FIGURE 4 from Anti-Vα24Jα18 TCR Antibody Tunes iNKT Cell Responses to Target and Kill CD1d-negative Tumors in an FcγRII (CD32)-dependent Manner

6B11 mAb treatment induces degranulation of iNKT cells toward K562 cells in a CD32-dependent manner. A, K562 cells were stained with anti-FC receptor (CD16, CD32, and CD64) mAbs and assessed by flow cytometry. B, E, G, iNKT cells sorted by using Vα24-FITC Ab/FITC MicroBeads were maintained in the presence of IL2 overnight, and then iNKT cells were washed twice and cultured in fresh medium with IL2 for 4–5 days. B, iNKT cells treated with the 6B11 mAb (50 ng/mL) overnight were washed and cocultured with K562 cells at an E/T ratio of 2:1 in the presence of an anti-CD32 mAb or isotype (mIgG1, 10 µg/mL). An CD107a assay was then performed. C, The indicated cell lines were stained with an anti-CD32 mAb and assessed by flow cytometry. D, CD32 was constitutively overexpressed via lentiviral transduction in A549 cells. A549 cells, which express CD32 at various levels, were stained with an anti-CD32 mAb and then assessed by flow cytometry. E, iNKT cells treated with the 6B11 mAb (50 ng/mL) overnight were washed and cocultured with A549 cells overexpressing CD32 at an E/T ratio of 2:1. A CD107a assay was then performed. F, U937 cells were stained with anti-CD16 and anti-CD64 mAbs and then assessed by flow cytometry. G, iNKT cells treated with the 6B11 mAb (50 ng/mL) overnight were washed and cocultured with U937 cells at an E/T ratio of 2:1 in the presence of anti-CD32, anti-CD64, or isotype control Abs. A CD107a assay was then performed. Data are representative of two independent experiments. Data represent the mean ± SEM. ***, P < 0.001. HD, healthy donor.