Anti cd3 percp cy5

The following antibodies (Abs) were used for short-term culture or surface marker and intracellular cytokine staining for flow cytometry (all Abs were from Biolegend): anti-CD3-PerCP-cy5.5 (Clone UCHT1), anti-CD8-APC-Cy7 (Clone SK1), anti-CD4-PE-Cy7 (Clone RPA-T4), anti-Vδ₁-APC (Clone REA173), anti-Vδ₂-PE (Clone B6), anti-Vγ₂-FITC (Clone 7A5), anti-TNF-α-PE (Clone MAb11), anti-IFN-γ-PE-Cy7 (Clone 4S.B3), anti-interleukin-17A (IL-17A)-PE-Cy7 (Clone BL168), anti-Granzyme A-PE (Clone CB9), anti-Perforin-APC (Clone dG9), anti-granulocyte macrophage colony-stimulating factor (GM-CSF)-APC (Clone BVD2-21C11). The isotype control mAbs were purchased from the related company, respectively. The reagents listed below were all commercial products: brefeldin A (GolgiPlug, BD Biosciences), Cytofix/Cytoperm, and Perm buffer (BD Biosciences). The isotype control mAbs were purchased from the related company, respectively.

For mouse cell phenotype analysis, anti CD3-percp/cy5.5, anti CD4-FITC, anti CXCR5-allophycocyanin, anti ICOS-PE, and anti PD-1-PE were purchased from BioLegend (San Diego, CA, USA). Anti CD19-FITC, anti CD138-PE, anti IgD-allophycocyanin, anti CD27-percp/cy5.5, and relevant IgG isotypes were purchased from eBioscience (San Diego, CA, USA).
For human cell analysis, anti CD3-FITC, anti CD4-percp/cy5.5, anti CXCR5-allophycocyanin, anti PD-1-PE, and anti CD19-PE were purchased from BioLegend. Anti-IL-21 neutralizing antibody (eBioscience) and anti-CD40 (BioLegend) were used for functional analysis in vitro.

CB-NK cells were cryopreserved using a freezing medium consisting of 50% Plasmalyte (Baxter International Inc.), 40% human AB serum and 10% DMSO (Thermo Scientific) at 2x10⁶ cells per ml per vial. NK cells were thawed in a bath at 37°C, centrifuged at 362g for 5 minutes and resuspended in “NK medium”. The next day, cells’ viability was checked by flow cytometry. In order to test their functionality, thawed CB-NK cells were co-cultured with K562 target cells at a ratio of 1:1 in a 24-well plate for 4 h at 37°C. At the beginning of the assay, anti-CD107a-BV421 (BD Biosciences, clone H4A3) was added in order to detect the degranulation activity of the effector cells against the target cells. Golgi Stop (BD Biosciences) (monensin) was added following the manufacturer’s protocol. After the incubation, cells were collected, washed, and labeled with anti-CD3-PerCP/Cy5.5 (Biolegend, clone SK7), anti-CD56-APC (Miltenyi Biotec, clone REA196) and analyzed using flow cytometry. Degranulating NK cells, characterized by the expression of CD107a were determined in the CD56+/CD3− cell population.

Human T cells were surface stained with anti-CD3 Percp-cy5.5, anti-CD4 FITC, anti-CD8 BV510, anti-CD27 APC, anti-CD45RA PE-Cy 7 (Biolegend). CD4⁺/CD8⁺ T cells were sorted as CD3⁺CD4⁺/CD3⁺CD8⁺ cells. Central memory T cells were stained with CD27⁺CD45RA⁻. Effector memory T cells were stained with CD27⁻CD45RA⁻. Naïve T cells were stained with CD27⁺CD45RA⁺. Terminally T cells were stained with CD27⁻CD45RA⁺.

Fresh PBMCs were isolated from whole blood by density-gradient centrifugation using the BD Vacutainer CPT Mononuclear Cell Preparation Tubes with Sodium Heparin (BD Biosciences, Franklin Lakes, NJ, USA). The cells were first stained using the Zombie Yellow™ Fixable Viability Kit (BioLegend, San Diego, CA, USA) and then with combinations of the following monoclonal antibodies against human cell-surface antigens for 30 min on ice: anti-CD11c-Alexa700, anti-HLADR-V500, anti-CD19-APC-H7 (all from BD Biosciences), anti-CD14-ECD, anti-CD56-APC, (both from Beckman Coulter, Brea, CA, USA), anti-CD123-FITC, anti-CD3- PerCPCy5.5, anti-CD56-BV421 (all from BioLegend), and anti-CD19-PE (TONBO Biosciences, San Diego, CA, USA). pDCs were identified as CD3^-CD19^-CD14^-CD56^-HLADR⁺CD11c^-CD123⁺(Additional file 2: Figure S1). Data were acquired on a FACS LSR Fortessa (BD Biosciences) and the percentages of each cell population and mean fluorescence intensity were analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA).