Tnfr2 apc

Cells were phenotypically analyzed using a multicolor flow cytometer Navios (Beckman Coulter, Mijdrecht, Netherlands). The following conjugated mAb were used: CD25/Pe-Cy7 (M-A251), HLA-DR/FITC (L243) (both from BD Bioscience); TIGIT/PE (MBSA43, eBioscience, Vienna, Austria), CD3/ECD (UCHT1), CD4/PE-Cy5.5 (1388.2), CD8/APC-AF700 (B9.11) (all from Beckman Coulter), TNFR2/APC (#22235; R&D), and Fixable Viability Dye eFluor780 (eBioscience). To detect the expression of mTNFα, cells were first stained with biotin-labelled Infliximab followed with APC-conjugated streptavidin (eBioscience). For intracellular staining, EZH2/PE (11/EZH2, BD Bioscience), FOXP3/eFluor 450 (PCH101), and Helios/AlexFluor 647 (22F6) (both from eBioscience) were used after fix-perm-treatment of cells, according to the manufacturer’s instructions. Isotype matched control antibodies were used to define marker settings. Data were analyzed using the software Kaluza (Beckman Coulter).

Peripheral blood samples from all participants were collected into ethylenediamine tetraacetic acid-containing tubes. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque (LiankeBio, China) gradient centrifugation, and then CD4⁺ T cells were isolated by human CD4 MicroBeads (Miltenyi Biotec, Germany) according to the manufacturer’s protocols. After blocking FcR, CD4⁺ T cells were incubated with appropriately diluted antibodies in the dark for 30 min. The antibodies used for surface staining were CD4-FITC, CD25-PerCP/Cy 5.5 (BioLegend, USA), TNFR1-PE, and TNFR2-APC (R&D systems, USA) antibodies. Isotype controls were utilized to enable correct compensation and to confirm antibody specificity. Stained cells were analyzed by flow cytometric analysis (BD science Pharmingen, San Jose, CA, USA).