Anti pd 1 pe cy7

PD1 expression on T-cells was blocked by incubating with 20 µg/mL Nivolumab (anti-PD1, MedChemExpress, NJ, USA) for 30 min at 37 °C. After incubation, cells were washed twice with PBSx1 supplemented with 0.5% BSA and 0.1% sodium azide, then washed once with PBSx1 before being centrifuged at 1500 rpm for 5 min. Next, cells were incubated with Fc Block (Milteny Biotech, Bergisch Gladbach, Alemania) for 30 min at 4 °C and then washed with PBSx1 supplemented with 0.5% BSA and 0.1% sodium azide before being centrifuged at 1500 rpm for 5 min. The cells were then subjected to surface staining using anti-Nivolumab PE (Anti-Human IgG4 pFc, Abcam, Cambridge, United Kingdom), anti-PD1 PE-Cy7 (Clone: EH12.1, BD Pharmingen, San Jose, CA, USA), and CD3 PERCP (Clone: HIT3a, BD Pharmingen) [27 (link)]. The corresponding isotypes and tube with cells without previous Nivolumab incubation were used as controls. PD1 expression and Nivolumab binding through human IgG4 were evaluated by flow cytometry using FACSCanto II (BD Biosciences) and analyzed using FlowJo (Tree Star, Ashland, OR, USA).

Thymocytes and peripheral T lymphocyte subsets from NOD, with or without MK626 treatment, were stained using an optimized panel of fluorochrome-conjugated monoclonal antibodies (mAb) (shown in Table 1). Specifically for T cell regulatory subset, T cells from spleen and PLNs were stained with anti-CD4-APCCy5.5 (BD), anti-CD25-PerCP (BD) and anti-FoxP3-eF450 (EBiosciences). Also T cells were stained with anti-CD8-V500 (BD), anti-PD1-PeCy7 (BD) and anti-CD122-biotin (BD). SA-APC (BD) was used as a secondary antibody. Analyses were run on a FACS Canto II flow cytometer (BD).

EDTA-treated whole blood (50 μL) was stained with 20 μL of BD Multitest 6-color TBNK reagent in Trucount tubes (BD Biosciences. San Jose, CA, USA) for 15 min. After lysis of red blood cells, T, B and NK lymphocyte subsets were enumerated with a FACSCanto II flow cytometer, and data were analyzed using FACSDiva software (BD Biosciences) [55 (link)].
For the analysis of T helper cell subsets after vaccination, EDTA-treated whole blood was incubated with: anti-CD3-PerCPy5.5 and anti-CD4-KO (Beckman Coulter); anti-CXCR3-PE, anti-CCR6-PB and anti-PD-1-PECy7 (BD Biosciences). Acquisition was performed with a Navios flow cytometer (Beckman Coulter), and data were analyzed with FlowJo software v10.6.2. Th1 cells were defined as CD4+CXCR3+CCR6-, Th2 cells as CD4+CXCR3-CCR6- and Th17 cells as CD4+CXCR3-CCR6+. PD-1 was considered as a recent activation marker.

