Ary005b

GSC40 cells transduced with TIM-3 overexpression or control lentiviral vectors were seeded into 6-well plates at a density of 1×10⁶ cells in 1.5 mL F12 medium without B27 supplement, EGF, basic-FGF, penicillin/streptomycin (Gibco) per well. After culture at 37°C with 5% CO₂ for 24 hours, the medium was collected by centrifugation and then measured with proteome profiler human cytokine array method (ARY005B, R&D), following manufactures’ instructions. Protein expression was quantified using Image J software.

CD19-CAR-T cells were washed with RPMI medium supplemented with 5% human serum, 1% glutamax, 1% MEM-NEAA, 1x penicillin/streptomycin (R-5 medium), then resuspended in R-5 medium at a concentration of 1 million/mL. Stage 7 β-like cells differentiated from INS: tdT, INS:CD19, or INS:CD19; PDL1 PSCs were washed with PBS-. Then R-5 medium with different numbers of CD19-CAR-T cells were added to β-like cells based on designated E/T ratios. After overnight coculture, medium samples were collected and centrifuged at 1500 g for 5 min, and the supernatant were analyzed with human cytokine array (R&D ARY005B) based on the manufacturer’s instructions. Cytokines showing difference between CD19-CAR-T cells cocultured with INS:tdt β-like cells and INS:CD19 β-like cells were selected to for quantification with a Luminex assay (custom assay from R&D to analyze IL2, TNFalpha, INFgamma, GM-CSF, CCL3, CCL4, and CCL5) with a Bio-Rad Bioplex3D Luminex equipment with the cytokine standards and suggested settings provided by the manufacturer.