The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.
The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.
Natural killer (NK) cells are the major lymphocytic population of the innate immune system1. NK cells function as the first-line defenders of the host immune response against tumors and infections2,3,4. NK cells also play a central role in the development of tolerance through the secretion of potent cytokines and chemokines5. Due to their potent ability to target and eliminate tumor cells, multiple clinical trials are being conducted to evaluate donor-derived human NK cells as an adoptive immunotherapy for cancer6,7. In contrast to T cells, the developmental biology of NK cells has yet to be well-characterized8. This lack of knowledge is partially due to the absence of efficient techniques that deliver genes of interest to mouse or human primary NK cells. For these reasons, most NK-cell studies have been conducted in cell lines, rather than in primary cells. Therefore, the need for a reliable and efficient protocol to transduce primary NK cells with genes of interest is crucial.
The overall goal of this study was to formulate a consistent and reliable method by which primary human or murine NK cells could be transduced with lenti- or retroviruses.
Earlier studies that attempted to address this problem have been performed, largely using the transient transformation of primary NK cells. This includes plasmid transfection9,10, Epstein-Barr Virus (EBV)/retroviral hybrid vector11, vaccinia vectors12,13, and Ad5/F35 chimeric adenoviral vectors14. Despite the modest efficiency of these techniques, the transient nature of transduction makes them unsuitable for the long-term utilization of the genetically modified NK cells. A few recent studies have used retroviral vectors to transduce NK cells, requiring multiple cycles of infection to achieve an acceptable level of gene expression11,15. In contrast to retroviral vectors, lentiviral vectors can use host-cell nuclear import machinery to translocate the viral pre-integration complex into the nucleus. This is a major limiting factor in the replication of the virus in non-dividing cells, which include primary NK cells.
Interactions between different cell-surface receptors and viral particles permit viral uptake into the cell. The initial engagements between the viral envelope proteins and their cognate host receptors could be limited because of the potential negative charges existing between these two. The rationale behind many transduction techniques is that the addition of cationic polymers, such as polybrene (Pb), protamine sulfate (PS), or dextran, could give a positive charge to the cell-surface receptors and thereby augment the binding of viral envelope proteins. This will increase the fusion efficiency and the uptake of the viral particles by the cells16. Although it has been reported that Pb or PS can improve gene transfer in T cells17, their application did not have any effect in the transduction efficiency of primary NK cells. Moreover, a comparative analysis between these reagents using primary NK cells has not been performed. In this study, the transduction efficiencies of the three cationic polymers were compared. The results show that, among these three cationic polymers, only dextran significantly enhances efficient viral transduction into both mouse and human primary NK cells.
All animal protocols followed the humane and ethical treatment of animals and were approved by the Institutional Animal Care and Use Committee (IACUC) within the Biomedical Research Center (BRC) of the Medical College of Wisconsin (MCW), Milwaukee, WI. The use of human peripheral blood mononuclear cells (PBMCs) was approved by the Institutional Review Board (IRB) of the Blood Research Institute of the Blood Center of Wisconsin, Milwaukee, WI.
1. Mice, cell lines, and vectors
2. Preparation and titration of lentiviral vectors
3. Purification and expansion of murine primary NK cells
4. Purification and expansion of human primary NK cells
5. Transduction of murine and human primary NK cells with lentivirus
Dextran induces the efficient gene transfer of lentiviral vector in primary human and murine NK cells
Human NK cells were isolated and purified from PBMC (with a purity of more than 85%) and incubated overnight with rIL-2 300 U/mL. These primary NK cells were then transduced with GFP lentivirus at varied multiplicities of infection (MOI; 3, 10, and 20 IU per cell) in 24-well plates in the presence of 8 µg/mL Pb, PS, or dextran. Cells were centrifuged at 1,000 × g for 60 min and cultured (in the presence of virus) overnight at 37 °C in a CO2 incubator. Cells were washed and resuspended in fresh RPMI1640 complete medium with rIL-2 300 U/mL for seven days. The transduction efficiency was evaluated by flow cytometry for GFP expression seven days after transduction. Results show that dextran enhances the efficiency of lentiviral transduction in human NK cells and increases the viral titer, which can also improve the percentage of transduced NK cells (Figure 1).
Murine NK cells were isolated and cultured as described in the earlier section. The above protocol was used to analyze and compare the transduction efficiency of Pb, Ps, and dextran. Results in Figure 2 show that dextran can augment the efficiency of lentiviral vectors compared to Pb or PS.
