Monocyte purity, proper Mo-DC differentiation and the basal state of activation of both cells were assessed by flow cytometry using the following anti-human monoclonal antibodies: CD14 (M5E2, Biolegend, San Diego, CA, USA), CD1b (SN13, Biolegend), CD1d (51.1, Biolegend), CD11c (3.9, eBioscience), CD80 (2D10, Biolegend), HLA-DR (LN3, eBioscience).
iNKT and T cell determinations were performed in total PBMCs or in CD14− fractions, using CD1d-PBS57 tetramers (NIH Tetramer Core Facility, Emory University, Atlanta, GA, USA) and the following anti-human monoclonal antibodies: CD3 (OKT3, eBioscience), CD4 (OKT4, Biolegend), and CD8 (RPA-T8, eBioscience). The purity of T cell clones VM-D5 and JS63 was assessed by using CD1d-PBS57 tetramers (NIH Tetramer Core Facility, Emory University, Atlanta, GA, USA) together with anti-human CD3 (OKT3, eBioscience) monoclonal antibody. The purity of T cell clones s33d, GG33A, and DS1C9b was assessed by using anti-human TCR Vβ13.1 (IMMV222), Vβ18 (BA62.6), and Vβ7.1 (ZOE), monoclonal antibodies from Immunotec (Immunotec Research Inc, Canada). Cells were acquired in a FACS Canto II (BD Biosciences, San Diego, CA, USA) using the BD FACSDiva™ software (BD Biosciences). Data analysis was performed with FlowJo® v10 (FlowJo LLC, Ashland, OR, USA).
iNKT and T cell determinations were performed in total PBMCs or in CD14− fractions, using CD1d-PBS57 tetramers (NIH Tetramer Core Facility, Emory University, Atlanta, GA, USA) and the following anti-human monoclonal antibodies: CD3 (OKT3, eBioscience), CD4 (OKT4, Biolegend), and CD8 (RPA-T8, eBioscience). The purity of T cell clones VM-D5 and JS63 was assessed by using CD1d-PBS57 tetramers (NIH Tetramer Core Facility, Emory University, Atlanta, GA, USA) together with anti-human CD3 (OKT3, eBioscience) monoclonal antibody. The purity of T cell clones s33d, GG33A, and DS1C9b was assessed by using anti-human TCR Vβ13.1 (IMMV222), Vβ18 (BA62.6), and Vβ7.1 (ZOE), monoclonal antibodies from Immunotec (Immunotec Research Inc, Canada). Cells were acquired in a FACS Canto II (BD Biosciences, San Diego, CA, USA) using the BD FACSDiva™ software (BD Biosciences). Data analysis was performed with FlowJo® v10 (FlowJo LLC, Ashland, OR, USA).