Cd127 bv421

Cell surface staining was processed using whole blood lysis method. Freshly drawn heparinized peripheral blood samples were labeled with monoclonal antibody (mAb) panel for T lymphocyte subsets [anti-CD3-AlexaFlour700, -CD4-APC, -CD8-APC/CY7, -CD45-BV510, -CD45RA-FITC, -CD45RO-PE, -CCR7-PE and -HLA-DR-BV711 (all from Biolegend, San Jose, CA, USA)], mAb panel for B lymphocyte subsets [anti-CD19-APC, -CD24-PE, -CD27-PE/CY7, -CD38-AlexaFlour700, -CD45-BV510, -CD138-BV421 and -IgD-FITC (all from BD-Biosciences, San Jose, CA, USA)], and with mAb panel for Treg [anti-CD3-AlexaFlour700, -CD4-APC, -CD25-PE, -CD45-BV510, -CD127-BV421 (all from BD-Biosciences, San Jose, CA, USA)]. Following incubation, erythrocytes were lysed using FACS Lysing Solution (BD Biosciences, San Jose, CA, USA), and at least 10.000 cells were collected in CD45⁺ lymphocyte gate. The data were acquired on a NovoCyte flow cytometer (Agilent Technologies, USA) and analyzed using the NovoExpress operating system software (Agilent Technologies, USA).

Treg cells were isolated from human PBMCs and expanded as described by Ding et al.^{21 (link)}. Briefly, CD4+ T cells were negatively sorted using CD4+ T Cell isolation kit (130-096-533, Miltenyi Biotech). Enriched CD4+ T cells were stained with conjugated antibodies CD4 PerCP (1:10, 550,631, BD Bioscience San Jose, CA, USA), CD127 BV421 (1:10, 562,436, BD Bioscience), CD25 PE-Cy7 (1:10, 557,741, BD Bioscience) and CD45RO APC (1:10, 559,865, BD Bioscience) before Tregs (CD4+ CD25highCD127lowCD45RO-) were sorted on a BD Aria FACS II (BD Biosciences). The gating strategy for sorting naïve CD4⁺CD25^highCD127^lowCD45RO⁻ Treg cells is shown in the supplementary (Fig. S6A). For all donors, the purity of FACS sorted naïve CD4⁺CD25^highCD127^lowCD45RO⁻ Treg cells was above 90%. Sorted Tregs were expanded for 7 days at 37 °C using Treg expander beads (11129D, ThermoFisher) at a bead to cell ratio of 4:1 in expansion culture media (RPMI 1640 Glutamax-1, ThermoFisher + 10% heat inactivated FCS + 100 U/mL PEST + 1 mM sodium pyruvate) supplemented with 10 ng/mL recombinant human IL-2 (rhIL-2) (200-02, Peprotech, Rocky Hill, NJ) and 100 nM Rapamycin (R8781, Sigma Aldrich). After 7 days of expansion, beads were magnetically removed and Rapamycin was washed away. Tregs were expanded for another 7 days in 10 ng/mL rhIL-2 containing culture media.

For the study of the cell populations, the following monoclonal antibodies were used: Granulocyte/macrophage-colony stimulating factor (GM-CSF)-PE, CD197-PE (CCR7-PE), CD24-PE, Interferon (IFN)-gamma-FITC, CD14-FITC, CD8-FITC, CD27-FITC, CD8-APC-H7, CD4-APC-H7, CD56-APC, CD45RO-APC, CD3-BV421, CD127-BV421, CD45-V500 TNF-alpha-PerCP-Cy5.5, CD38-PE-Cy5.5, CD19-PE-Cy7, CD25-PE-Cy7, CD3-PerCP, (BD Biosciences, San Diego, CA, USA) and IL-17-APC (R&D Systems, Minneapolis, MN, USA).