Golgi plug protein transport inhibitor brefeldin a

IRU CAR-T cells/conventional CAR-T (made by lentivirus infection) were cocultured with irradiated recipient PBMCs (30 Gy) for 4 days in at 1:1 in X-vivo medium supplemented with 10% FBS and 50 U/ml of recombinant human IL-2 for 4 days. The Golgi Plug protein transport inhibitor brefeldin A (BioLegend) was added for the last 4 hrs of FACS detection.

All the antibodies for flow cytometry were purchased from BioLegend unless otherwise stated. Single cells from paired INT and TB tissues were pre-incubated with blocking buffer (TruStain FcX from Biolegend) for 20 min on ice, followed by stained with antibody cocktails for another 30 min on ice. The following antibodies, coupled to the appropriate fluorochromes, were used: anti-human CD3ε (OKT3), CD4 (A161A1), CD45 (HI30), CD19 (SJ25C1), CD11b (ICRF44), CD8 (DAKO; DK25), PD-1 (A17188B), CD103 (Ber-ACT8), CD14 (HCD14), CD56 (5.1H11), CD137 (4B4-1), IFNγ (4S.B3). life/dead Zombie Aqua™ Fixable Viability Kit (Biolegend) were used as viability dyes. For tetramer staining, PE-conjugated streptavidin-HLA-peptide tetramer were used. For cell sorting, Auqa⁻CD45⁺CD19⁻CD14⁻CD56⁻CD11b⁻CD3⁺CD4⁻CD8⁺CD103⁺ and PD-1⁺ cells were sorted into CSB for RNA isolation. For intracellular staining, the Golgi Plug protein transport inhibitor brefeldin A (BioLegend) was added for the last 4 h of stimulation. Cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD) and stained with antibodies against intracellular antigens for 30 min on ice. Flow Cytometry was performed on BD LSRFortessa or CytoFLEX and DxFLEX flow cytometer (BECKMAN COULTER). Cell sorting was performed on Sony SH800. Data were analyzed using FlowJo v.10 software.

For in vitro cytokine assay, 1 × 10⁵ sorted CAR-T cells were co-cultured with 5-fold irradiated Nalm6-FFLuc-GFP cells (HepG2-FFLuc-GFP for GPC3 WT/IRU CAR-T cells) in U-bottom 96-well tissue culture plates in 200 μl X-vivo medium supplemented with 10% FBS for 24 hrs. The Golgi Plug protein transport inhibitor brefeldin A (BioLegend) was added for the last 4 hrs of stimulation (n = 3 biological samples). For ex vivo cytokine assay, 0.5–5 × 10⁶ extracted bone marrow cells were stimulated by PMA/Ionomycin for 4 hrs (50 ng/ml, 1μg/ml) in the same condition as above (n ≥ 7 animals per group).