Anti cd3 percp

Lymphocytes were labeled for 30 min with 5 µL of the respective monoclonal antibodies: anti-CD3 PerCP, anti-CD4 FITC, and anti-CD8 P.E. (B.D. Biosciences, San Jose, CA, USA). An isotype control (IgG1-FITC or IgG1-PE (B.D. Biosciences, San Jose, CA, USA) was used in all analyses, and cells underwent flow cytometry (FACSCalibur, BD Bioscience, San Jose, CA, USA).

NK cells were treated with the following antibodies to analyze the surface expression: anti-CD56 FITC (BD), anti-NKp46 PE (BD), anti-CD3 PerCP (BD), and anti-CD16 PerCP (Biolegend). To analyze the intracellular expression of phosphorylated NF-κB p65 peptide, NK cells were stained with surface antibodies, fixed and permeabilized according to the BD Cytofix/Cytoperm™ protocol, and were stained with PE mouse anti-NF-κB p65 (BD) antibody for 30 min. NK cell purity was monitored by NKp46⁺ and CD3⁻ gating and was consistently over 95% (21 (link), 22 (link)). For analysis of actin dynamics in live cells, cells were stained in 1 μM Live Cell Fluorogenic F-actin Labeling Probe (SiR-actin 647, Spirochrome) for 50 min. Relative CD56 and F-actin values were calculated with equations (1 (link)) and (2 (link)). Flow cytometric analysis was performed with a FACSCalibur (BD), and data were analyzed by FlowJo software (TreeStar).

Activation-induced markers were quantified via flow cytometry (FACSLyric, BD Biosciences). A surface staining on PBMC was performed with anti-CD3^PerCP (BD, clone SK7), anti-CD4^V450 (BD, clone L200), anti-CD8^FITC (DAKO, clone DK25), anti-CD45RA^PE-Cy7 (BD, clone L48), anti-CCR7^APC (R&D Systems, clone 150503), anti-CD69^APC-H7 (BD, clone FN50) and anti-CD137^PE (Miltenyi, clone 4B4-1). T-cell subsets were identified via the following gating strategy: LIVE CD3⁺ were selected and divided in CD3⁺CD4⁺ and CD3⁺CD8⁺. Within the CD4 and CD8 subsets, memory subsets were gated as CD45RA⁺CCR7⁺ (naive, T_N), CD45RA^-, CCR7⁺ (central memory, T_CM), CD45RA^-CCR7^- (effector memory, T_EM) or CD45RA⁺CCR7^- (terminally differentiated effectors, T_EMRA). T cells specifically activated by SARS-CoV-2 were identified by up-regulation of CD69 and CD137. An average of 500,000 cells was always acquired, the gating strategy is schematically represented in (Fig. S1A-J). In analysis, PBMC stimulated with MP_CD8_A and MP_CD8_B were concatenated and analyzed as a single file for SARS-CoV-2-specific responses to MP_CD8.

PFMC or PBMC were stained with LIVE/DEAD yellow fixable dead cell stain kit (Life Technology, Eugene, OR) and with the following two monoclonal antibody staining panels: In Panel 1, cells were surface-stained by anti-CD3-PerCP, CD4-PE-Cy7, CD45RO-FITC, CD69-APC, CD38-AF700 (BD Bioscience, San Jose, CA), CD8-PE-TR (Invitrogen, Frederick, MD), CCR7-APC-Cy7 and HLADR-Pacific Blue (BioLegend, San Diego, CA). Cells were then fixed, permeabilized, and washed with Transcription Factor Buffer Set (BD Pharmingen) according to the manufacturer and stained with anti-HIV-1 p24-PE (KC57) (Beckman Coulter, Indianapolis IN). In Panel 2, monoclonal antibodies included: anti CD25-APC (BioLegend), CCR5-V450 and intracellular staining for Ki67-BV711 (both from BD Bioscience) in addition to anti-CD3, CD4, CD8, CCR7, CD45RO, and HIV-1 p24 as in panel 1. Gates were set using Fluorescent-Minus-One controls for each sample. T cells were identified as naïve (CD45RO-CCR7+), central memory (Tcm) (CD45RO+CCR7+), effector memory (Tem) (CD45RO+CCR7-), and terminally differentiated effector memory (TemRA) (CD45RO-CCR7-). Stained samples were analyzed by a LSRII cytometer (BD). Data were analyzed using FlowJo (Tree Star, Ashland, OR).

