FIGURE 2 from Anti-Vα24Jα18 TCR Antibody Tunes iNKT Cell Responses to Target and Kill CD1d-negative Tumors in an FcγRII (CD32)-dependent Manner

6B11 mAb induces degranulation of iNKT cells toward K562 cells. PBMCs isolated from healthy donors were cultured in the presence of α-GalCer and IL2 for 9–10 days, followed by sorting using Vα24-FITC Ab/FITC MicroBeads. Sorted iNKT cells were maintained in the presence of IL2 overnight and then washed. iNKT cells were then maintained in the presence of IL2 until analysis. A, Sorted iNKT cells were maintained overnight and then the culture supernatant was collected. The culture supernatant depleted with anti-mouse Ig beads or the control was added to iNKT cell cultures overnight prior to coculture with K562 cells for the CD107a assay. B, Sorted iNKT cells treated with the 6B11 mAb (10, 50, and 100 ng/ mL) or isotype mAb (mIgG1, 50 ng/mL) were cocultured with K562 cells for CD107a assays. C, Sorted iNKT cells treated with the 6B11 mAb (50 ng/mL) or isotype mAb (50 ng/mL) were cocultured with K562 cells at E/T ratios of 10:1, 5:1, and 2.5:1. An LDH release assay was then performed. D, Sorted iNKT cells treated with the 6B11 mAb (50 ng/ mL) or isotype (mIgG1, 50 ng/mL) were cocultured with K562 cells. Culture supernatants were collected for Cytometric Bead Array to assess IFNγ and TNFα production. E, iNKT cells were treated with the 6B11 mAb and anti-CD3e (UCHT1), anti-CD28, anti-CD2, or isotype control Abs overnight. iNKT cells were then washed and cocultured with K562 cells. Data are representative of two independent experiments. Data represent the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. HD, healthy donor.