*2.5. Cytokine Assay*

To stimulate cytokine production, separated PBMCs were mixed with phytohemagglutinin (PHA) at a concentration of 5μg/mL (Merck, Darmstadt, HE, Germany) before they were incubated at 5% CO2, 37 ◦C for 48 h [21]. Supernatants were harvested from each well and stored at −80 ◦C until they were measured for levels of cytokines, namely interleukin-4 (IL-4) and interferon-γ (IFN-γ), by ELISA human cytokine development kits (PeproTech, Rocky Hill, NJ, USA). Each sample was analyzed in triplicate, and controls and unstimulated blanks were analyzed simultaneously.

### *2.6. Proliferation Assay*

A total of 2 × 10<sup>6</sup> cells/mL of PBMCs in complete medium were grown in 96-well plates and stimulated with 5 μg/mL PHA [13] and incubated at 37 ◦C, 5% CO2 for 48 h. Following incubation, 3-(4-5-dimethylthaizolyl-2)-2,5-diphenyltetrazolium bromide (Merck, Darmstadt, HE, Germany) was added and the cells were incubated at 37 ◦C for a further 4-h period. Then, 100 μL of dimethyl sulfoxide (DMSO) was added and samples were mixed thoroughly by repeated pipetting and incubated at room temperature in the dark for 2 h. The absorbance wavelength of 570 nm and the reference wavelength of 630 nm were measured by a Thermo ScientificTM MultiskanTM GO microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). Each sample was analyzed in triplicate, and controls and unstimulated blanks were analyzed simultaneously.

#### *2.7. Determination of T Cell Subpopulations by the Flow Cytometry*

Cells were stained before they were incubated with FACS lysing solution, containing paraformaldehyde for fixing cells (https://www.bdbiosciences.com/ds/is/tds/23-1358.pdf). To control for staining variability, negative isotype control was used for every sample tested. The negative isotype control ensured that the observed staining was due to specific antibody binding to the target rather than an artefact or background. The utility of Fc receptor blocking reagents was unnecessary when the negative isotype was used. For CD4 and CD8, at least 10,000 event cells were collected as the concentrations of Th and Tc cells were 800 and 500 cells/μL, respectively. For Treg cells, at least 20,000 events were collected as there was 1–2% of CD4 T cells.

#### 2.7.1. Helper T Lymphocytes and Cytotoxic T Lymphocytes

To a 5-mL FalconTM polystyrene tube (Becton Dickinson and Company, Franklin Lakes, NJ, USA), fifty microliters of EDTA blood was added per 10 μL of BD Tritest CD4/CD8/CD3 (Becton Dickinson and Company, Franklin Lakes, NJ, USA). The method followed the manufacturer's manual. The negative

control tube was reacted with the BD FastImmuneTM γ1 PE/CD45 PerCP control (Becton Dickinson and Company, Franklin Lakes, NJ, USA). Samples were analyzed by BD FACSCaliburTM flow cytometry (Becton Dickinson and Company, Franklin Lakes, NJ, USA). Cytotoxic T (Tc) cells are CD3+CD8+, while helper T (Th) cells are CD3+CD4+. The data were analyzed by BD CellQuest software version 5.0, 2002 (Becton Dickinson and Company, Franklin Lakes, NJ, USA).

#### 2.7.2. Regulatory T Lymphocytes

To a 5-mL FalconTM round-bottom polystyrene tube (Becton Dickinson and Company, Franklin Lakes, NJ, USA), fifty microliters of bu ffy coat of EDTA blood was added per 10 μL of isotype control (PE-CyTM 7 Mouse IgG1κ isotype control (Becton Dickinson and Company, Franklin Lakes, NJ, USA) and cocktail of human regulatory T cells (CD4/CD25/CD127) (Becton Dickinson and Company, Franklin Lakes, NJ, USA) antibody in each sample. The regulatory T (Treg) cells (CD4+CD25brightCD127dim) were analyzed by flow cytometry. Events collected using a FACSCaliburTM (Becton Dickinson and Company, Franklin Lakes, NJ, USA) were analyzed using CellQuest version 5.0, 2002 (Becton Dickinson and Company, Franklin Lakes, NJ, USA).
