The combination of anti-CD3 mAb, IFN-r, and IL-2 enhances radiation-induced activating ligand expression in human PBMCs
Our previous study developed a large-scale NK cell expansion method using irradiated autologous PBMCs [26]. In this study, PBMCs isolated from healthy donors were activated by treatment with anti-CD3 mAb, IFN-r, and IL-2 and then irradiated with 25 Gy. Radiation alone and activated/irradiated PBMCs were cultured for 0, 24, 48, and 72 h, and then NKG2D, 2B4, and DNAM-1 ligand were analyzed by flow cytometry (FCM) at each time point. The cell surface expression levels were quantified using median fluorescence intensities (MFIs). Relative expression ratios were calculated by dividing the 24 h, 48 h, and 72h samples’ MFI by the untreated PBMCs’ (0h) MFI. As shown in Fig. 1, the various activating ligands that stimulate the sensitivity of NK cells showed different patterns depending on the donor and time Irradiated PBMCs significantly increased the expression levels of MICA, MICB, ULBP1, ULBP2/5/6, ULBP3, and CD155 compared to untreated PBMCs. Activated/irradiated PBMCs also significantly increased the expression of MICA, MICB, ULBP1, ULBP2/5/6, CD48, and CD155 compared to untreated PBMCs. In particular, the expression levels of MICA, MICB, ULBP1, ULBP2/5/6, and CD155 on activated and irradiated PBMCs were significantly increased compared to radiation alone. Therefore, radiation alters activating ligand expression levels in human PBMCs, and the combination of anti-CD3 mAb, IFN-r, and IL-2 further promotes these alterations.
Activated and irradiated PBMCs potently induces expansion of NK cells
To determine the expansion efficiency of NK cells by activated and irradiated PBMCs, NK cells isolated from the whole blood of healthy donors were expanded using activated and irradiated autologous PBMCs. As shown in Fig. 2, isolated NK cells expanded effectively in vitro during the culture period and expanded more than 10,000-fold at 3 weeks (Fig. 2A and 2B). In particular, T cell contamination, which can induce graft-versus-host disease (GVHD), was hardly observed in the expanded NK cells finally obtained (Fig. 2C and 2D). In addition, the expanded NK cells showed high expression of various activating receptors (Fig. 2C and 2D). Therefore, these results indicate that the combination of anti-CD3 mAb, IFN-r, and IL-2 with radiation potently promotes the expansion of NK cells by inducing the expression of various activating ligands of PBMCs.
Radiation increases expression levels of various ligands associated with NK cells sensitivity in human colon cancer cells
We evaluated the expression levels of various activating and inhibitory ligands in SW480 and HT-29 human colon cancer cells following radiation. SW480 and HT-29 cells were harvested at 0, 24, or 48 h after irradiation at 4 or 8 Gy and analyzed using flow cytometry. The cell surface expression levels were quantified using mean fluorescence intensities (MFIs). Relative expression ratios were calculated by dividing the 24 and 48 h samples’ MFI by the untreated samples’ MFI. As shown in Fig. 3, the HLA-ABC and HLA-E expression levels were increased by radiation in both SW480 (Fig. 3A and 3B) and HT-29 (Fig. 3C and 3D) cells. The EGFR expression level was increased only at 24 h after 8 Gy radiation and then the expression decreased in both SW480 and HT-29 cells. The expression levels of CD112, CD155, MICA, MICB, ULBP2/5/6 were significantly increased by radiation in both SW480 and HT-29 cells. In particular, the expression levels of MICA, MICB, and ULBP2/5/6 showed a higher increase at 8 Gy of radiation in both SW480 and HT-29 cells, and there were more significant differences at 24 h after 8 Gy radiation in HT-29 cells. Therefore, these results indicate that radiation strongly increases the various ligands that modulate the sensitivity of NK cells in SW480 and HT-29 colon cancer cells.
The combination treatment using radiation and cetuximab along with expanded NK cells efficiently enhances cytotoxic activity against human colon cancer cells
We confirmed the cytotoxic activity of expanded NK cells in the presence or absence of cetuximab and/or radiation in SW480 and HT-29 human colon cancer cells. Expanded NK cells were co-cultured with indicated and/or cetuximab-coated human colon cancer cells at various ratios for 4 h. As shown in Fig. 4, despite the high expression of MHC class Ⅰ of SW480 and HT-29 cells (Fig. 4), the expanded NK cells effectively lysed these colon cancer cells, and this antitumor cytotoxic activity was significantly enhanced by the combination treatment of radiation. The combination of expanded NK cells and radiation showed higher cytotoxic activity compared to NK alone in HT-29 (Fig. 4B) cells. However, the cytotoxic activity of the combination of expanded NK cells and radiation against SW480 (Fig. 4A) cells was relatively lower than that of HT-29 cells. This result may be due to the higher expression of MHC class Ⅰ (HLA-ABC and -E) molecules in SW480 cells by irradiation compared to HT-29 cells. Importantly, the combination treatment of radiation and cetuximab with expanded NK cells showed the strongest antitumor cytotoxic activity among all the treatments for SW480 and HT-29 cells. Taken together, these results indicate that expanded NK cells are capable of effectively remove human colon cancer cells, and the antitumor cytotoxic activity of expanded NK cells is further enhanced by cetuximab-mediated ADCC and radiation-induced activating ligands.