*4.5. Flow Cytometry Analysis*

For flow cytometry analysis 0.5 <sup>×</sup> <sup>10</sup><sup>6</sup> cells were labelled with 1 mg/mL of a respective antibody for 30 min at 4 ◦C, as described in detail elsewhere [23,58]. In brief, for evaluation of the purity of isolated CD4<sup>+</sup> T cells the cells were labelled with FITC-conjugated anti-CD4 monoclonal antibodies (BD Biosciences, San Jose, CA, USA). For evaluation of Treg cells in CD4<sup>+</sup> T cell cultures the harvested cells were labelled with PerCP-conjugated anti-CD4 and APC-conjugated anti-CD25 monoclonal antibodies (both from BD Biosciences) followed by a permeabilization-fixation procedure and intracellular staining with Phycoerythrin (PE) Anti-Human Foxp3 Staining Set (eBioscience Inc., San Diego, CA, USA) according to the detailed protocol provided by the manufacturer. For identification and evaluation of Th17 cells cultured CD4<sup>+</sup> T cells were labelled with FITC-conjugated anti-CD161 monoclonal antibody (BD Biosciences) followed by intracellular staining with Phycoerythrin (PE) conjugated Anti-Human ROR-γ antibody (eBioscience Inc.). As a negative control served nonspecific isotype IgG antibodies conjugated with the respective fluorochrome.

Cell samples were analyzed on the FACSCalibur using CellQuest / BD FACS DivaTM software (BD Biosciences). The cells were specifically analyzed by selective gating, based on the parameters of forward and side scatter as described elsewhere [23,58]. The results were based on analysis of at least 100,000 cells and were shown as the percentage of positively labelled cells. The gating strategy for identification and evaluation of Treg and Th17 cells is shown in Supplementary Figures S2 and S3, respectively.
