Pe anti human cd25

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation. For surface staining, PBMCs were stained for 30 min in the dark at 4°C with the monoclonal antibodies PE-CF594 mouse anti-human CD4 (BD Biosciences, San Diego, CA, USA), PE anti-human CD25 (BioLegend, San Diego, CA, USA), FITC anti-human CD161 (BioLegend), 7AAD (BD Pharmingen, San Diego, CA, USA), and CD45RO PE-Cy7 (BD Biosciences) or with FITC Mouse IgG1 κ isotype control (BioLegend) and Mouse IgG1 κ isotype control PE (eBioscience, San Diego, CA, USA). For intracellular staining of cytokines, incubate PBMCs in RPMI 1640 medium (Gibco, Life Technologies, Shanghai, China) in 5% CO₂ at 37°C, then stimulate these cells for 5 h, PBMCs were stimulated for 5 h with 50 ng/mL phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, Steinheim, Germany) and 1 μg/mL ionomycin (BD Pharmingen) in the presence of 10 μg/mL Brefeldin-A (BFA; BioLegend) and subsequently fixed and permeabilized by Foxp3/Transcription Factor Staining Buffer Set (eBioscience). Then the cells were stained for 30 min away from the light at 4°C with anti-human IL-17A APC (eBioscience) or Mouse IgG1 κ Isotype Control APC (eBioscience).

Fresh human PBMCs were stained with monoclonal antibodies, such as PercP-Cy5.5-anti-human CD3, APC/Cy7-anti-human CD4, PE-anti-human CD25, and Brilliant Violet 510-anti-human CD127 (all purchased from BioLegend, San Diego, CA, USA), in 0.5% BSA + 2 mM EDTA in PBS. Splenocytes were obtained from the spleens of mice by mechanical disruption and through a 200-gauge steel mesh. Splenocytes were lysed with 1_X red blood cells (RBCs) lysis buffer to remove RBCs. First, magnetic microbeads (Miltenyi Biotec) bead-based enrichment of CD4⁺T cells was performed. Then, cells were stained with monoclonal antibodies, such as PE-anti-mice CD4, APC/Cyanine7-anti-mice CD25, and Alexa-Fluor647-anti-mice CCR6 (all purchased from BioLegend), in 0.5% BSA + 2 mM EDTA in PBS. Human Treg cells and murine Treg cells were sorted using a SH800S cell sorter (SONY, Japan) and analyzed with the SH800 software (SONY, Japan). Human Treg cells were gated as CD3⁺CD4⁺CD25⁺CD127^- within the lymphocyte gate, which excluded cell debris, doublets, and dead cells (Supplementary Figure 2). Mice Treg cells were gated as CD4⁺CD25⁺ within the lymphocyte gate which excluded cell debris, doublets, and dead cells (Supplementary Figure 2). The purity of the sorted cell population was above 95% for human and 91% for mice, which was verified using post-sorting flow cytometry.

The PBMC cryovials were removed from storage and immediately thawed in a water bath at 37 °C. For analysis of the cell surface molecules, PBMC suspensions were prepared and incubated with the following antibodies: PreCP-Cyanine 5.5-anti-human CD3 (317336, Biolegend, San Diego, CA, USA), FITC-anti-human CD4 (357406, Biolegend, San Diego, CA, USA), APC-Cyanine 7-anti-human CD8 (344714, Biolegend, San Diego, CA, USA), PE-anti-human CD25 (302606, Biolegend, San Diego, CA, USA), PE-Cyanine 7-anti-human CD45RA (304126, Biolegend, San Diego, CA, USA), APC-anti-human CD127 (351316, Biolegend, San Diego, CA, USA), BV421TM-anti-human CCR7 (353208, Biolegend, San Diego, CA, USA) and BV785-anti-human HLA-DR (307624, Biolegend, San Diego, CA, USA). Flow cytometry data were acquired using a BD LSR Fortessa X-20 cell analyzer (BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo software version 10.10.0 (Tree Star, OR, USA). The cells were sorted into CCR7+CD45RA+ (Tnaïve), CCR7−CD45RA+ (Teff), CCR7+CD45RA− (TCM), CCR7−CD45RA− (TEM), and CD4+CD25+CD127− (Treg) subpopulations, as shown in the gating strategy (Figure 1B). Since it is difficult to make comparisons based on absolute counts, the analysis of subpopulations was based on the percentage of each population. Spanning Tree Progression of Density Normalized Events (SPADE) analysis [15 (link)] was implemented using the R package.