Cd56 bv510

NK cell phenotype of melanoma patients enrolled in the trial was examined using fluorochrome-conjugated antibodies against the following cell-surface markers: CD56-FITC, CD3-PC7, CD16-APC, CD69-BV421, NKp30-BV711, CXCR3-BV421, CCR3-BV510 (BD Biosciences; San Diego, CA), NKp44-PerCP eFluor 710 (eBioscience; San Diego, CA), CXCR1-PE (R&D Systems; Minneapolis, MN), CCR7-BV711 (BioLegend; San Diego, CA), and matching IgG isotype controls from the same vendors. The immune checkpoint and NK cell activation receptor panel included the following markers: Zombie NIR Fixable Viability Dye (BioLegend; San Diego, CA), CD3-PE-Vio770 (Miltenyi Biotec; San Diego, CA), ANK-1-PE (Santa Cruz Biotechnology; Dallas, TX), TIGIT-PerCP eFluor 710 (eBioscience), CD45-BUV395, CD56-BV510, CD16-BUV737, NKG2D-APC, NKp46-BV711, CD69-BV421, and PD-1-BV650 (BD Biosciences).

To analyse expression of surface markers, MCs were incubated with fluorochrome‐labeled monoclonal antibodies for 30 min in the dark on ice and fluorescence intensity was measured using a LSR II flow cytometer (BD Biosciences). CD3 V500, CD3 Alexa Fluor 647, CD4 PerCP‐Cy5.5, CD8 APC‐H7, CD14 V500, CD14 Pe‐Cy7, CD27 BV510, CD43 FITC, CD45RA PeCy‐7, CD56 BV510 (BD Biosciences) and Siglec‐1 Alexa Fluor 647 (Sanbio) were used. Relative mean intensity fluorescence (MFI) of CD43 was calculated as follows: MFI stained/MFI unstained. GFP fluorescence was used to determine replication of (GFP)‐labeled RSV.

One-step staining of cell-surface antigens was performed using fluorochrome-conjugated primary antibodies as previously described (10 (link), 18 (link), 33 (link)). For the analysis of blood NK cells two antibody panels were constructed around CD56-FITC, CD16-APC, and CD3-PE-Cy7 (BD Bioscience, San Jose, CA) antibodies. NK cell activation receptors were evaluated with CD69-BV421, NKp30-BV711 (BD Bioscience), and NKp44-PerCP eFluor 710 (eBioscience, San Diego, CA) antibodies. Chemokine expression levels were tested using CXCR1-PE (R&D Systems, Minneapolis, MN), CXCR3-BV421, and CCR7-BV510 (BD Bioscience) antibodies. For the TINK analysis, Zombie NIR (BioLegend), CD45 BUV395, CD56 BV510, CD16 BUV737 (BD Bioscience), and CD3 PE-Vio770 (Miltenyi Biotec) antibodies were used. Suitable IgG controls were acquired from the same vendors. FACS analyses were performed using the BD LSRFortessa™ cell analyzer, and analyzed using FlowJo v10 (FlowJo, LLC; Ashland, OR) software.