T Cell Proliferation Assay with Toxins

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.