Transduction of NK cells with dextran does not affect their ability to mediate effector functions
The cytotoxic capacity of transduced human and murine NK cells was examined by 51Cr-release assays against K562 and YAC-1 as the target cells. Results presented in Figure 3a and b reveal that the transduction of NK cells by dextran does not negatively alter the killing potential of transformed NK cells compared to non-transduced NK cells. Additionally, the cytokine production from transduced primary NK cells was analyzed. IFN-γ generation was measured using an ELISA. Transduced human primary NK cells were co-cultured with K562 for 24 h, and the supernatants were collected to measure IFN-γ. Results presented in Figure 4a reveal that dextran has no impact on the ability of transduced NK cells to produce cytokines. As an independent validation, transduced NK cells were activated with titrated concentrations of plate-bound anti-NKG2D (A10) mAb for 18 h. Culture supernatants were collected, and IFN-γ generation was measured by ELISA. Results shown in Figure 4b reveal that the transduction of murine NK cells with dextran has no effect on their ability to produce cytokines.
Transduction of NK cells with dextran does not affect their cell viability
The influence of Pb, PS, or dextran on the viability of transduced NK cells was evaluated using a propidium iodide (PI) assay and subsequent flow analyses. Results in Figure 5a and b demonstrate that, although transducing primary human and mouse NK cells with viruses can induce necrosis in about 20% of NK cells, the addition of dextran did not augment this necrosis compared to Pb or PS.
Statistical analyses were performed with a two-tailed unpaired Student's t-test. P-values ≤ 0.05 were considered significant.
Figure 1: Dextran has a higher efficiency of transduction of human primary NK cells.
Primary human NK cells were transformed with MOI of 3, 10, or 20 IU/cell and were cultured for seven days in the presence of Pb (8 µg/mL), PS (8 µg/mL), or dextran (8 µg/mL). Percentages of GFP-positive NK cells were determined by flow cytometry on day seven following the transduction. One of three independent experiments is shown. Please click here to view a larger version of this figure.
Figure 2: Dextran has a higher efficiency of transduction in murine NK cells.
Primary murine NK cells were transformed with an MOI of 30 IU/cell and were cultured for seven days in the presence of Pb (8 µg/mL), PS (8 µg/mL), or dextran (8 µg/mL). Percentages of GFP-expressing cells were determined by flow cytometry at seven days following the transduction. One of three independent experiments is shown. Please click here to view a larger version of this figure.
Figure 3: Dextran does not modify the NK cell-mediated cytotoxic activity of transformed cells.
Primary NK cells were transformed with an MOI of 10 IU/cell and were cultured for seven days. YAC-1 and K562 were used as target cells to determine the cytotoxic potentials of murine (a) and human (b) NK cells, respectively. The data shown are representatives of two independent experiments. The data shown are averages with SEM. Please click here to view a larger version of this figure.
Figure 4: Dextran does not alter the IFN-γ production of primary NK cells.
a) Murine NK cells were transduced with an MOI of 30 IU/cell and cultured for seven days. NK cells were stimulated with 2.5 µg/mL of plate-bound anti-NKG2D antibody for 18 h, and IFN-γ was quantified in culture supernatants using an ELISA. b) Transduced human NK cells were cultured with K562 cells for 24 h, and the IFN-γ production was measured in culture supernatants. The data presented are representative of three independent experiments. The data shown are averages with SEM. Please click here to view a larger version of this figure.
Figure 5: Transduction of NK cells with dextran does not alter their cell viability.
The viability of transformed human (a) and mouse (b) NK cells was quantified after staining for Annexin-V (PE)/7-AAD positive cells. The data shown are averages with SEM. Please click here to view a larger version of this figure.
This study demonstrates that use of dextran as a cationic polymer agent enhances the lentiviral transduction efficiency of both murine and human primary NK cells. Additionally, other cationic agents, such as Pb or PS, have no discernible effect on the delivery of viral vectors into human primary NK cells. Previously, it has been demonstrated that Pb can augment gene transduction in human T cells17. These results, however, suggest that neither Pb nor PS have a similar efficiency on human primary NK cells. In this study, Pb improved the transduction efficacy only modestly in murine NK cells. It has been shown that the inhibition of intracellular antiviral defense mechanisms using BX795, an inhibitor of TBK1/IKKɛ, and PS as an enhancer can increase the lentiviral transduction efficiency25. Nevertheless, the results showed that this inhibitor has no effect on the transduction efficacy in murine or human primary NK cells (data not shown).
The results demonstrate that dextran can induce the efficient transduction of primary human and mouse NK cells, while PS and Pb have no effect. The results also show that dextran does not alter the effector functions of NK cells, such as anti-tumor cytotoxicity and the production of pro-inflammatory cytokines. Thus, these results prove that the viability of these primary NK cells was not altered by the use of dextran.
