Cd127 percp cy5

Cell surface proteins on (CAR-)T cells were stained with the following antibodies: annexin V-PE, CCR7-PerCP/Cy5.5, CD3-allophycocyanin (APC), CD3-BV421, CD4-APC/H7, CD8a-PE/Cy7, CD25-PE/Cy7, CD69-fluorescein isothiocyanate (FITC), CD95-PE, CD127-PerCP/Cy5.5, CXCR3-AF488 (all from BD Biosciences, San Jose, CA); CD8a-APC, CD8a-FITC, CD27-PE, CD28-FITC, CD45RA-APC, LAG-3-PE, TIGIT-PerCP/Cy5.5, TIM-3-FITC (all from BioLegend, San Diego, CA); and PD-1-APC (eBioscience, Thermo Fisher Scientific). Monocytes were stained with CD14-PE (eBioscience). To determine CAR expression, cells were labeled with a recombinant Fc-tagged BCMA protein (Enzo Life Sciences, Farmingdale, NY) followed by a BV421-conjugated anti-Fc antibody (BioLegend). Samples prepared from mouse organs were treated with Fc block (BD Biosciences) prior to staining. Cells were washed and resuspended in fluorescence-activated cell sorting (FACS) buffer (1% FCS in PBS) or 1% paraformaldehyde prior to acquisition. In experiments to assess viability and/or apoptosis, cells were resuspended in FACS buffer containing 1 μg/mL 7-aminoactinomycin D (7-AAD). In all other experiments, live cells were gated based on forward and side scatter.
All flow cytometry data were acquired on a FACSCanto II (BD Biosciences) and analyzed using FlowJo software version X.0.7 (Treestar, Ashland, OR).

To determine the percentage and the absolute count of CD3 and CD4 T cell subsets, 50 μL of whole marrow blood was stained with CD45 PerCP-Cy™5.5, CD3 FITC, CD4 PE-Cy7™, CD8 APC-Cy7, CD16 and CD56 PE, and CD19 APC monoclonal antibodies (MoAbs) (BD Multitest 6-color TBNK) in a calibrated number of fluorescent beads (Trucount, BD Pharmingen). For Treg identification, 100 μL of marrow blood was incubated with a lyophilised pellet of CD45RA FITC, CD25 PE, CD127 PerCP-Cy 5.5, HLA-DR PE-CY™7, CD39 APC, and CD4 APC-H7 MoAbs (BD Pharmingen). Samples were processed according to the manufacturer's guidelines and acquired on a DB FACSCanto II Flow Cytometer. The absolute number (cells/μL) of positive cells was calculated by comparing cellular events to bead events using BD FACSCanto clinical software (version 3).

We analyzed the magnitude, breadth, polyfunctionality, and memory phenotype of the adaptive and memory HCV-specific CD4⁺ and CD8⁺ T cell immune responses by Intracellular Cytokine Staining (ICS), as previously described [24 (link),25 (link)]. Briefly, spleens were collected from immunized mice and processed to obtain splenocytes. Then, splenocytes were stimulated for 6 h at 37 °C with 1 μg/mL of different HCV peptide pools of the HCV genotype 1a, H77 strain and stained with the appropriate fluorochrome-conjugated antibodies against different surface markers: CD4-APC-Cy7, CD8-V500, CD62L-Alexa700, CD127-PerCP-Cy5.5, and CD107a-FITC (all from BD Biosciences, Franklin Lakes, NJ, USA). Subsequently, cells were fixed and permeabilized with Cytofix/Cytoperm kit (BD Biosciences, Franklin Lakes, NJ, USA) and the intracellular cytokines were stained using the appropriate fluorochrome-conjugated antibodies: IFN-γ-PECy7 (BD Biosciences, Franklin Lakes, NJ, USA), TNF-α-PE (eBioscience, San Diego, CA, USA) and IL-2-APC (BD Biosciences, Franklin Lakes, NJ, USA). Cells were passed through a Gallios flow cytometer (Beckman Coulter, Brea, CA, USA) and data analysis was carried out using the FlowJo (version 8.8.7) program.

The immunochemical reagents used were as follows: anti-human IgG, Fcγ-PE (1/200 dilution, #109-116-170; Jackson Immunoresearch, West Grove, PA, USA), CD3-BV711 (1/250 dilution, #300463; BioLegend, San Diego, CA, USA), CD4-BV510 (1/100 dilution, #344633; BioLegend), CD25-BV421 or CD25-BV605 (1/40 dilution, #302629 or #302631; BioLegend); CD127-PerCP-Cy5.5 (1/40 dilution, #560551; BD Biosciences, Franklin Lakes, NJ, USA), TNFR2-PE (1/70 or 1/100 dilution, #22235; R&D Systems, Minneapolis, MN, USA), and Foxp3-AlexaFluor 647 (1/100 dilution, #320213; BioLegend). Cells were treated with PBS containing 0.2% sodium azide and 5% FBS. For the antibody-binding analysis of TNFR2-expressing Ramos-Blue and HEK293T cells, primary antibodies were first incubated in a dilution series for 30 min on ice and then labeled with anti-human IgG-PE. For quantitation, BD Quantibrite™ PE Phycoerythrin Fluorescence Quantitation Kit (BD Biosciences, lot #76536) was used. For the multi-color labeling experiment, fluorescent intensities were compensated using BD™ CompBeads Anti-Mouse Ig, κ/Negative Control Compensation Particles Set (BD Biosciences), and the performance of two anti-CD25 antibodies was confirmed to be equivalent. The cells were analyzed using a BD LSRFortessa Cell Analyzer (BD Biosciences) and data was analyzed using FlowJo_v10.8.1 (FlowJo, LLC).

CD127 was measured by flow cytometric assays. Lymphocytes were stained according to manufacture instructions for CD3-FITC (clone: HIT3a), CD127-PercP-Cy5.5 (clone: HIL-7R-M21), CD4-APC (clone: RPA-T4) from BD Pharmingen and corresponding isotype controls. Briefly, the staining for surface markers CD3, CD4 and CD127 was performed and cells were incubated for 30 minutes followed by fixation using BD FACS Lysing solution BD Biosciences.