Cd335

The mice were subcutaneously inoculated with 5 × 10⁵ EL-4 cells on the right side of h4-1BB KI mice (supplied by Shanghai Model Organisms Center, Inc). When the average size of the tumor reached 71 mm³, randomization was performed on the basis of the tumor size. All of mice were injected intraperitoneally every 2 days. The first treatment was done in the grouping day, which was defined as day 0. On day 10, all groups (8 mice per group) were treated with PBS (vehicle control), IgG (isotype control, 10 mg/kg), and ATG-101 (1, 3.25, 13 mg/kg), and mouse tumors were collected for flow cytometry analysis. All samples were processed into single-cell suspensions and stained cells followed by flow cytometry data collecting. Tumor cells were analyzed using viability dye for live/dead cells and fluorochrome-conjugated antibodies against human CD45 (BioLegend, 103138) as a leukocyte marker. Expression of T-cell and natural killer (NK) cell markers was analyzed using fluorochrome-conjugated antibodies against CD3 (BD Biosciences, 740268), CD8 (BioLegend, 100705) and CD4 (BioLegend, 100438), CD25 (BioLegend, 102012), CD69 (BioLegend, 104536), CD44 (BioLegend, 103032), CD62 L (BioLegend, 104440), Foxp3 (eBiosciences, 12-5773-82), Ki67 (eBiosciences, 56-5698-82), and CD335 (BioLegend, 137619). The markers for different immune cell population were shown in Supplementary Table S2.