Cell lines
K562, THP-1 cells were purchased from Procell (Wu Han, China). OCI-AML3, U937, MOLM-13, Hep-G2, OVCAR3, 293T cells were provided by the Hematology Lab or Gene and Cell Therapy Research Institute at The First Affiliated Hospital of Xi'an Jiaotong University. K562, THP-1, OCI-AML3, U937 cells were maintained in PRMI-1640 medium (HyClone, USA) with 10% fetal bovine serum (FBS, ExCell Bio, China). Hep-G2 cells were maintained in MEM medium (HyClone, USA) with 10% FBS. OVCAR3 were maintained in PRMI-1640 medium and 10 µg/mL insulin with 20% FBS. 293T cells were used for lentiviral packaging and cultured in Dulbecco's Modified Eagle's Medium (DMEM, HyClone, USA) with 10% FBS. Mycoplasma testing was performed on all cell lines once every two weeks using the MycoAlert Mycoplasma Detection Kit (Lonza, Germany).
Plasmid construction
To construct mbIL-21, the coding sequence of mature human IL-21 was directly fused to the human CD8a hinge, transmembrane and cytoplasmic domains. The mbIL-15 was created by linking human IL-15 with its receptor IL-15RA. The codon-optimized sequences for mbIL-21 and mbIL-15 were synthesized and linked using the T2A self-cleaving peptide. This synthetic fusion sequence of mbIL-21/-15 was then cloned into the pLVX-EF1α-IRES-Neo vector (Miaoling Biology, China) using the EcoR1 and BamH1 restriction sites, resulting in the recombinant plasmid pLVX-EF1α-mbIL-21/-15-IRES-Neo (Fig. 1a). Given that human 4-1BBL functions as a membrane protein, its codon-optimized sequence was also synthesized and subsequently inserted into the pSIN-EF1α-IRES-Puro vector (Miaoling Biology, China) to create the overexpression plasmid pSIN-EF1α-4-1BBL-IRES-Puro (Fig. 1a).
K562 feeder cells co-expressing 4-1BBL, mbIL-21 and mbIL-15 construction
Lentiviral particles were produced by co-transfecting 293T cells with the respective lentiviral transfer plasmids (pLVX-EF1α-mbIL-21/-15-IRES-Neo or pSIN-EF1α-4-1BBL-IRES-Puro) along with the packaging plasmids (psPAX2 and pMD2.G). Three days following transfection, the viral supernatants were collected and concentrated by ultracentrifugation at 100,000 g for 2 hours. The concentrated viral particles were resuspended in RPMI-1640 medium and stored at -80°C until further use. To determine the viral titer, 293T cells were transduced with varying volumes of the viral solutions. After three days, the percentage of 4-1BBL + or IL-21/IL-15 + cells were estimated by flow cytometry. Titers was calculated using the following formula: Titer (IU/mL) = (Number of infected cells × Percentage of positive cells) / Volume of virus used (mL).
K562 cells were seeded into 6-well plates at 2×105 cells/mL and subsequently transduced with the concentrated virus stocks containing mbIL-21/-15 and 4-1BBL at a multiplicity of infection (MOI) of 2 in the presence of protamine sulfate (10 µg/mL; Solarbio, China). Three days later, the transduced cells were selected with puromycin (2 µg/mL) and G418 (600 µg/mL) for two weeks. The transduction efficacy was analyzed by flow cytometry using APC-conjugated anti-human 4-1BBL monoclonal antibodies (mAb) (Biolegend, USA), PE-conjugated anti-human IL-21 and IL-15 mAbs (eBioscience, USA). The stable feeder cell line co-expressing 4-1BBL and mbIL-21/-15 (K562-4-1BBL-mbIL-21/-15) was maintained in RPMI 1640 with 10% FBS and low concentration of puromycin and G418. Upon reaching sufficient cell numbers, these feeder cells were irradiated with 100 Gy gamma rays by Cs-137 gamma to inhibit further cell division, then cryopreserved with 2×107 cells/vial in liquid nitrogen, creating a ready-to-use irradiated K562-4-1BBL-mbIL-21/-15 cell bank for future NK cell expansion endeavors.
NK cells expansion using K562-4-1BBL-mbIL-21/-15 feeder cells
PBMCs were isolated form the peripheral blood of healthy donors by Ficoll gradient centrifugation (GE Healthcare, USA). 2×107 PBMCs were directly co-cultured with frozen-thawed irradiated K562-4-1BBL-mbIL-21/-15 feeder cells at a ratio of 1:1 in a T75 flask containing 20 mL of fresh medium. The medium was composed of a serum-free medium (Hangzhou Zhongying Bio-Medical Technology, China) supplemented with 5% heat-inactivated autologous plasma and recombinant human IL-2 (200 U/mL; Jiangsu Kingsley Pharmaceutical Co., Ltd, China). Three days post-culture, the cell suspension was centrifuged at 350 g for 5 minutes, and then the supernatant was discarded and equal amount of fresh medium was added. Up until day 7, fresh medium was supplemented every two days to maintain the cell density between 1.0-1.5×106 cells/mL. On day 7, a second of frozen-thawed K562-4-1BBL-mbIL-21/-15 feeder cells were added for re-stimulation. Subsequently, the percentage of heat-inactivated autologous plasma in the medium was reduced from 5–1%, and the cell densities were adjusted to be within 1.5–2.5×106 cells/mL. Cells were harvest on day 13 or 14, and the expansion fold was calculated by the absolute number of NK cells on day 14 by the respective number on day 0.
