Anti human leukocyte antigen

The expression of GD2 on the tumor cell lines was determined by staining with PE‐conjugated human anti‐GD2 Ab (BioLegend). Allophycocyanin (APC)‐conjugated human anti‐PD‐L1 and anti‐human leukocyte antigen (HLA) A, B, C Abs (all from BioLegend) were used for the evaluation of PD‐L1 and HLA class I on the tumor cells, respectively. Cell surface expression of GD2‐CAR on PB‐GD2‐CAR‐T cells, which has the IgG CH2CH3 spacer region, was determined using an FITC‐conjugated goat anti‐human IgG Fc fragment‐specific Ab (Merck Millipore), or FITC‐conjugated goat F(ab′)₂ anti‐mouse IgG(Fab′)₂ Ab (Abcam) for the CAR‐T cells lacking the IgG CH2CH3 spacer region. The FITC‐conjugated Ab against CD45RA, APC‐conjugated Abs against CD3, CCR7, PD‐1, and Tim3, and Alexa Fluor 647 anti‐LAG3 Ab (all from BioLegend) were used for characterizing the phenotype of CAR‐T cells. Detailed information of recombinant proteins and Abs used is provided in Table S2. All flow cytometry data were acquired using BD FACS Accuri C6 Plus (BD Biosciences) and analyzed using FlowJo Software (Tree Star).

Primary tumor specimens, xenograft tumors, and the liver of the PDX mice were dissociated using collagenase III (Worthington) and analyzed as previously described.¹⁴ Dissociated cells were stained with monoclonal antibodies conjugated to fluorescent dyes. Antibodies used in this study are anti‐human CD44‐allophycocyanin (APC, clone IM7, 1:20; Biolegend), anti‐human leukocyte antigen (HLA) A, B, C‐Alexa488 (clone W6/32, 1:20; Biolegend), anti‐mouse H‐2K^d/H2‐D^d‐biotin (clone 34‐1‐2S, 1:40; eBioscience), and anti‐mouse Cd45‐biotin (clone 30‐F11, 1:40; BD Biosciences) antibodies. Dead cells were depleted using 4,6‐diamidino‐2‐phenylindole (DAPI).