Anti cd27 apc

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⁺.

Flow cytometry analyses were performed on an LSR Fortessa flow cytometer (BD Biosciences). Automatic compensation was performed using CompBeads (BD Biosciences). Fluorescence minus one controls were performed to define gates of positivity. Following antibodies and reagents were used: biotinylated anti-CD11b, anti-CD33 PE-Cy7, anti-HLA-DR PE-Cy7, anti-CD15 FITC, anti-CD8 PE, anti-CD4 APC-Hy, anti-CD127 FITC, anti-CD16 FITC, anti-CD45 RO PE, andi-CD45 RA FITC, anti-CD95 PD-CF594, anti-PD-1 APC, anti CD62L APC (BD Biosciences); anti-CD14 APC-Cy7, anti-CD3 BV 605, anti-CD3 PE/Dazzle 594, anti-CD4 PE-Cy7, anti-CD4 PerCPCy5.5, anti-CD56 PE-Cy7, andi-CD28 FITC, anti-CD27 APC, anti-ICOS APC-Cy7, anti-CD137 PE, anti-CD137 APC, Zombie Yellow Fixable Viability Kit (BioLegend); eFluor 450 labeled streptavidin, anti-CD8 APC-H7, anti-Foxp3 APC, anti-CCR7 APC, anti-TIM3 eFluor 450, anti-TIGIT PE, anti-LAG3 PerCPeFluor710 (eBioscience), anti-CD25 PE (Myltenyi Biotec) and anti-OX40 APC (R&D Systems).

PBMCs (1 × 10⁶ cells) were incubated with anti-CD19-PEDy647, anti-CD38-PE, anti-CD41a-FITC, anti-CD41a-PE (Immunotools, Friesoythe, Germany), anti-CD19-PECy7, anti-CD27-APC, anti-BAFFR-FITC, anti-TACI-PE (BioLegend, San Diego, USA), anti-CD86-PE, anti-IgG-PE, anti-IgD-FITC (BD), anti-IgA-FITC, and anti-IgM-PE (Dako, CA, USA) mAbs and the corresponding isotype controls. Whole blood (100 μl) was incubated with anti-CD4-PECy7 (BioLegend), CD5-PE (BD Biosciences), CD19-PEDy647, and CD41a-FITC (Immunotools). Red blood cells were lysed, and white cells fixed using BD FACS lysing solution (BD Biosciences) to be analyzed by flow cytometry.

Fresh CSF was transported at 4°C and centrifuged at 1,200 rpm for 10 min immediately. The cells were suspended with 500 μl cryopreservation solution (90%FBS +10%DMSO) and stored at −80°C. Recombinant Protein NR1 (OriGene) was labeled by lightning-link ®FITC (Expedeon) through incubation for 3 h in dark conditions. Before flow cytometry, the cells were thawed into 1 ml FBS (4°C) +15 ml 1640 medium (4°C) and centrifuged at 250 g for 10 min. The supernatant was discarded. Then 1 ml Cell Staining Buffer (Biolegend) and fluorescent antibodies were added. All kinds of antibodies (anti-CD20-percp/Cy5.5, anti-CD27-APC, anti-CD38-PE, Biolegend; NR1-FITC) were added with 1 μl to the cell suspension.
4',6-diamidino-2-phenylindole (DAPI) (3 μmol/L, add 500 μl/tube) staining for about 5 min. Cells were sorted into 8-strip PCR tubes, and each tube contained 4 μl ice-cold cell lysis buffer: 1.86 μl nuclease-free water, 1 μl Oligo dT18 (10 μmol/L), 0.1 μl RNase inhibitor (4U, Applied Biosystems), 0.04 μl Triton X-100 (100 ml/L, Sigma), and 1 μl dNTPmix (10 mm). Using BD FACSAria IIIu flow cytometry, target cells (CD20+NR1+) with negative and weak positive DAPI (indicating the cells were still alive) were selected and placed in liquid nitrogen immediately.

Polyclonal antibody (pAb) Goat anti-human Ig-PE (cat# 2040-09, Southern Biotech, Birmingham, AL, USA), monoclonal antibodies (mAb): anti-CD27-APC (cat# 302810, clone O323, BioLegend, San Diego, CA, USA) (also used as CD27 mAb), anti-EGFR-FITC (cat# sc-120 FITC, clone 528, Santa Cruz Biotechnology, Dallas, TX, USA), anti-CD25-APC (cat# 302610, clone BC96, BioLegend), anti-CD4-FITC (cat# 300506, clone RPA-T4, BioLegend), anti-CD8-Brilliant Violet 421 (cat# 344748, clone SK1, BioLegend), mouse (IgG2A) (mAb 425) (Cat# EWI020, Kerafast, Boston, MA, USA), anti-Myc mAb Alexa Fluor 647 (cat# 2233, clone 9B11, Cell Signaling, Danvers, MA, USA). Atezolizumab was obtained from the pharmacy of the UMCG (Groningen, the Netherlands).

The following antibodies were used for the flow cytometric analysis: anti-CD57-FITC, anti-CD28-PE, anti-CD4-PerCP, anti-CD8α-PerCP, anti-CCR7-PE-Cy7, anti-CD27-APC, anti-CD3-APC-Cy7, anti-CD45RA-APC-Cy7, anti-CD3-Pacific Blue, and anti-CD3-Pacific Blue (BioLegend, U.S.). A LIVEDEAD™ Fixable Aqua Dead Cell Stain kit (Thermo Fisher Scientific, U.S.) was used to monitor the cell viability. All flow data were acquired using FACS Canto II (Becton Dickinson), and the analysis was performed using FlowJo software ver.10.8.0 (Tree Star, U.S.).