Anti humancd161 apc

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.

Samples were analyzed on the Gallios flow cytometer (Beckman Coulter), Accuri C6 flow cytometer (BD Biosciences) or LSRII flow cytometer (BD Biosciences). For HER2 testing on pancreatic cancer lines, 500, 000 cells were stained with IgG₁ PE isotype antibody (2μl, BD 340761) or HER2-PE (20 μl, BD 340552) according to the manufacturer’s instructions. For CAR T cell staining, cells were incubated for 30–60 minutes at 4° C with recombinant human HER2 Fc chimera protein (3 μl @ 0.1 μg/μl, R&D systems 1129-ER-050) and then stained with goat anti-human IgG₁ Fc-PE (0.5 μl, eBioscience 12–4998-82), anti-human CD161-APC (5μl, BioLegend, 339912, Clone HP-3G10), CD3-Flourescein isothiocyanate (FITC) (20 μl, BD Biosciences 56180) or CD8-Phytoplankton (PerCP) (20 μl, BD Biosciences 347314) according to the manufacturer’s instructions. To ensure proper compensation for the panels, Versacomp antibody capture beads (Beckman Coulter) were stained with the same antibodies (51 (link)). Data analysis was conducted on ≥10, 000 events with FlowJo version 10.0.00003 (Tree Star Inc.) for iOS.

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.