Flow cytometry analysis
The purity of NK cells on days 0, 7 and 14 was examined by flow cytometry using FITC-conjugated anti-human CD3 mAb and APC- conjugated anti-human CD56 mAb (Biolegend, USA). The expression levels of various NK cell receptors were analyzed using PE- conjugated anti-human NKG2D, NKp30, NKp46 mAbs (BD Biosciences, USA), PE-Cy7 conjugated anti-human NKp44, CD69, CD158b mAbs (Biolegend, USA), and APC-Cy7 conjugated anti-human NKG2A and CD16 mAbs (Biolegend, USA). For all samples, PBMC or the expending cells (1.0×106 cells) were washed with FACS buffer (MACS rinsing solution with 0.2% BSA; Miltenyi Biotec, Germany) and resuspended with 100 µL FACS buffer in a 96 well U-bottom plate. To block non-specific binding, Human Fc-receptor Blocking Solution (Biolegend, USA) was applied to each sample for 5 minutes at room temperature. Subsequently, the samples were stained with the designated mAbs for 20 minutes at 4°C in the dark. Following staining, cells were washed twice with FACS buffer and then were resuspended with 100 µL FACS buffer. To assess cell viability, 10 µL Propidium Iodide (PI) were added to each sample, followed by incubation for 5 minutes at 4 ℃ in the dark. The data was required and analyzed using NoveExpress software.
Cytotoxicity assay
NK cell cytotoxicity was assessed using the One Glo Luciferase Assay System (Promega, USA). The stable luciferase-expressing cell lines (K562-Luc, Molm-13-Luc, THP-1-Luc, OCI-AML3-Luc, U937-Luc, Hep-G2-Luc, OVCAR-Luc) were prepared to serve as target cells. The unexpanded and expanded NK cells were co-cultured with each targeted cells at effector-to-target (E:T) ratio of 5:1, 2:1, 1:1 and 0.5:1 in a 384 well plate with total amount of 30 µL for 12–16 hours in triplicate. After co-culture, 30 µL of the luciferase ONE-Glo Reagent was added to each well containing the NK/target cell mixture. The plate was incubated at room temperature for 3 minutes to allow complete cell lysis. Finally, the luminescence was measured in a luminometer. The cytotoxicity was calculated using the following formula:
Cytotoxicity (%) = [((Luminescence (target cells only) - Luminescence (effector cells only))- ((Luminescence (target cells with effector cells) - Luminescence (effector cells only))] / [Luminescence (target cells only) - Luminescence (effector cells only)] ×100%.
CD107a degranulation assay
The unexpanded and expanded NK cells were washed with phosphate-buffered saline (PBS) once and re-suspended into completed NK cells medium. Subsequently, they were seeded into a U-bottom 96-well plate at the concentration of 2×10^4 cells/well. APC-Cy7-conjugated CD107a antibody (Biolegend, USA) were added in and thoroughly mixed with the cells. NK cells were then cocultured at a 1:1 E:T ratio with target cells for 5 h. After 1 h of incubation, Golgi stop containing monensin (BD Biosciences) was added. Following 4 additional hours of incubation, the cells were used for anti-CD56 APC and anti-CD3 FITC antibody staining. The cells were resuspended with 100 µL FACS buffer for the detection of the CD107a surface expression on NK cells by NoveExpress software.
In vivo functional studies of expanded NK cells in xenografted mice
To evaluate the potential therapeutic effect of expanded NK cells for cancer therapy, an acute leukemia mouse model was employed. Female NSG mice, aged 6–8 weeks, were procured from the Saiye Model Biology Research Center Co. The mice were housed and maintained in individual ventilated cages under specific pathogen-free conditions, with adequate access to food and water.
The mice were injected via the tail vein with U937 cells (1×106 U937 cells/mouse) mixed with either PBS or NK cells (1×107). Seven days post-injection, mice were injected with D-fluorescein potassium salt (150 mg/kg) and the bioluminescence signal was analyzed using an IVIS® Lumina II Multispectral Imaging System (PerkinElmer, USA) to verify the tumor cell engraftment. Tumor progression was monitored weekly through bioluminescence imaging (BLI).
Statistics
Group differences were analyzed using one-way analysis of variance (ANOVA) or Student’ s t-test. Each experiment was conducted with a minimum of three independent replicates. The survival distributions were estimated by the Kaplan-Meier method and compared with the use of log-rank test between groups. The data were performed using SPSS software (version 23.0). The following denotations for significance levels were used: *p < 0.05, **p < 0.01.