjected for endodermal differentiation. At the first day, differentiation medium was X-VIVO 20 medium (Lonza) supplemented with 100 ng/ml Activin A (Peprotech), 50 ng/ml WNT3A (Peprotech) or 3 μM CHIR99021(Selleck), 10 μM ROCK inhibitor Y-27632 (Selleck). The following day, the medium was changed to X-VIVO 20 (Lonza) medium containing 100 ng/ml Activin A, 0.5% B27 (Gibco). At day 4 after the differentiation, endodermal cells were dissociated with TrypLE (Gibco), and reseeded on Matrigel (Corning)-coated plates with DMEM-F12 medium (Gibco)or EGM2 medium (Lonza), supplemented with 0.5% B27, 1 μM RA (Sigma), 10 μM SB431542 (MCE), 20 ng/ml BMP4 (Peprotech), 2.5μM IWP2 (Selleck) or 2.5μM IWR1 (Selleck). The medium was changed daily, and the differentiation lasted until day 9 for the induction of 3PPE.

After isolation and sorting out CD56^dim NKs, the process was followed by expansion of cells in XVIVO-20 medium (Lonza, Barcelona, Spain) supplemented with 10% fetal bovine serum (FBS, Gibco, UK), 1,000-IU/ml IL-2 (Promokine, Germany), 10 ng/ml IL-12 (Promokine, Germany), 50 ng/ml IL-15 (Promokine, Germany), and finally 10 ng/ml IL-21 (Promokine, Germany) for activation of cells. The process lasts for about 7 days.

Each T-cell subset was plated into anti-CD3- or CD3/CD28-coated wells at 3 ×
10⁵ cells per well in serum-free X-Vivo-20 medium (Lonza) for
the determination of TGF-β1 and 2, or the RPMI medium described above
for TGF-β3,
respectively. All cultures were incubated at 37 °C for
72 h, and the supernatants were collected and stored at
−80 °C before the measurement of TGF-β family members, unless
otherwise mentioned. TGF-β1, 2 and 3 levels in supernatants were
determined with the TGF-β1 Emax ImmunoAssay System (Promega),
TGF-β2
Quantikine ELISA Kit (R&D Systems) and TGF-β3 ELISA Kit
(Mybiosource), respectively, according to the manufacturer’s
protocol. TGF-β3
levels in the RPMI medium supplemented with 10% FBS were lower than the minimum
detectable levels of the TGF-β3 ELISA Kit.

Cytotoxicity was measured using CytoTox-Glo (Promega) following the manufacturer's protocol with minor modifications. Primary T cells expanded from healthy donor PBMC as described above were used as effector cells and IGROV-1, SKOV-3, or JeKo-1 cells were used as target cells. The cells were incubated at an effector-to-target (E:T) ratio of 10:1 in X-VIVO 20 Medium (Lonza) with 5% (v/v) off-the-clot human serum AB (Innovative Research). The target cells (2 × 10⁴) were first incubated with the biAbs prior to adding the effector cells (2 × 10⁵) in a final volume of 100 μL/well in a 96-well tissue culture plate. The plates were incubated for 16 h at 37 °C with biAb concentrations ranging from 0.08 to 500 nM. After centrifugation, 50 μL of the supernatant was transferred into a 96-well clear bottom white walled plate (Costar 3610; Corning) containing 25 μL/well CytoTox-Glo. After 15 min at room temperature, the plate was read using a SpectraMax M5 instrument with SoftMax Pro software. The same supernatants (diluted 10-fold) used for the CytoTox-Glo assay were also used to determine IFN-γ, IL-2, and TNF-α secretion with Human IFN-γ, IL-2, or TNF-α ELISA MAX™ Deluxe kits (BioLegend), respectively, following the manufacturer's protocols.