Cxcr5alexa647

The following fluorochrome conjugated antihuman monoclonal antibodies (MoAbs) were used for flow cytometry studies: ICOSBV421, ICOSAlexa488, CD40LBV605, CD69BV650, HLA-DRFITC, CD38APCCy7, and TNF-αAPCCy7 from BioLegend (San Diego, CA); CD3BUV395, CD4PerCPCy5.5, CD8Alexa-Fluor700, CCR7PECF594, IL-2BV711, CXCR5Alexa647, IFNγPE-Cy7, PD1BV650, CD45ROAPCH7, CD21PECy5, CD27PerCPCy5.5, IgDFITC, CD10PECy7, and CD20Alexa700 from BD Bioscience (San Jose, CA); IL-21PE, CD27PECy5, and IL-17Alexa488 from e-Biosciences (San Diego, CA); and CD45ROPE-TexasRed from Beckman Coulter (Fullerton, CA). Live/Dead Fixable Aqua Dead Cell Stain Kit and CellTrace Violet Cell Proliferation Kit were from ThermoFisher (Boston, MA). Recombinant human IL-21 (Cat #8879-IL-010) from R&D systems, purified antihuman IL-2 (Cat #3440-ON-500) and antihuman-IL-21 (Cat #MT216G/21.3m) from Mabtech, and antihumanTNF-α (Cat #502922) from BioLegend were used. Samples were acquired on a BD LSRFortessa (BD Biosciences, CA) flow cytometer and analyzed by FlowJo V10 (TreeStar, Inc).

Peripheral blood mononuclear cells (PBMCs) were acquired and then were stained for surface markers for 20 min in PBS containing fluorescent antibodies. Fluorescently labeled antibodies included CD3-PerCP-Cy5.5, CD25-PE, CD45RA-FITC, CD8-PerCP-Cy5.5, CD19-PerCP-Cy5.5 and CD56-PE-Cy7 (Tianjin Three arrows, China); CD4-APC-H7, CD8-BV510, CD127-BV421, CCR7-AF647, CD28-PE-Cy7, CD3-APC-H7, CD4-PE-Cy7, CXCR3-Alexa488, CXCR6-BV510, CXCR5-Alexa647, CCR4- BV421, PD-1-PE, CD45-APC-H7, CD27-BV421, IgD-BB515, IgM-BV510, CD38-APC, CD24-PE, and CD21-PE-Cy7 (BD, United States). The instrument settings and gating strategies were adopted from previous works (Supplementary Figure S1) (Yang et al., 2020 (link); Zhu et al., 2020 (link)). All experiments, including cell separation and sample preparation, were performed according to standardized experimental manuals. Samples were analyzed using CytoFLEX flow cytometer (Beckman, United States). Results are expressed as the proportion of cells expressing particular markers. T cell subsets, including cytotoxic T (Tc) cells, helper T (Th) cells, T follicular helper (Tfh) cells, regulatory T (Treg) cells, B cells and NK subsets were identified (Supplementary Table S1).