Cxcr3 bv421

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).

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.

To evaluate CD4 T-naïve differentiation, the lymphocyte component of the ascites was analyzed in all culture conditions from 3 patients. Specifically, after 72 h of co-culture with hA-MSCs, the maturation status (naïve, central memory, CM; effector memory, EM; terminal effector memory, TEM) of CD4+ cells was analyzed and compared to the control lymphocytes. We phenotyped T cells using a panel of markers that distinguish lymphocyte cell subsets in T helper CD4+ Th1/Th2/Th17. In detail, we analyzed the CD4+ T cells’ differentiation states using different marker combinations, CD4+ naïve (CD45+CCR7+), CD4+ CM (CD45RA-CCR7+), CD4+ EM (CD45RA-CCR7-), and CD4+ TEM (CD45RA+CCR7-), and the lymphocytes cells subsets in T-helper CD4+ using specific marker combinations, Th1 (CD4+CXCR3+CCR4-), Th2 (CD4+CCR4+CCR6-), and Th17 (CD4+CCR4+CCR6+). The cells were labeled with CD45 APC-Cy7, CD3 BV510, CD4 BV786, CD45RA PE Cy-7, CCR7 BV711, CXCR3 BV421, CCR4 APC, and CCR6 BB515—all from BD Biosciences (BB: Brilliant Blue, BV: Brilliant Violet). Analyses were conducted using a BD FACS Celesta SORP instrument, FACS Celesta SORP flow cytometer, and FACS Diva software version 9.0 (BD Biosciences, CA, USA).