Lymphocytes from human thymi, cord blood, lymph nodes (mesenteric and pancreatic), spleen and blood samples were cell surface stained with anti‐CD3‐AF700 (557943)/anti‐CD3‐PE‐CF594 (562280), anti‐CD19‐BV570 (BioLegend, San Diego, CA, USA; 302236), anti‐CD4‐APC‐H7 (560158)/anti‐CD4‐BV650 (563875), anti‐CD8‐BV421 (BioLegend, 301035)/anti‐CD8‐BV650 (563875)/anti‐CD8‐PerCP‐Cy5.5 (565310), anti‐CD27‐BV711 (563167), anti‐CD45RA‐FITC (555488), anti‐PD‐1‐PE‐Cy7 (561272) antibodies (all from BD Biosciences, San Jose, CA, USA), unless otherwise stated with their catalogue number in brackets) and LIVE/DEAD (Aqua, Near‐IR, Molecular Probes, Eugene, OR, USA) or 7‐amino‐actinomycin D (Sigma‐Aldrich, Darmstadt, Germany) on ice for 30 min in PBS. Human PBMCs were sorted for CD27⁺CD45RA⁺ naïve or CD27⁺CD45RA⁻ T_CM CD4⁺ and CD8⁺ T cell populations using FACS Aria III (BD Biosciences). Alternatively, cells were fixed and permeablized using the eBioscience Foxp3 staining kit (ThermoFisher Scientific, Waltham, MA, USA) then intracellularly stained with anti‐SATB1‐AF647 (BD Biosciences, 562378) and anti‐GzmB‐AF700 (BD Biosciences, 560213). Samples were acquired using a BD LSR Fortessa (BD Bioscience) and analyzed using FlowJo software (V10.4.2 and 10.5, Treestar, Ashland, OR, USA) and GraphPad Prism (GraphPad Software, La Jolla, CA, USA, V7.02).

Whole blood was permeabilized and fixed using cytofix/cytoperm (BD Pharmingen, San Jose, CA, USA) according to manufacturer’s protocol followed by staining for 20 min at room temperature in the dark with cocktail of antibodies including anti-CD4-APC, anti-CD25-FITC, anti-FOXP3-PE, anti-PD1-PeCy7, anti-IL10-APC, anti-CD127-APC, anti-CD8PeCy5, anti-NOTCH1 PE, and anti TGF-β APC (BD Pharmingen, CA, USA). After staining, RBCs were lysed using BD FACS lysing solution (BD Pharmingen, San Jose, CA, USA) as per manufacturer’s instructions.
Anti-Notch1 PE antibody (clone mN1A) was procured from eBiosciences, CA, USA; mN1A antibody reacts with the intracellular domain of human Notch1. The mN1A antibody has a low affinity for the full-length (unprocessed or heterodimeric cell surface) forms of Notch1. Therefore, Notch1 expression was considered intracellular not surface expression.
More than 50,000 cells were acquired for flow-cytometric analyses on BD FACS Caliber and the results were analyzed using the TreeStar Flow-Jo software version 8.8.7.

Probe-specific single B cells were sorted as previously described [27 (link)]. Briefly, one tube of PBMCs was thawed in water at 37 °C and resuspended in a prewarmed RPMI-1640 (Corning, Corning, NY, USA) containing 10% fetal bovine serum (Sera Pro, Gremlingen, BW, Germany) and 50 IU/mL benzonase (Sigma, St. Louis, MO, USA). After washing with PBS, the PBMCs were stained with a LIVE/DEAD dye (Invitrogen, Carlsbad, CA, USA) on ice for 20 min. After washing, the PBMCs were stained with an antibody cocktail containing anti-CD3-Pacific Blue, anti-CD8-Pacific Blue, anti-CD19-BV510, anti-IgM-PercpCy5.5, anti-IgG-FITC, anti-CD27-APC-Cy7, anti-PD-1-PECy7, anti-CXCR5-APC-R700, anti-CXCR3-PECy5, anti-CD45RA-BV650, anti-CD4-BV605 (above antibodies are all from BD Biosciences), anti-CD14-eflur450 (ebioscience, USA), anti-CD20-ECD (Beckman Coulter, Bria, CA, USA) and RBD-PE. After filtering through a cell strainer (Corning, Corning, NY, USA), the PBMCs were sorted on BD FACS Aria SORP. RBD-specific single memory B cells were gated as CD3⁻, CD8⁻, DAPI⁻, CD14⁻, CD19⁺, CD20⁺, CD27⁺, IgM⁻, IgG⁺, RBD-PE⁺, and were sorted into a 96-well PCR plate containing cell lysis buffer. Then, the PCR plate was frozen immediately at −80 °C and stored overnight.