Cd127 pe

Immune activation and differentiation were quantified as previously described [54] (link). In brief, one million thawed PBMC were stained with either an activation or differentiation panel for 15 minutes at 37°C prior to fixation in formaldehyde. Both panels included CD3 V450 (Becton Dickinson); CD4 PE-Texas Red (Invitrogen); CD8 Qdot605 (Invitrogen). Activation panel included HLA-DR FITC; PD-1 AF647; CD38 PE; CCR5 PE-Cy5; 45RA PE-Cy7 (all Becton Dickinson); CCR7 APC eFluor-780 (eBioscience). Differentiation panel included CD45RA PE; CD28 PE-Cy5; CCR7 PE-Cy7; CD31 FITC (all Becton Dickinson); CD57 AF647 (Biolegend); CD27 AF780 (eBioscience). For Tregs, PBMC were surface stained with CD4 PerCP, CD127 PE and CD25 FITC (all Becton Dickinson) followed by intracellular staining using eBioscience FoxP3 staining kit and FoxP3 APC as per manufacturer's instructions. Data was acquired on a BD LSR-Fortessa and analysed using FlowJo version 10.

Peripheral Blood was collected in 5 ml EDTA tubes at cycles: 1, 2, 3, 6, 9, and 12. Blood samples were taken at day 1 of each cycle before the administration of IL2 and at day 8. The phenotype of T-cells was evaluated with the following monoclonal antibodies: CD57-FITC, PE‐γδ TCR, CD3 Per-Cp, CD45RA PE-Cy7, CD45RO, CD45APC-Cy7, CD27 V500, CD25 PE-Cy7, CD4 FITC, CD69 Per-Cp, CD3 V500, CD127 PE, CD56 APC, CD8 V500 (Becton Dickinson, San Jose, CA, USA). Corresponding irrelevant isotype‐matched mouse monoclonal antibodies were used as negative controls. Briefly, 100 μl of peripheral blood are incubated for 20 min with the respective monoclonal antibodies (7 μl for FITC and PE, 5 μl for the other fluorochromes). Then 2 ml of lysant (NH4Cl) were added for erythrocyte lysis and then centrifuged at 2.1 rpm for 5’. The resulting pellet, after removal of the supernatant, was re-suspended with 1 ml of PBS. Finally, 30 × 10⁴ total events or 10 × 10⁴ events in the lymphocyte gate were acquired on a FacsCanto II, equipped with three lasers (violet 405 nm, blue 488 nm, and red 633 nm) and analyzed with FacsDiva software (BD Bioscience, Erembodegem, Belgium).

CD3 cells were isolated from 10 million previously frozen PBMCs using the EasySep Human T Cell Isolation Kit (STEMCELL Technologies). The isolated CD3 cells were stained with antibodies CD3-AF488 (BD, catalog 557694), CD4-PErCP (BD, catalog 345770), CD25-APC (BD, catalog 555434), and CD127-PE (BD, catalog 557938) and sorted with the BD FACSAria III cell sorter to isolate CD8 (CD3⁺CD4^–) and CD4 (CD4⁺CD127⁺) effector cells and Tregs (CD4⁺CD127^–CD25^hi).
The sorted CD4⁺ or CD8⁺ T cells were stained with the CellTrace Violet Cell Proliferation Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Labeled effector cells (10,000 cells/well) were seeded in a 96-well, U-bottomed plate. To induce proliferation, the Gibco Dynabeads Human T Activator CD3/CD28 kit (Thermo Fisher Scientific) was used at a 1:20 Teff cell ratio. The proliferation of Teff cells was carried out for 72 hours before 10,000 freshly isolated Tregs were added either for 24 or 48 hours. To measure the proliferation after 96 or 120 hours of incubation, the cells were stained with a CD4-PE (BD, catalog 561841) and CD8-APC (BD, catalog 561953) antibody mix. The suppressive capacity of Tregs was calculated by comparing the division of Teff cells in the presence of Tregs with the control wells without Tregs.

All chemicals and reagents were purchased from Sigma Aldrich (St. Louis, MO, United States) unless otherwise mentioned. For culturing of cells, tissue culture grade plastic ware was purchased from BD Biosciences (Bedford, MA, United States). RPMI-1640 media and fetal bovine serum (FBS) were purchased from GIBCO (Grand Island, NY, United States) for cell culture. L-pyruvate, L-glutamine, streptomycin and penicillin were from Serva (Heidelberg, Germany). Anti–mouse fluorochrome labeled antibodies (Abs): CD4-PB, CD62L-APC, CD44-PerCP-Cy5.5, CD127-PE, and Abs for ELISA were procured from BD Pharmingen (San Diego, CA, United States) or otherwise mentioned.