Cd56 bv605

The Raji cell line was used as target to measure ADCC capacity of PBMCs of patients and donors, as described before [39 (link)]. Briefly, Raji cells were previously labeled with PKH67 Green Fluorescent Cell Linker (Merck KGaA, Darmstadt, Germany) and then coated with rituximab (50 μg/mL) (Selleckhem, Houston, TX, USA) for 4 h. Labeled Raji cells were then cocultured for 18 h with PBMCs (1:2 ratio) from the recruited patients and healthy donors. Apoptosis of Raji cells was determined by staining with Annexin V conjugated with phycoerythrin (PE) (Immunostep, Salamanca, Spain). Cytotoxic cell populations such as natural killer (NK), NKT-like and TCRγδ+ cells were analyzed in the supernatants using specific conjugated antibodies: CD3-PE, CD56-BV605, CD16-PercP, CD8-APC H7, CD107a-PE-Cy7, and TCRγδ-FITC (BD Biosciences). Data acquisition was performed in a BD LSRFortessa X-20 flow cytometer and FACS Diva software (BD Biosciences). FlowJo software (Tree Star Inc.) was used for data analysis.

ASPCs were cultured in 10 cm dishes until they reached 90% confluence. Cells were released from the plate using Tryple (Invitrogen) and counted followed by a wash with PBS and heat inactivated FBS (FACS buffer). Cells were resuspended to a concentration of 1 × 10^{^6} cells/mL in FACS buffer and 50,000 cells were transferred to each well of a 96-well plate. Cells were stained for surface markers using the following antibodies: anti-human CD31-FITC (BD Biosciences Catalogue number: 557508), CD90-PerCP-Cy5.5 (BD Bioscience, Catalogue number: 561557), CD166-PE (BD Bioscience, Catalogue number: 559263) and CD56-BV605 (BD Bioscience, Catalogue number: 562779). Prior to the final experiment, a titration was performed to identify the optimal antibody dilution. Antibodies were combined as indicated in the figures with a final dilution of 1:50. 100 μl of the antibody mix (containing 2ul of each antibody diluted into FACS buffer) was added to each well and incubated for 20 min. Cells were washed and resuspended in wash buffer (PBS containing 2% heat inactivated FBS and 0.5 mM EDTA). A compensation plate was prepared with positive anti-mouse IgG beads together with individual antibodies for single staining including unstained cells and beads. Data was analyzed using FCS Express 7 Flow version 7.04.0014.

Due to D614 SARS-CoV-2 viruses were the majority of the earliest variants detected in Spain within clade 19B [36 (link)], a mutant clone with D614G change was created by site-directed mutagenesis in pNL4-3Δenv_SARS-CoV-2-SΔ19(G614)_Ren pseudovirus [37 (link)]. For analysis of direct cellular cytotoxicity (DCC), Vero E6 cells were infected with equal amounts of both one cycle pseudotyped viruses D614 and G614 (100 ng p24 Gag/well) and then plated onto 48-well plates. After 48 h of incubation, Vero cells were co-cultured for 1 h with PBMCs from patients and healthy donors (ratio 1:10). After detaching Vero monolayer with trypsin-EDTA solution (Sigma Aldrich-Merck, Darmstadt, Germany), caspase-3 activity was measured by luminescence using Caspase-Glo 3/7 Assay system (Promega). Cytotoxic cell populations such as Natural Killer (NK), NKT and TCRγδ+ cells were analyzed in the supernatants using specific conjugated antibodies: CD3-PE, CD56-BV605, CD16-PercP, CD8-APC H7, and TCRγδ-FITC (BD Biosciences). Data acquisition was performed in a BD LSRFortessa X-20 flow cytometer and FACS Diva software (BD Biosciences). FlowJo software (Tree Star Inc.) was used for data analysis.

For the analysis of direct cellular cytotoxicity (DCC), Vero E6 cells were infected with equal amounts (100 ng p24 Gag/well) of the most important variants of SARS-CoV-2 within clade 19B that were circulating in Spain at the time of the study: D614 and G614 [21 (link)]. After 48 h of incubation, Vero cells were co-cultured for 1 h with PBMCs from CML individuals and healthy donors (ratio 1:1). After detaching the Vero monolayer with the trypsin-EDTA solution (Sigma Aldrich-Merck, Darmstadt, Germany), caspase-3 activity was measured via luminescence using a Caspase-Glo 3/7 Assay system (Promega). Cytotoxic cell populations such as NK, NKT-like, and Tγδ cells were analyzed in the supernatants using specific conjugated antibodies: CD3-PE, CD56-BV605, CD16-PercP, CD8-APC H7, CD107a-PE-Cy7, and TCRγδ-FITC (BD Biosciences). Data acquisition was performed in a BD LSRFortessa X-20 flow cytometer and FACS Diva software v7.0 (BD Biosciences). FlowJo software v10 (Tree Star Inc.) was used for data analysis.

Frozen PBMCs were thawed in Dulbecco’s Phosphate Buffered Saline. Antibody panels and staining protocols were established prior to the experiments. Each sample was incubated with fixable viability dye (Fixable Viability Stain 780, BD Biosciences) at room temperature for 10 min to rule out dead cells. After washed twice, Fc block (BD Biosciences) was added to significantly reduce potential non-specific antibody staining. Immune cell subset distribution was assessed by flow cytometry using two panels of antibodies against cell surface markers. Lymphocyte panel: CD3 PerCP-Cy5.5 (BD Biosciences), CD4 FITC (BD Biosciences), CD8 APC (BD Biosciences), CD25 BV421 (BD Biosciences), CD127 PE (BD Biosciences), CD19 APC-R700 (BD Biosciences), CD56 BV605 (BD Biosciences). MDSC panel: CD33 APC (BD Biosciences), CD11b FITC (BD Biosciences), HLA-DR PE (BD Biosciences), CD14 PerCP-Cy5.5 (BD Biosciences), CD15 BV421(BD Biosciences). For lymphocyte panel, after incubation of 30 min at 4 °C, cells were washed and permeabilized with fixation/permeabilization buffer (Transcription Factor Buffer Set, BD Biosciences) for 45 min at 4℃, followed by intracellular staining with Ki67 PE-Cy7 (BD Biosciences) for 45 min at 4℃ and two washes. Fluorescence minus one (FMO) controls were used for Ki67 PE-Cy7, CD11b FITC and HLA-DR PE.