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

<p>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. <b>A,</b> 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>B,</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. <b>C,</b> 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. <b>D,</b> 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. <b>E,</b> 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. *, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001; ****, <i>P</i> < 0.0001. HD, healthy donor.</p>