Flow cytometry antibodies
tumor, peripheral blood myeloid
TUMOR, SECONDARY
Both tumor cell suspensions and PBMCs were stained with two different panels of antibodies (“Lymphoid” and “Myeloid” panels).For the tumor lymphoid panel, the following mouse-anti-human antibodies were used: anti-CD3 (AF700, clone: UCHT1, Biolegend), anti-CD4 (BV650, clone: OKT4, Biolegend), anti-CD8 (PE-Texas Red, clone: 3B5, TermoFisher Scientific), anti-CD25 (BV605, clone: BC96, Biolegend), anti-CD27 (PerCP-Cy5.5, clone: M-T271, Biolegend), anti-CD45 (APC-Cy7, clone: 2D1, BD), anti-CD45RA (BV786, clone: HI100, BD), anti-CD56 (BUV395, clone: NCAM16.2, BD), anti-CD127 (AF488, clone: A01905, Biolegend), anti-ICOS (BUV737, clone: DX29, BD), anti-OX40 (PE, clone: Ber-ACT35, Biolegend), and anti-PD1 (AF647, clone: EH12.2H7, Biolegend).For the blood lymphoid panel, the following surface antibodies were used: anti-CD3 (AF700, clone: UCHT1, Biolegend), anti-CD4 (BV650, clone: OKT4, Biolegend), anti-CD8 (PE-CF594, clone: 3B5, Thermo Fisher), anti-CD25 (BV605, clone: BC96, Biolegend), anti-CD27 (PerCP-Cy5.5, clone: M-T271, Biolegend), anti-CD45RA (BV786, clone: HI100, BD), anti-CD56 (BUV395, clone: NCAM16.2, BD), anti-ICOS (BUV737, clone: DX29, BD), and anti-OX40 (PE, clone: Ber-ACT35, Biolegend). For intracellular detection of Ki67 (BV421, clone: Ki67, Biolegend), the FOXP3 fixation-permeabilization kit was used (eBioscience). For PD-1 detection at baseline, cells were directly stained with anti-PD1 (AF647, clone: EH12.2H7, Biolegend). At week 3 and week 6, cells were first incubated with an anti-IgG4 secondary antibody (Biotin, clone: HP-6025, Sigma), recognizing the Fc portion of Nivolumab, and then with AF647- conjugated Streptavidin (Biolegend).The following antibodies were used as isotype controls: BUV737 Mouse IgG1, κ (clone: X40, BD), and PE Mouse IgG1, κ (clone: MOPC-21, Biolegend).For the tumor myeloid panel, the following mouse-anti-human antibodies were used: anti-CD3 (AF700, clone UCHT1, Biolegend), anti-CD11c (BUV395, clone: B-ly6, BD), anti-CD14 (FITC, clone: M5E2, BD), anti-CD16 (BUV737, clone: 3G8, BD), anti-CD19 (AF700, clone: HIB19, Biolegend), anti-CD45 (APC-Cy7, clone: 2D1, BD), anti-CD56 (AF700, clone: B159, BD), anti-CD141 (PerCP-Cy5.5, clone: M80, Biolegend), anti- CD163 (AF647, clone RM3/1, Biolegend), anti-CD206 (PE-CF594, clone 19.2, BD), anti-EpCAM (BV786, clone 9C4, Biolegend), anti-HLA-DR (BV650, clone: L243, Biolegend), anti-ICOS-L (PE-Cy7, clone: 2D3, Biolegend), and anti-PD-L1 (BV421, clone MIH1, BD).For the blood myeloid panel, the following mouse-anti-human antibodies were used: anti-CD1c (BV421, clone: L161, Biolegend), anti-CD3 (APC-y7, clone: UCHT1, Biolegend), anti-CD11c (BUV395, clone: B-ly6, BD), anti-CD14 (AF488, clone: M5E2, BD), anti-CD16 (BUV737, clone: 3G8, BD), anti- CD19 (APC-Cy7, clone HIB19, Biolegend), anti-CD56 (AF700, clone: B159, BD), anti-CD123 (AF647, clone: 6H6, Biolegend), anti-CD141 (BV786, clone: M80, Biolegend), anti-HLA-DR (BV650, clone: L243, Biolegend), anti-ICOS-L (PE-Cy7, clone: 2D3, Biolegend), and anti-PD-L1 (PerCP-eFluor710, clone MIH1, eBioscience).The following antibodies were used as isotype controls: PE-Cy7 Mouse IgG2b, κ (clone: MOPC-11, Biolegend), PerCP-eFluor710 Mouse IgG1, κ (clone: P3.6.2.8.1, eBioscience), and BV421 Mouse IgG1, κ (clone: MOPC-21, Biolegend).In all panels, PBS(1×) + 2% FBS-diluted LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) was used for dead cell exclusions, according to the manufacturer’s instructions. For both tumor and peripheral blood myeloid panels, a 10 min incubation at +4 °C with 1:25 PBS(1x)-diluted Fc receptor binding inhibitor (eBioscience) was performed, to prevent aspecific antibody binding of myeloid cells, prior to the direct addition of the antibody mix. The samples were acquired on an LSR Fortessa Cytometer (BD) and analyzed with FlowJo software (TreeStar, version 10.8.0). Details regarding antibody dilutions, catalog numbers, validations, and other FC reagents are provided in Supplementary Table 