Overview of NK Cells:

 

Natural Killer (NK) cells belong to the family of innate lymphoid cells and are typically defined in humans as CD3^- CD56⁺ cells, accounting for 5% to 15% of circulating lymphocytes. NK cells serve as the first line of defense against viral infections and cellular mutations, capable of killing cancer cells, allogeneic cells, and cells infected by foreign pathogens without prior sensitization. They are considered key effector cells in cancer immune surveillance, transplant rejection, and early viral immunity.

Figure: NK Cells (Yellow) and Cancer Cells (Red)

 

NK Cell Immunotherapy:

 

The success of CAT immunotherapy has sparked interest in utilizing other immune cells for cancer treatment. Simply put, cell immunotherapy involves collecting the body's own immune cells, expanding their numbers in vitro, and then reinfusing them into the body. This process aims to eliminate cancer cells and mutated cells while activating and enhancing the body's immune system to control cancer metastasis and recurrence. Compared to CAT-T therapy, NK therapy can target multiple pathogenic antigens, exhibits stronger cytotoxicity, and does not secrete the primary cytokines that trigger cytokine release syndrome (CRS), significantly reducing the risk of adverse reactions and holding great promise.

 

Main Strategies of NK Cell Therapy:

Strategies include in vitro activated autologous or allogeneic NK cells, combination of NK cells with monoclonal antibodies (such as immune checkpoint inhibitors) to induce antibody-specific cytotoxicity (e.g., cetuximab against EGFR), CAR-NK cell immunotherapy, and more.

 

Currently, no NK cell therapy has been approved for market use, but clinical trials are in full swing. As of November, the search results for "NK Cell" on the clinicaltrials website have reached 720 cases. NK cell therapy has shown promising therapeutic effects in the treatment of acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndromes, neuroblastoma, as well as solid tumors such as gastric cancer, liver cancer, lung cancer, and colorectal cancer.

Figure: Search Results for "NK Cell" on clinicaltrials

 

Sources of In Vitro Expanded NK Cells:

 

NK cells can be obtained not only from autologous or allogeneic peripheral blood but also from umbilical cord blood and stem cell differentiation. Additionally, due to the complexity of obtaining cells through the above three methods, many researchers use NK-92 for further modification in CAT-NK studies, with preclinical studies yielding favorable results. It is important to note that cells need to be irradiated before infusion.

 

Isolation and Purification Protocol for Human Peripheral Blood NK Cells:

 

1. Obtaining PBMC

1) Prepare sterile 15mL centrifuge tubes or conical tubes;
2) Add 5mL of lymphocyte separation medium; (Note: The lymphocyte separation medium should be brought to room temperature before use, 18-25℃)
3) Dilute the anticoagulated whole blood sample with Hank's solution (abs9257) or PBS (abs962) at a 1:1 ratio (if the whole blood sample is viscous, the ratio can be appropriately increased);
4) Carefully layer 4mL of the freshly diluted whole blood sample over the lymphocyte separation medium by slowly pipetting along the tube wall; (Note: Be careful not to disturb the lymphocyte separation medium)
5) Carefully place the tube in a centrifuge (horizontal rotor) and centrifuge at 4℃ for 20-30 minutes at 400g-1000g; (Note: The centrifuge brake should be turned off, allowing it to stop naturally)
6) Carefully remove the tube, ensuring not to shake it;
7) The upper layer will be a light red transparent plasma, followed by a thin, dense white ring, which is the PBMC layer;
8) After removing the supernatant, gently transfer the white layer to a new centrifuge tube;
9) Resuspend in PBS, centrifuge at 300g for 10 minutes at room temperature, repeat twice, discard the supernatant, and resuspend in a small amount of PBS.

 

2. Purification of NK Cells

 

The most common method for NK cell purification is magnetic bead sorting, including positive and negative selection. The developmental stages of human NK cells are primarily based on the expression levels of CD34, CD117, CD56, and CD94. Increased CD56 expression is key to NK cell maturation, and CD56⁺ and CD16⁺ can identify and isolate NK cells without contamination from other lymphocytes. Additionally, the interleukin-2/15 receptor β (CD122) is also an important marker in the later stages of NK cell development.

 

3. NK Cell Culture

 

1) Resuspend the purified NK cells in serum-free immune cell culture medium (abs9772) and seed them at a certain density in culture dishes or flasks.

2) Add 100-200IU/mL of IL-2 to the serum-free immune cell culture medium to ensure NK cell proliferation and activation, and to enhance cytotoxicity. Combined use with IL-15 can promote NK cell proliferation and activation.

3) Incubate the cells at 37℃ for at least 48 hours before proceeding with subsequent functional assays.

 

Glossary:

 

Positive Selection: Magnetic beads bind to surface markers of the target cells to achieve purification.

Negative Selection: Magnetic beads or other methods are used to remove non-target cells from a mixed cell population to achieve purification.

 

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