Recombinant porcine il 2

PBMCs were isolated by density gradient centrifugation using Ficoll-Paque PLUS to exclude polymorphonuclear cells. The cells were incubated with BD Pharm Lyse Buffer (BD Bioscience) to remove erythrocytes. The cells were labeled with a PE-conjugated anti-porcine CD16 antibody (AbD Serotec). The labeled cells were isolated with anti-PE paramagnetic microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacture's instructions. The isolated cells were labeled with an FITC-conjugated anti-porcine CD3 antibody (BD Bioscience). Using a Cell Sorter SH800 (SONY, Tokyo, Japan), the fraction of CD3-negative and CD16-positive cells was collected. The collected cells were used as porcine NK cells. C1 iPS cells were incubated as target cells with NK cells at ratios of 1∶30 for 6 h at 38.5°C in DMEM supplemented with 1% FBS, 0.1 mM 2-mercaptoethanol and 1 ng/ml recombinant porcine IL-2 (R&D systems). The cytotoxicity of C1 iPS cells to NK cells was assessed by lactate dehydrogenase (LDH) release assay. Percent cytotoxicity was calculated as follows: cytotoxicity (%) = (Experimental value – Effector control – Target control)×100/(High control – Target control). The High control was obtained after incubating C1 iPS cells with 2% Triton X-100. The Effector control and the Target control were obtained after incubating NK cells alone and C1 iPS cells alone, respectively.

Sorted naïve T cells and total PBMCs were labelled using the CellTrace^TM Violet Cell Proliferation Kit (Thermo Fisher Scientific, Waltham, MA, USA), as described above and in [26 (link),50 (link)]. After the violet proliferation labelling, sorted CD8α⁻CD27^high CD4⁺ T cells and total PBMCs were counted and 2 × 10⁵ cells per well were plated in triplicates in 96-well plates (Greiner Bio-One, Kremsmünster, Austria). The cells were resuspended and seeded either in cell culture medium alone (RPMI 1640 supplemented with 10% heat-inactivated FCS, 100 IU/mL penicillin, and 100 µg/mL streptomycin) or stimulated with one of the following stimulation conditions: (i) ConA alone (3 µg/mL, Amersham Biosciences), (ii) ConA + DON 0.2 μM, (iii) ConA + DON 0.4 μM, (iv) ConA + DON 0.8 μM, and (v) ConA + DOM-1 16 μM. In addition, sorted CD172a⁺ APCs were added to all wells with sorted CD8α⁻CD27^high CD4⁺ T cells in a 1:10 ratio (2 × 10⁴ cells/well), as well as recombinant porcine IL-2 (50 ng/mL, R&D Systems, Minneapolis, MN, USA). Final total volume per well was 200 μL. After four days of in vitro cultivation cells were harvested, washed in PBS + 3% FCS, stained with the same Abs as prior to sorting (CD4, CD8α, and CD27), and analyzed on a FACSAria (BD Biosciences). At least 1 × 10⁵ cells were recorded per sample.