Figure 3.

NKR-2 T cells fratricide is modulated by a PI3K inhibitor or a blocking antibody. (A) MFI of NKG2D expression on NKR-2 T cells treated or not with increasing concentrations of LY294002. Data shown are mean ± SD of N = 3 independent donors. (B) Transduced T cells were cultured in complete X-vivo (100 IU/ml IL-2) supplemented or not with increasing concentrations of LY294002 for expansion. T cells were analyzed 96 h after seeding for fold expansion relative to the initial cell seeding density. Data shown are mean ± SD of N = 3 independent donors. (C) Viability of cells after cryopreservation. NKR-2 T cells were produced in presence of increasing concentration of LY294002. After production, cells were harvested, washed, and formulate for cryopreservation. After cryopreservation, cells were thawed using a water bath and resuspended in Plasmalyte/Human Serum Albumin (HSA) 5%. Cells viability was directly assessed after thawing (T0h) or after 6 h at 4°C in Plasmalyte/HSA5% (T6h) (N = 3) (D) NKR-2 T cells were produced in presence of increasing concentration of LY294002. After production, cells were harvested, washed, transferred in Plasmalyte/HSA1% (50 × 10⁶ NKR-2 T cells/ml) and stored at 4°C for 48 h. After 48 h, cell viability was assessed using trypan blue staining (N = 3) and normalized for the number of cells at cryopreservation (E) tCD19 T cells were co-cultured with NKR-2 T cells generated from the same donor in presence of increasing concentrations of blocking Ab [from 0 (–) to 10 μg/ml]. Flow cytometry analysis of CD19 positivity and viability was acquired after 44 h of incubation. Data are normalized with CD19 positivity of Mock cultured without NKR-2 T cells (N = 3). (F) Thawed NKR-2 T cells were cocultured with either PANC-1 cells or K562 cells at a 1:1 ratio, in the presence of CD314 blocking Ab, an isotype control or no Ab. Following a 24 h incubation, supernatants were harvested and IFN-γ measured (N = 3). (G) Thawed NKR-2 T cells were left in culture for 24h in the presence of an isotype control, the CD314 blocking Ab (or no Ab) and IFN-γ levels measured. Data shown are mean ± SD of N = 3 independent donors. A two-tailed unpaired t-test was used to assess statistical significance. A p < 0.05 was considered significant (*) and **p < 0.01.