Multiple studies analyzed and compared the transduction efficiency of retroviral vectors using different cationic polymers, with varied outcomes26,27,28. One earlier study compared the transduction efficacy of these polymers using lentiviral vectors; however, this was tested in CD4+ T cells16. In one of these studies, it has been shown that Pb has a better capacity of transduction on transformed B cells and dendritic cells compared to dextran26. In another study, it has been shown that dextran facilitates a higher transduction efficiency than other enhancers in human B cells and T cells16. The current study is the first of its kind, to our knowledge, that analyzed and compared the transduction capacity of different polymers in both human and murine primary NK cells.
Dextran-based gene transductions may require a minimum of two rounds of transductions. These results show that one round of transduction is enough to reach up to 40% of positively transduced cells, which is increased up to 100% following two rounds of transduction (data not shown). One of the major challenges in transducing NK cells is the ability of these cells to preserve the expression of the transduced gene. The current results demonstrate that the transduction of primary NK cells with dextran is stable and that the NK cells that are cultured in the presence of IL-2 can retain the vector and maintain transgene expression for four weeks (data not shown). Additional experiments are required to analyze the transduction efficiency and to examine the concurrent expression of multiple transgenes.
The clinical efficacy of immune cell-based cancer therapies is being validated by multiple institutions. CAR-transduced T cells provide renewed promise for the disease- and relapse-free recovery of cancer patients compared to conventional therapies. As part of innate immune responses, NK cells do not require prior sensitization to mediate their effector functions, including anti-tumor cytotoxicity. Due to their rapid response, NK cells are an ideal effector lymphocyte subset to mediate an efficient cell-based cancer immunotherapy. Despite their positive attributes in tumor recognition and elimination, the technical hurdles regarding the genetic manipulation to deliver transgenes limit the fullest clinical utilization of NK cells.
Electroporation of mRNA for exogenous gene expression is highly efficient and has better outcomes. However, mRNA utility is limited, as they allow only the transient expression of genes of interest and thereby are not suitable for clinical applications. The retroviral transduction of NK cells is less efficient as it requires multiple rounds of transduction; moreover, retroviral vectors cannot transduce into non-dividing cells. The only promising approach for stable transgene delivery is the use of lentiviral vectors.
Altogether, this study provides an efficient method to deliver transgenes into primary NK cells, without impairing their effector functions. This protocol makes NK cell-based cancer immunotherapy highly efficient and applicable to emerging clinical trials. Future experiments are needed to validate the efficacy of this method in NK cell-based cancer immunotherapies.
The authors have nothing to disclose.
We thank Lucia Sammarco and her Lulu's Lemonade Stand for inspiration, motivation, and support. This work was supported in part by NIH R01 AI102893 and NCI R01 CA179363 (S.M.); NHLBI-HL087951 (S.R.); NIH-CA151893-K08 (M.J.R.); NCI 1R01CA164225 (L.W); the Alex Lemonade Stand Foundation (S.M.); the HRHM Program of the MACC Fund (S.M.; S.R.; M.S.T); the Nicholas Family Foundation (S.M.); the Gardetto Family (S.M.); the Hyundai Scholars Program (M.S.T.); Hyundai Hope on Wheels (S.R.); the MACC Fund (M.S.T. and S.M.); the Children's Research Institute, MCW (S.R.); and the Kathy Duffey Fogerty Award (M.J.R.).
Dextran | Sigma-Aldrich | 90-64-91-9 | |
polybrene (Pb) | Sigma-Aldrich | TR-1003 | |
protamine sulfate (PS) | Sigma-Aldrich | p3369 | |
Trypsin | Corning | 25-052-CI | |
RPMI1640 | Corning | 10-040-CV | |
Fetal Bovine Serum | ATALANTA | S11150 | |
Penicillin | Corning | 30-001-CI | |
B-mercaptoethanol | SIGMA | M3148 | |
sodium pyruvate | Corning | MT25000CI | |
Interferon gamma (IFN-γ ) | eBioscience | 14-7311-85 | |
Propidium lodding staining solution | BD | 51-66211E | |
Lipofectamine 3000 | Thermo Fisher | L3000015 | |
Isoflurane | PHOENIX | NDC 57319-559-05 | |
NK cell negative selection kit | Stem Cell | 19855 | |
Yac-1 | ATCC | TIB-160 | |
K562 | ATCC | CCL-243 | |
Mice | Jakson | 664 | |
293T cells | ATCC | CRL-3216 | |
T75 flasks | Cornnig | 430641U | |
antibody-based negative selection kits | Stem Cell | 19055 | |
51Chromium (Cr)-release assays | perkin elmer's | NEZ030 | |
ELISA kits | Ebioscience | 00-4201-56 | |
Sodium Butyrate | Sigma | 5887-5G | |
Linear polyethylenimine | polysciences | 23966-2 | |
Ficoll | GE Life Science | 17-1440-03 | |
HBSS | Corning | 21-022-CV |