We thank Lucia Sammarco and her Lulu&#39;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&#39;s Research Institute, MCW (S.R.); and the Kathy Duffey Fogerty Award (M.J.R.).

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
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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...

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.

<table>
	<tbody>
		<tr>
			<td>Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>90-64-91-9</td>
			<td></td>
		</tr>
		<tr>
			<td>polybrene (Pb)</td>
			<td>Sigma-Aldrich</td>
			<td>TR-1003</td>
			<td></td>
		</tr>
		<tr>
			<td>protamine sulfate (PS)</td>
			<td>Sigma-Aldrich</td>
			<td>p3369</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Corning</td>
			<td>25-052-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640</td>
			<td>Corning</td>
			<td>10-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ATALANTA</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>Corning</td>
			<td>30-001-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>B-mercaptoethanol</td>
			<td>SIGMA</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium pyruvate</td>
			<td>Corning</td>
			<td>MT25000CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Interferon gamma (IFN-&#947; )</td>
			<td>eBioscience</td>
			<td>14-7311-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium lodding staining solution</td>
			<td>BD</td>
			<td>51-66211E</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 3000</td>
			<td>Thermo Fisher</td>
			<td>L3000015</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>PHOENIX</td>
			<td>NDC 57319-559-05</td>
			<td></td>
		</tr>
		<tr>
			<td>NK cell negative selection kit</td>
			<td>Stem Cell</td>
			<td>19855</td>
			<td></td>
		</tr>
		<tr>
			<td>Yac-1</td>
			<td>ATCC</td>
			<td>TIB-160</td>
			<td></td>
		</tr>
		<tr>
			<td>K562</td>
			<td>ATCC</td>
			<td>CCL-243</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Jakson</td>
			<td>664</td>
			<td></td>
		</tr>
		<tr>
			<td>293T cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 flasks</td>
			<td>Cornnig</td>
			<td>430641U</td>
			<td></td>
		</tr>
		<tr>
			<td>antibody-based negative selection kits</td>
			<td>Stem Cell</td>
			<td>19055</td>
			<td></td>
		</tr>
		<tr>
			<td>51Chromium (Cr)-release assays</td>
			<td>perkin elmer&#39;s</td>
			<td>NEZ030</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA kits</td>
			<td>Ebioscience</td>
			<td>00-4201-56</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Butyrate</td>
			<td>Sigma</td>
			<td>5887-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear polyethylenimine</td>
			<td>polysciences</td>
			<td>23966-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll</td>
			<td>GE Life Science</td>
			<td>17-1440-03</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Corning</td>
			<td>21-022-CV</td>
			<td></td>
		</tr>
	</tbody>
</table>

dextran enhances the lentiviral transduction efficiency of murine and human primary nk cells

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.

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 &#181;g/mL Pb, PS, or dextran...

Watch this Scientific Journal Video about Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells at JoVE.com

