Anti hla dr apc clone g46 6

For surface HLA-DR and PD-L1 expression, single cell suspensions were initially stained with LIVE/DEAD® Fixable Near-IR Dead Cell Stain Kit (Invitrogen) to allow exclusion of non-viable cells. Cells were then stained on ice for 30 min with combinations of test (0.25 μg per antibody) or isotype-matched control antibodies in cold FACS buffer [0.5% BSA (Sigma, St Louis, MO, USA) and 0.02% sodium azide (Sigma) in PBS]. Anti-HLA-DR-APC (clone G46-6) was from BD Biosciences (San Jose, CA, USA) and anti-PD-L1-BV421 (clone 29E.2A3) was from Biolegend (San Diego, CA, USA). Cell acquisition was performed on an LSR Fortessa (BD, San Jose, CA, USA) and data analysed with FlowJo software (TreeStar, Ashland, OR, USA).

Multiparameter flow cytometry was performed using peripheral blood, as described previously (Ma et al., 2012 (link); Xia et al., 2009 (link); Zhang et al., 2017b (link); Zheng et al., 2014a (link)). For the frequency of HLA-DR⁺CD8⁺ T cells, blood was treated with lysing buffer (BD Biosciences, CA, USA) for 10 min and then incubated with a mixture of flow cytometry antibodies at 4 °C for 30 min. For staining with ki67, surface-labeled cells were further treated with fixation and permeabilization solution (BD Biosciences, CA, USA), followed by perm/wash buffer (BD Biosciences, CA, USA). Cross-reactive flow cytometry human antibodies anti-CD3 APC-Cy7 (clone SP34-2), anti-CD8 PE-Cy7 (clone RPA-T8), anti-HLA-DR APC (clone G46-6), and anti-ki67 PE (B56) were purchased from BD Pharmingen (Franklin Lakes, NJ, USA). Anti-CD4 PerCP-Cy5.5 (clone OKT4) was obtained from Biolegend (San Diego, CA, USA). Flow cytometry acquisition was performed on a BD FACSVerse^TM flow cytometer (BD, Franklin Lakes, NJ, USA) and flow cytometric data analysis was performed using FlowJo vX.0.7 (TreeStar, Ashland, OR, USA).