Anti cd4 clone rpa t4

To determine by flow cytometry the phenotype and cytokine profiles of vaccine-specific CD4+ and CD8+ T-cells 12 days after in vitro stimulation (IVS), patient PBMCs were re-challenged with individual peptides overnight at 37 °C, 5%CO2 in presence of Brefeldin A (51-2301KZ, BD Bioscience). The next day, the cells were washed with PBS and stained for 20 min on ice with anti-CD3 (clone SK7; # 344840, Biolegend), anti-CD8 (clone RPA-T8; #301042, Biolegend), anti-CD4 (clone RPA-T4; #558116, BD Biosciences), and a viability dye Zombie UV (#77474, Biolegend). After fixation and permeabilization for 20 min at 4 °C (BD Bioscience Cytofix/Cytoperm Kit), anti-CD154 (CD40-L, clone 24–31; #310826, Biolegend), anti-Granzyme B (clone GB11; #GRB17, Invitrogen), anti-Perforin (clone B-D48; #353310, Biolegend), anti-IL-2 (clone MQ1-17H12; #559334, BD Biosciences), anti-TNF-α (clone MAb11; #557647, BD Biosciences), and anti-IFN-γ (clone B27; #554702, BD Biosciences). All samples were acquired on a 5-laser BD FORTESSA instrument equipped with the FACS DiVa software. Analyses were performed with FlowJo v10.5 (FLOWJ,.LLC, Ashland, OR, USA) and SPICE 6 software.

Cells were stained with anti-CD4 (clone RPA-T4) and anti-CD3 (clone SK7) conjugated antibodies (BD Bioscience). Cells were then fixed and permeabilized with Cytofix/Cytoperm (BD Bioscience) followed by intracellular staining with phycoerythrin (PE)-conjugated KC57 anti-p24 antibody (Beckman Coulter).
CR-1 (E11), DARC (2C3), CD71 (OX-26), CD235a (GA-R2), CCR5 (3A9), and CXCR4 (12G5) all were purchased from BD. ROS staining (Sigma) and staining with the MitoSOX red mitochondrial superoxide indicator (ThermoFisher Scientific) were performed per the manufacturers’ protocols. Paraformaldehyde-fixed cells were acquired using a BD LSR Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo (version 10) software.

All clinical flow cytometry (FACS) was performed following good clinical practice at the Department of Clinical Immunology, Addenbrooke’s Hospital, Cambridge, UK, within 4 h of phlebotomy. Operators were blinded to the aldesleukin dose allocated. A 2.6-ml sample of peripheral whole blood was collected into EDTA tubes, and 50 μl was stained with specific fluorochrome-conjugated antibodies at room temperature for 15 min to identify Tregs as CD3⁺CD4⁺CD25^highCD127^low T cells. The clones used were anti-CD3 (clone SK7, phycoerythrin [PE]-Cy7-labelled; BD Biosciences), anti-CD4 (clone RPA-T4, FITC-labelled; BD Biosciences), anti-CD127 (clone HIL-7R-M21, PE-labelled; BD Biosciences), anti-CD25 (clone M-A251 and 2A3, allophycocyanin [APC]-labelled; BD Biosciences), anti-CD45RA (clone HI100, APC-Cy7-labelled; BioLegend), and anti-CD62L (clone DREG-56, PerCP/Cy5.5-labelled; BioLegend). Red cells were then lysed (BD FACS Lysing Solution); the cells were washed and resuspended in BD Cell Fix and then immediately analysed on a BD FACSCanto II flow cytometer utilising FACSDiva software (BD Biosciences). In parallel, a whole-blood BD Multitest 6-Color TBNK assay using BD Trucount Tubes according to the manufacturers’ instructions (BD Biosciences) was run to determine the relative and absolute concentration of lymphocyte subpopulations, including T, B, and NK cells.