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

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 overall goal of this protocol is to formulate technologies that allow successful gene transduction in primary natural killer cells. This method can help answer key questions in the Immunotherapy field, like the effect of genetic modification on effector lymphocytes, such as natural killer, or NK cells. The main advantage of this technique is the efficient expression of a gene of interest in primary mouse or human NK cells.After preparing and titrating Lentiviral vectors according to the text protocol, purifying murine primary NK cells by passing single cell spleen suspensions through a nylon wool columns to deplete the adherent populations consisting of B-cells and macrophages. Use 1, 000 units per milliliter of Interleukin-2 in RPMI1640 complete medium to culture the non-adherent population that contains NK cells. On day four of culture, replace the medium using 20 milliliters of RPMI1640 complete medium, and 1, 000 units per milliliter of IL-2 to remove the non-adherent T and NKT cells.On day seven, use MK cell-specific markers to check the purity of the murine NK cells by flow cytometry using preparations with a greater than 95%CD3 negative and NK1.1 positive population. To isolate peripheral blood mononuclear cells, or PBMCs, carefully layer 35 milliliters of half diluted cells over a 15 milliliter density gradient in a 50 milliliter conical tube. Centrifuge the gradient in a swinging-bucket rotor at 400 times G and 20 degrees Celsius for 30 minutes without a break.After purifying human primary NK cells according to the text protocol, determine the purity of the cells by adding two micrograms per milliliter of anti-CD3, and two micrograms per milliliter of anti-CD56 antibodies. Incubate the cells at four degrees Celsius for 20 minutes. Use PBS to wash the cells twice.Then, analyze the cells by flow cytometry. The NK cell population will be positive for CD56 on the cell's surface, and negative for CD3. Suspend mouse or human primary NK cells in the wells of a 24-well plate at 0.5 times to the 5th per milliliter of medium with GFP Lentivirus supernatant, at an NLY of five, 10, and 20.Then in the presence of polybrene, protamine sulfate, or dextran. centrifuge the plates at 1, 000 times G for 60 minutes. Then, without decanting the supernatant, culture the cells in a 37 degree Celsius incubator infused with 5.2%carbon dioxide overnight.After the cells have been washed with 10 milliliters of PBS, use two milliliters of RPMI1640 medium with IL-2 to re-suspend the cells. The day before harvesting the cells, coat 96-well highly protein absorbent polystyrene plates with 2.5 micrograms per milliliter of anti-MKG2D monoclonal antibodies. and incubate the plates at room temperature or in the fridge overnight.The following day, use 100 microliters of PBS to wash each well three times. Harvest the transduced NK cells by first gently tapping the plates, then add 100 microliters of the cells to each well of the 96-well plates. 16 to 18 hours post-activation, use a multi-channel pipette to collect the supernatants.Then using the recombinant cytokine from an ELISA kit, generate a standard curve and quantify the cytokines, such as Interferon Gamma in the supernatant. To test the NK cell viability, four days after transduction, wash the cells with 10 milliliters of cold PBS two times. Then, harvest the cells and use an XM5 7-aminoactinomycin D to stain them.Use flow cytometry to determine the percent of necrotic cells among the transformed NK cells, and analyze the data according to the text protocol. This figure shows that human NK cells incubated with recombinant IL-2 and then transduced with GFP Lentivirus achieved a greater transduction efficiency and increased the viral titer when dextran was added to the culture, as compared to the addition of polybrene or protamine sulfate. As seen here, similar results were demonstrated in murine NK cells, where dextran augmented the efficiency of Lentiviral vectors, compared to polybrene or protamine sulfate.In this experiment, the cytotoxic capacity of transduced primary NK cells was examined by a CR-release assay against K562 and YAC-1 as target cells. The transduction of NK cells by dextran does not negatively alter the killing potential of transformed NK cells compared to non-transduced NK cells. Here, transduced human primary NK cells were co-cultured with K562 for 24 hours, and the supernatants were collected to measure Interferon Gamma.The results indicate that dextran has no impact on the ability of transduced NK cells to produce cytokines. In addition, an independent validation showed that dextran-treated NK cells activated with plate-bound anti-NKG2DA10 monoclonal antibodies were still able to produce the cytokine Interferon Gamma. While attempting this procedure, it's important to remember to spin the transduced cells.Following this procedure, other methods like transfection, can be profound in order to answer additional questions, like how to improve gene delivery. After its development, this technique paved the way of researchers in the field of Immunology, for explore gene manipulation in NK cells. After watching this video, you should have a good understanding of how to transduce NK cells.

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Research

JoVE Journal

Immunology and Infection

10.8K visualizações.  The Blood Center of Wisconsin. The overall goal of this protocol is to formulate technologies that allow successful gene transduction in primary natural killer cells. This method can help answer key questions in the Immunotherapy field, like the effect of genetic modification on effector lymphocytes, such as natural killer, or NK cells. The main advantage of this technique is the efficient expression of a gene of interest in primary mouse or human NK cells.After preparing and titrating Lentiviral vectors according t...