Peripheral blood mononuclear cells (PBMCs) were isolated using Lymphoprep (Axis Shield, Oslo, Norway) centrifugation and stored in liquid nitrogen until further use. Cells were defrozen and subsequently fixed and permeabilized using the Foxp3 / Transcription Factor Staining Buffer Set (eBioscience, San Diego, CA, USA) and stained for flow cytometric analysis with anti-CD3-PerCP (BD Biosciences, San Jose, CA, USA), anti-CD8-APC-eFluor780 (eBioscience), anti-CD127-Alexa647 (Biolegend, San Diego, CA, USA), anti-CD25-PE (eBioscience), anti-CD45RO-Alexa700 (Biolegend), anti-CD45RA-Alexa605 (BD Biosciences) and anti-FoxP3-Alexa488 (eBioscience). Cells were measured on an LSR-II flow cytometer (BD Biosciences) and the data were analyzed with Kaluza 1.2 Analysis Software (Beckman Coulter, Woerden, The Netherlands).
Absolute numbers of lymphocytes in EDTA anti-coagulated blood were determined using the BD MultiTest TruCount method with sixcolor MultiTest reagents detecting CD45, CD3, CD4, CD8, CD19, CD16 and CD56 (BD Biosciences) according to the manufacturer’s instructions. Percentages of (functional) Treg subsets were characterized as described before [7 (link),16 (link)] and CD4+ T-cell counts were used to calculate the absolute numbers of these subsets.

To evaluate the ability of the selected epitopes to trigger the immune cells by CD8+ T cell dependent manner, PBMCs (1 × 10⁶/ml) from five HLAA2 positive VL treated subjects were incubated for overnight in the presence or absence of peptide at 37 °C and 5% CO₂. Additionally, six HLA A2 negative VL treated subjects were also considered for this study. Stimulation with SLA was run in parallel to all experiments as described previously^{50 (link)}. After overnight incubation followed by 6 h incubation with brefeldin-A (1 mg/ml), cells were harvested, washed with PBS, and stained with anti-CD3-PerCP (BD Biosciences) and anti-CD8-FITC(BD Biosciences) conjugated antibodies for 30 min at 4 °C. The cells were then washed with stain buffer, fixed and permeabilized Cytofix-Cytoperm buffer (BD Biosciences) for 20 minutes at 4 °C. The intracellular level of IFN-γ was stained with anti-IFN-γ PE-conjugated antibody (BD Biosciences) for 30 minutes and washed with perm wash buffer (BD Biosciences). A logical gate set using was used to measure the co-expression of intracellular CD8+ ve IFN-γ. At least 30 000 cells were acquired for each analysis, and the results are shown in percentage gated value (% gated).

To determine maturation status, DCs were harvested two days after addition of peptides and maturation factor LPS. Cells were stained in FACS buffer (PBS (GIBCO) containing 0.5% BSA (Sigma) and 0.5 mM EDTA (ICN Biomedicals)) for 30 minutes at 4°C with either one of two panels that contained the following maturation markers: anti-CD80-FITC, anti-CD14-PE, anti-DC-SIGN-APC, anti-HLA-DR-Pacific Blue and Live/dead-AmCyan (Invitrogen) (panel 1) or anti-CD83-FITC, anti-CD40-PE, (BD Biosciences), anti-PD-L1-APC (eBioscience), anti-CD86-Pacific Blue (BioLegend) (panel 2). Live/dead-AmCyan (Invitrogen) was included in both panels. For analysis of the co-culture, the following markers were used: anti-CD8-FITC (Sanquin), anti-CD3-PerCP, anti-TNFα-PE-Cy7, anti-IFN-γ-APC (BD Biosciences), anti-CD4-Pacific Blue (eBioscience) and Live/dead-AmCyan (Invitrogen). Four hours prior to staining, Brefeldin A (BD Biosciences) was added to the culture; then cells were stained using the Cytofix/Cytoperm kit from BD Biosciences according to manufacturer’s recommendations. Cells were measured using a FACS Canto II (BD Biosciences) and results were analyzed using FlowJo version 9.7.5 software. First, lymphocytes were gated, followed by gating of live cells, then CD3⁺ cells and finally CD8⁺ or CD4⁺ cells were placed in a quadrant with TNF-α⁺ or IFN-γ⁺ cells.