Anti humancd26 percp cyanine5

CD161⁺CD26⁻Tregs (CD4⁺CD25⁺CD127lowCD161⁺CD26⁻), CD26⁺CD161⁻Tregs (CD4⁺CD25⁺CD127lowCD161⁻CD26⁺), double-negative Tregs (DNTregs; CD4⁺CD25⁺CD127lowCD161⁻CD26⁻), CD26⁺Tconv cells (CD4⁺CD25⁻CD127⁺ CD26⁺) and CD26-T conv cells (CD4⁺CD25⁻CD127⁺ CD26⁻) were sorted by flow cytometry. Subsequently, different Treg subsets were incubated with or without IL2 (50 ng/mL; R&D Systems) in 96-well plates at 37°C in the presence of 5% CO₂. Cells were stained with anti-humanCD161-APC (BioLegend), anti-humanCD26-PerCP/Cyanine5.5 (BioLegend) and anti-humanKi67-BV421(BD Horizon) and then analyzed on a flow cytometer. DN Tregs (CD4⁺CD25⁺CD127lowCD161⁻CD26⁻) were sorted by flow cytometry. Purified DN Tregs were incubated with or without IL2 in the present or absent of Dynabeads Human T-Activator CD3/CD28. STAT5 inhibitor-573108 (Millipore) was used to block STAT5 phosphorylation. See Supplementary Tables S4 and S5 for details of the flow cytometry antibodies and other reagents used.

For surface marker detection, 1 × 10⁶ or 1 million cells were washed in PBS and incubated with fluorochrome-conjugated antibodies (Abs) against and analyzed on a flow cytometer: anti-humanCD4-FITC (BD Bioscience), anti-humanCD25- PE-Cy7 (BD Bioscience), anti-humanCD127- BV421 (BD Bioscience), anti-humanCD161-APC (BioLegend), anti-humanCD26-PerCP/Cyanine5.5 (BioLegend). For apoptosis detection, Treg subsets were cultured in 96-well plate at 37°C in the presence of 5% CO₂. After 72 hours, cells were stained with PI/annexin V-APC and analyzed by flow cytometry. For Ki67 expression detection, Treg subsets were cultured in 96-well plate at 37°C and 5% CO₂ in the presence of Dynabeads Human T-Activator CD3/CD28 (Gibco). After 72 hours, cells were fix and permeabilized with reagents from Fixation/Permeabilization Solution Kit (BD Bioscience). Cells were then stained with anti-humanKi67-BV421 (BD Horizon) and analyzed by flow cytometry. See Supplementary Tables S4 and S5 for details of the flow cytometry antibodies and other reagents used.