Supercharged NK cells: a unique population of NK cells capable of differentiating stem cells and lysis of MHC class I high differentiated tumors

Abstract

This study highlights the significance of supercharged NK (sNK) cells in inducing the lysis and differentiation of tumors at much higher levels compared to primary activated NK cells. sNK cells-induced higher release of growth factors, cytokines, and chemokines when compared to primary activated NK cells. When we used a similar level of IFN-γ from primary activated NK cells and sNK cells, the IFN-γ secreted from sNK cells exhibited greater potential to induce differentiation in both oral and pancreatic tumors. It is long known in the field of NK cells that primary NK cells induce significant lysis of stem-like/poorly differentiated tumors, but differentiated tumors are generally resistant to primary NK cell-mediated lysis. sNK cells, unlike primary activated NK cells, are found to highly target stem-like as well as differentiated tumors, indicating sNK cells can target not only tumors specific to NK cells but also those targeted by CD8+ T cells. Differentiation by sNK cells was inhibited less by the antibodies to IFN-γ and TNF-α when compared to that mediated by the primary activated NK cells, suggesting the role of other unexplored mechanisms in sNK cell-induced tumor differentiation. Overall, this study suggests the role of sNK cells in targeting the heterogeneous population of tumors, likely mediating the functions of both NK cells and T cells in controlling tumors, and inducing them to be effectively targeted by chemotherapy.

Fig. 1. Illustration to show the generation process of supercharged NK cells.

Human peripheral blood mononuclear-derived NK cells were activated with rh-IL-2 (1000 U/ml) and anti-CD16 mAb (3 µg/ml) for 18–20 h before they were co-cultured with OCs and probiotic bacteria sAJ2 (OCs:NK:sAJ2; 1:2:4). The medium was refreshed every three days with RPMI containing rh-IL-2 (1500 U/ml).

Fig. 2. sNK cells secreted much higher levels of cytokines, chemokines, and growth factors compared to primary NK cells.

OCs were generated as described in “Materials and methods” section. NK cells (1 × 10⁶ cells/ml) from healthy individuals were treated with a combination of IL-2 (1000 U/ml) and anti-CD16 mAb (3 µg/ml) for 18 h before they were cultured with the healthy individuals’ OCs and sAJ2 at a ratio of 1:2:4 (OCs:NK:sAJ2). On day 15 of sNK cell cultures, another set of NK cells was isolated from the same healthy individuals and were treated with IL-2 (1000 U/ml). The supernatants were harvested from primary IL-2-treated NK cells and sNK cells to determine secretion levels of cytokines, chemokines, and growth factors using a multiplex assay (n = 4).

Fig. 3. sNK cells induced a significant level of killing in both stem-like and differentiated tumors.

OSCSCs (A, C), OSCCs (D, F), MP2 (G, I), and PL12 (J, L) were cultured on eSight plates for 20–24 h before primary untreated, primary activated, and sNK cells were added at 2.5:1 E:T ratio, and co-cultures were continued to 70–80 h. The graphs for normalized cell index were assessed by the eSight RTCA and RTCA pro software (A, D, G, J). Microscopic images of tumor and NK cell interactions, as shown in the figures, were captured by eSight after 24 h of incubation (scale: 0–200 µm) (C, F, I, L). 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 IL-2 (5000 U/ml) and with a combination of IL-2 (5000 U/ml) and anti-CD16 mAb (3 µg/ml) for 18 h. Primary activated NK cells and sNK cells were used as effectors against OSCSCs (B), OSCCs (E), MP2 (H), and PL12 (K) to measure NK cell-mediated cytotoxicity using a standard 4-h ⁵¹Cr release assay against tumor cells. The lytic units (LU) 30/10⁶ cells were determined using the inverse number of NK cells required to lyse 30% of target cells × 100.

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).

Fig. 5. Increased levels of IFN-γ secretion by sNK cells compared to primary activated NK cells.

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 (n = 4) (A). Fold change in IFN-γ was determined by using the formula: (left bar) IFN-γ of IL-2 + anti-CD16 mAbs-treated pNK cells/ IFN-γ of IL-2 + anti-CD16 mAbs-treated pNK cells, (right bar) IFN-γ of sNK cells/IFN-γ of IL-2 + anti-CD16 mAbs (n = 4) (B). The amounts of IFN-γ secretion shown in Fig. 5A were determined based on 1 × 10⁶ cells (n = 4) (C). Fold change in IFN-γ was determined by using the formula: (left bar) IFN-γ per million cells of IL-2 + anti-CD16 mAbs-treated pNK cells/IFN-γ per million cells of IL-2+anti-CD16 mAbs-treated pNK cells, (right bar) IFN-γ per million cells of sNK cells/IFN-γ per million cells of IL-2 + anti-CD16 mAbs (n = 4) (D).

Fig. 6. Antibodies against IFN-γ and TNF-α blocked the differentiation of tumors, an increased effect was seen in primary NK differentiated tumors compared sNK differentiated tumors.

Differentiation of OSCSCs (A) and MP2 (B) was conducted as described in Fig. 4. Primary IL-2 and anti-CD16 mAbs-treated and sNK cells’ supernatant-treated tumors were a combination of anti-TNFα mAbs (1:100) and anti-IFNγ mAbs (1:100) for six days. Freshly purified NK cells were treated with IL-2 (1000 U/mL) for 18 h and were used as effectors against untreated tumors, and primary IL-2 and anti-CD16 mAbs-treated, and sNK cells’ supernatant-treated tumors in the absence or presence of anti-TNFα mAbs and anti-IFNγ mAbs to measure NK cell-mediated cytotoxicity using a standard 4-h ⁵¹Cr release assay against tumor cells. The lytic units (LU) 30/10⁶ cells were determined using the inverse number of NK cells required to lyse 30% of target cells × 100 (A, B).

Fig. 7. Chemotherapeutic drugs induced higher killing in differentiated tumors.

Differentiation of OSCSCs (A) and MP2 (B) was conducted with an average total of 2000 pg and 5000 pg IFN-γ as described in Fig. 4. OSCSCs, OSCCs, primary NK cell-supernatant-treated OSCSCs, and sNK cell-supernatant-treated OSCSCs were treated with cisplatin (60 µg/mL) for 18–20 h, after which, the cells were stained with propidium iodide (PI) to determine percent cell death using flow cytometric analysis (A). MP2, PL12, primary NK cell-supernatant-treated MP2, and sNK cell-supernatant-treated MP2 were treated with paclitaxel (40 µg/mL) for 18–20 h, after which, the cells were stained with PI to determine percent cell death using flow cytometric analysis (B).

Fig. 8. Illustration to show the efficacy of sNK cells targeting and differentiating tumors.

sNK cells induce direct lysis of stem-like tumors, and differentiate remaining tumors via IFN-γ and TNF-α, and those differentiated tumors will be lysed by sNK cells. This process results in the complete eradication of the tumor.