*4.5. In Vitro Culture*

The spontaneous and stimulated T-lymphocyte subset expression of IFNγ, IL-17A and IL-4 was assessed by in vitro intracytoplasmic staining in the presence of 2mM monensin. The PBMCs were stimulated with 50 ng/mL phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich, MerkMillipore, Boston, MA, USA) plus 1μg/mL ionomycin (Calbiochem, MerkMillipore, Boston, MA, USA) for 6 h. Spontaneous cytokine expression was determined in parallel cultures in the absence of exogenous stimuli.

#### *4.6. Surface and Intracellular Lymphocyte Staining*

T-lymphocytes were studied in PBMCs by nine-color flow cytometry. PBMCs were incubated with the next surface-labeled monoclonal-antibodies, CD3-PercP, CCR7-PECY7 (Becton-Dickinson, BD, CA, USA), CD8-Alexa405, CD45RA-APC (Caltag, Carlsbad, CA, USA) and CD27-APCAlexa780 (eBioscience, San Diego, CA, USA).

For intracytoplasmic staining, cells were fixed and permeabilized (Fix and Perm, Caltag, Carlsbad, CA, USA), and cytokines were stained with IL-4-PE, IFNγ-Alexa700 and IL-17A-FITC (Becton-Dickinson, BD, CA, USA). All samples were stained with a dead cell-discriminator simultaneously with antibody addition (Fixable aqua dead cell stain kit for 405 nm excitation; Molecular Probes, Eugene, OR, USA).

Samples were acquired in a FacsAria-II flow cytometer and were analyzed using FacsDiva 5.0 and Flow-Jo 7.0 software (Becton-Dickinson, BD, San Jose, CA, USA).
