X-ray as Irradiation Alternative for K562 Feeder Cell Inactivation in Human Natural Killer Cell Expansion

Materials and Methods

Cell lines. K562 (human myelogenous leukemia cell line) and Raji (human Burkitt's lymphoma cell line) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). These cell lines were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum FBS (Gibco, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin (Lonza, Walkersville, MD, USA) at 37°C in a humidified incubator containing 5% CO₂.

Cytokine and antibodies. Recombinant human IL-2 and IL-15 (PeproTech, Rocky Hill, NJ, USA) were used to expand NK cells from PBMCs. AmCyan-conjugated CD45 (Cytognos, Salamanca, Spain), PE-Cy7-conjugated CD56, APC-Cy7-conjugated CD3, Pacific blue-conjugated CD16, fluorescein isothiocyanate (FITC)-conjugated CD57, APC-conjugated NKG2A, PE-conjugated NKG2C (R&D System, Minneapolis, MN, USA), PerCP-conjugated CD8a, Pacific blue-conjugated NKp46, PE-conjugated NKp30, FITC-conjugated CD62L, PerCP-conjugated NKG2D (eBioscience, San Diego, CA, USA), and APC-conjugated DNAM-1 (BD Biosciences, San Jose, CA, USA) antibodies were used to examine purity and the surface expression of NK cell receptors. (PE)-conjugated antibody to human HLA-ABC (BD Biosciences), MICA, MICB, ULBP1, ULBP2, ULBP3 antibodies and isotype controls (R&D System) were used to compare marker expression on K562 cells following 4 h and 24 h treatment with either X-ray or γ-irradiation.

Expansion of NK cells from PBMCs. Our Institutional Review Board (No. SMC 2017-11-051) approved this study and no data were used for personal identification of human PBMCs. PBMCs were isolated from healthy adult donors using density-gradient centrifugation with Ficoll-Hypaque (d=1.077, Lymphoprep™; Axis-Shield, Oslo, Norway) and washed twice with phosphate-buffered saline (PBS). The expansion process was performed as previously described (11). Freshly isolated PBMCs (3×10⁶ cells) were co-cultured with 50 Gy-γ-irradiated K562 cells or 50 Gy-X ray-irradiated K562 cells (0.5×10⁶ cells) in a 24-well tissue culture plate in the presence of 10 U/ml recombinant human IL-2 in complete RPMI-1640 medium (RPMI-1640 supplemented with 10% FBS, 4 mM-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin). The medium was replaced every 2-3 days with fresh medium containing 10 IU/ml human IL-2. After 7 days of culturing, the concentration of IL-2 was increased to 100 IU/ml, and 10 IU/ml IL-15 was added to the medium. The medium was replaced every 2–3 days.

NK cell receptors and K562 ligands analysis. The surface expression of NK cell markers after the expansion period was analyzed by flow cytometry. Briefly, 2×10⁵ cells were washed with 1X PBS and 1% FBS and stained with an appropriate combination of fluorochrome-conjugated monoclonal antibodies (CD45, CD56, CD3, CD16, NKp46, NKp30, CD8a, NKG2D, NKG2A, NKG2C, and DNAM-1) for 15 min. To detect NKG2D ligands on the cell surfaces following K562 treatment with either X-ray or γ-irradiation, K562 cells (1×10⁵) were stained for HLA-ABC, MICA, MICB, ULBP1, ULBP2, and ULBP3 as well as appropriate isotype controls. The cells were washed twice with 1X PBS and 1% FBS and fixed in 1X PBS and 1% formaldehyde before acquisition on the FACS Cano II (BD Biosciences). Data were analyzed by Kaluza (Beckman Coulter, Brea, CA, USA).

Evaluation of viability of K562 cells after X-ray or γ-irradiation. X-ray treated and γ-irradiated K562 cells were maintained in culture in complete RPMI-1640 medium without PBMCs for 10 days to evaluate apoptosis induction. Cell viability was examined on day 0, 3, 7, and 10 using the FITC Annexin V/Dead Cell Apoptosis Kit (Invitrogen, Eugene, OR, USA), according to the manufacturer's instructions. Briefly, irradiated K562 cells were washed in cold PBS. Cells were suspended in 100 ml of 1X annexin-binding buffer containing 5 μl of FITC annexin V and 1 μl of the propidium iodide (PI) solution. The cells were incubated at room temperature for 15 min; subsequently, 400 μl of 1X annexin-binding buffer was added to the samples for flow cytometry.

The percentage of non-replicating K562 feeder cells survived following X-ray treatment or γ-irradiation during NK cell expansion. To calculate the percentage of non-replicating K562 feeder cells survived following X-ray treatment or γ-irradiation during NK cell expansion, X-ray treated and γ-irradiated K562 cells were stained with 5 μM carboxyfluorescein diacetate succinimidyl ester (CFSE) prior to co-culturing with PBMCs. CFSE-positive K562 cells on day 0, 3, 7, and 10 were identified and counted by flow cytometry.

Flow cytometric cytotoxicity assay and antibody-dependent cell-mediated cytotoxicity (ADCC) assay. For the cytotoxicity assay, 1 μM CFSE-stained K562 were incubated in a 96-well U-bottom plate with expanded NK cells at effector-to-target (E:T) ratios 2:1; 1:1, and 0.5:1 for 4 h at 37°C in a 5% CO₂ incubator. To perform the ADCC assay, Raji cells were incubated with 1 μg/ml of rituximab for 4 h and then stained with 1 μM CFSE. The CFSE-stained rituximab-coated Raji cells were also co-incubated with expanded NK cells using the same E:T ratios described above for the cytotoxicity assay. The mixed cells were transferred to FACS tubes after incubation. Before acquisition (5-10 min), 1 μl of 1 mg/ml PI (Invitrogen) was added to each tube. The cells were acquired on a FACS Canto II and analyzed using Kaluza software.

Statistics. Statistical evaluation was performed using the Prism 5 software (GraphPad Software, San Diego, CA, USA). The Mann–Whitney test (non-parametric) was applied to analyze differences between groups in term of purity, fold expansion, cytotoxicity, and ADCC of expanded NK cells; viability and the frequency of K562 cells, with a p-value<0.05 considered as statistically significant.