Fig. 4: Changes in tumor cell surface proteins induced by supernatants from supercharged NK cells compared to primary NK cells.

Differentiation of OSCSCs (A, C) and MP2 (B, D) was conducted with an average total of 2000 pg and 5000 pg IFN-γ. sNK cells were generated as described in Fig. 1, on day 15 of sNK cell cultures, another set of NK cells was isolated from the same healthy individuals and were treated with a combination of IL-2 (1000 U/ml) and anti-CD16 mAb (3 µg/ml) for 18 h. The supernatants were harvested from primary IL-2 and anti-CD16 mAbs-treated NK cells and sNK cells to determine IFN-γ secretion using a single ELISA. The volume of NK cell supernatants to treat the tumor cells was determined based on the levels of IFN-γ in the supernatants as detected by ELISA and was divided over 4 days with daily treatment. On day 5, the surface expression levels of MHC class I and CD54 on OSCSCs (A) and MP2 (B) were assessed using flow cytometric analysis. Isotype control IgG2 and IgG1 were used for MHC class I and CD54, respectively, and a single isotype control was used for untreated, primary NK cell-supernatant-treated, and sNK cell-supernatant-treated OSCSCs (A) or MP2 (B). Fold change in the MFI of CD54 (C, E) and MHC class I (D, F) was determined for NK cells supernatants treated vs. untreated OSCSCs and MP2, respectively, using the formula: MFI of pNK or sNK supernatant/MFI of untreated tumors (n = 4) (C–F). Primary NK cells were treated with IL-2 (1000 U/mL) for 18 h and were used as effectors against untreated and primary or sNK cells’ supernatant-treated OSCSCs (G, H) and MP2 (I, J) to measure NK cell-mediated cytotoxicity using a standard 4-h ⁵¹Cr release assay against tumor cells. OSCCs and PL12 were used as positive controls, as differentiated oral and pancreatic tumors, respectively (G–J). The lytic units (LU) 30/10⁶ cells were determined using the inverse number of NK cells required to lyse 30% of target cells × 100 (n = 4) (G, I). The percentage killing of the tumor by NK cells at different effector-to-target ratios is shown in the figure (H, J). One of the representative experiment data (mean ± std dev.) is shown in (H and J).