Anti cd45ro pe

T/CAR-T cells were centrifuged at 300×g for 5 minutes and resuspended in 100 μL PBS with 2% FBS. The T/CAR-T cells were stained at 4°C for 30 minutes with the following surface marker-specific antibodies: APC/Cy7-anti-CD25 (BD Pharmingen, San Diego, CA, USA), APC-anti-CD69 (BioLegend, San Diego, CA, USA), PE-anti-CD4 (BD Pharmingen), PE/Cy7-anti-CD8 (BioLegend), PE-anti-CD45RO (BD Pharmingen), APC-anti-CD62L (BD Pharmingen), PE-anti-PD-1 (BioLegend), PE/Cy7-anti-CTLA-4 (BioLegend), APC-anti-TIM-3 (BioLegend), and APC anti-human CD127 (BioLegend). The cells were then washed twice and resuspended in 400 μL PBS with 2% FBS for flow cytometry.

Human peripheral blood mononuclear cells (PBMCs) were isolated from freshly drawn venous blood and BM mononuclear cells (BMMCs) were obtained from BM in both of OA and RA patients by Ficoll-Paque PLUS (GE Healthcare Biosciences AB, Uppsala, Sweden) density centrifugation. To examine the phenotype of regulatory T cells (Tregs), the following antibodies were used: anti-CD4-PerCP, anti-CD25-FITC, anti-CD127-PE, anti-CD45RA-FITC, anti-CD45RO-PE and anti-CD184 (CXCR4)-PE (all from BD Biosciences, San Jose, CA, USA). Next, cells were fixed and permeabilized using the Foxp3 Staining Buffer Set followed by intracellular staining with anti-Foxp3-APC antibody (PCH101; eBioscience, San Diego, CA, USA). Cells were acquired using FACSCalibur (BD Biosciences), and the results were analyzed using CellQuest (BD Biosciences) software. The gating strategy was based on the identification of lymphocytes according to FSC and SSC signal distribution. Then, CD4⁺ lymphocytes were gated, and next, appropriate cell populations were identified. Gates were settled according to the isotype control or fluorescence minus one (FMO) for the desired marker. The dot plots shown are representative for at least six experiments done on different patients. The described gating strategy is shown in Figure 2b and was used though all the analysis of the cell phenotype presented in this paper.

Human CB T-cells, including those which were deficient in PKCζ, were maturated as previously described using phytohaemagglutinin (PHA) and IL-2 [22 (link),28 (link)]. Maturation was gauged by flow cytometry, measuring the expression of CD45RA and CD45RO using anti-CD45RA-APC, anti-CD45RO-PE, anti-CD3-FITC or isotype control mix (IgG2b-APC, IgG2a-PE and IgG1-FITC) antibodies (all BD Biosciences). The data were analysed on a BD FACScan using BD CellQuest software (BD Biosciences). Cultures were set up such that at each time point, a count was made and cell concentrations were re-adjusted based on the number of viable cells as described under ‘Results’ section.

Lymphocyte separation solution (TBD, Tianjin, China) was used to extract lymphocytes from blood samples. Cells were surface-labeled with anti-CD4-FITC (BD, California, USA), anti-CD8-PE antibody (BD, California, USA), anti-CD45RO-PE (BD, California, USA), or anti-CD38-PC5.5 (BD, California, USA) at 4 °C. The results of detection were analyzed using Kaluza v2.1.1 software.

Anti-CD3 FITC, anti-CD3 APC, anti-CD4 PerCP-cy5.5, anti-CD8-PerCP-cy5.5, anti-CD8 FITC, anti-CD45RO FITC, anti-CD45RO PE, anti-IFN-γ FITC, anti-IFN-γ APC, anti-IL-4 PE and isotype-matched control mAbs were purchased from BD PharMingen (San Diego, CA, USA). Anti-IL-22 PE was purchased from R&D Systems (Abingdon, UK). Anti-IL-17 APC was purchased from eBioscience (San Diego, CA, USA). Phorbol myristate acetate (PMA), ionomycin, saponin and Brefeldin A (BFA) were purchased from Sigma-Aldrich (Fluka, Sigma, USA).

Anti-CD4 PerCP, Anti-CD4 PerCP-cy5.5, anti-CD3 APC, anti-CXCR5-Alexafluor 488, anti-CD45RO PE, anti-CCR7 PE, anti-CD28 PE, anti-CD69 PE, anti-HLA-DR PE, anti-IL-21 PE, anti-IL-4 PE, anti-IFN-γ APC and isotype-matched control mAbs were purchased from BD PharMingen (San Diego, CA, USA). Anti-IL-17 APC and ELISA for determining IL-21 was purchased from eBioscience (San Diego, CA, USA). Anti-IL-22 PE was purchased from R & D Systems (Abingdon, UK). PMA, ionomycin (INO), saponin and Brefeldin A were purchased from Sigma-Aldrich (Fluka, Sigma, USA).