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.
