NK Cell Overview:
Nature killer (NK) cells belong to the family of innate lymphoid cells, which are commonly defined as CD3-CD56+ cells in humans, accounting for 5% to 15% of circulating lymphocytes. NK cells are the first line of defense against viral infection and cell mutations, killing host-threatening cancer cells, allogeneic cells, and cells infected with foreign pathogens without prior sensitization, and are considered to be key effector cells for cancer immune surveillance, transplant rejection, and early viral immunity.
Figure NK cells (yellow) and cancer cells (red).
nkCellular immunity
The success of CAT immunity has stimulated the interest of researchers in using other immune cells for cancer, simply put, cellular immunity is to collect human autoimmune cells, multiply their number after in vitro culture, and then infuse them back into the human body, on the one hand, to eliminate cancer cells, mutant cells, on the other hand, to activate and enhance the body's immunity, control the metastasis of cancer cells and**. Compared with CAT-T**, NK** can target a variety of pathogenic antigens, has stronger cytotoxicity, and does not secrete major cytokines that trigger cytokine release syndrome (CRS), which can greatly reduce the risk of adverse reactions, and is highly expected.
The main strategy of NK cells**:
In vitro activated autologous or allogeneic NK cells, NK cells, and monoclonal antibodies (e.g., immune checkpoint inhibitors) are combined to induce antibody-specific cytotoxicity (e.g., anti-EGFR cetuximab), CAR-NK cell immunity**, and more.
At present, there are no NK cells** on the market, but its clinical trials are in full swing, as of November, the search results for "NK cell" have been as high as 720 cases, and the NK cells** have shown good results in acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, neuroblastoma, and solid tumors such as gastric cancer, liver cancer, lung cancer, and bowel cancer.
Figure Search results for "NK cell" in ClinicalTrials.
In vitro expanded NK cells**:
In addition to the isolation of NK cells from autologous or allogeneic peripheral blood, they can also be obtained from umbilical cord blood and stem cell differentiation, in addition, due to the cumbersome way of obtaining cells in the above three ways, in the study of CAT-NK, many researchers use NK-92 for further modification, and good results have been achieved in preclinical studies, it is worth noting that the cells need to be irradiated before infusion.
Isolation and purification protocol for human peripheral blood NK cells:
Preparation: 15 ml centrifuge tubes (ABS7102) or conical tubes, HANK'S solution (ABS9257) or PBS (ABS962), human lymphocyte separation solution (ABS930).
Figure Abics Human Lymphocyte Separation Solution (ABS930).
1. Obtain PBMC
1) Prepare sterile 15 ml centrifuge tubes or conical tubes;
2) Add 5ml of lymphocyte separation solution; (Note: Lymphocyte separator should be brought back to room temperature before use, 18-25).
3）hank'S solution (ABS9257) or PBS (ABS962) buffer 1:1 ratio (if the whole blood sample is viscous, the proportion can be appropriately increased) to dilute the anticoagulated whole blood sample;
4) Carefully add a fresh diluted 4 ml whole blood sample on top of the lymphocyte hydroselate very slowly along the wall tube, close to the separator layer; (Note: Remember not to muddy the lymphocyte separator).
5) Carefully put into the centrifuge (swing-out rotor), 4 centrifuge for 20-30min, the speed is 400g-1000g; (Note: The centrifuge brake should be removed and wait for the natural stop).
6) Carefully remove the tube, remember not to vibrate;
7) the upper layer is pale red transparent plasma, followed by a thin dense white ring, which is PBMC;
8) After aspirating the supernatant, gently aspirate the buffy coat layer and transfer it to a new centrifuge tube;
9) Resuspend with PBS, centrifuge at room temperature, 300g for 10min, repeat the operation 2 times, discard the supernatant, and resuspend with a small amount of PBS.
2. Purification of NK cells
The most commonly used method for NK cell purification is magnetic bead sorting, including positive sorting and negative sorting, and the development stage of human NK cells is mainly based on the expression levels of CD34, CD117, CD56 and CD94. Increased expression of CD56, CD56, is key to NK maturationand CD16+ can identify and isolate NK cells that are not contaminated with other lymphocytes. In addition, the interleukin 2 15 receptor (CD122) is also an important marker in the later stages of NK cell development.
3. NK cell culture
1) The purified NK cells were resuspended with immune cell serum-free medium (ABS9772) and seeded in a Petri dish or culture flask at a certain density.
2) Add 100-200 IU ml of IL-2 to serum-free medium for immune cells to ensure the multiplication and activation of NK cells and help to improve cytotoxicity, and the combination application with IL-15 can promote the proliferation of NK cells and facilitate the activation of NK cells.
3) The cells were cultured in an incubator at 37 for at least 48 hours, and then subsequent experiments such as cell function identification were carried out.
General explanation of nouns:
Positive screening:Magnetic beads bind surface markers that require screening of cells for purification purposes.
Negative Screening:Magnetic bead binding and other means are used to remove other cells in the mixture cells except the target cells to achieve the purpose of purification.
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References
1] Mi Jintao; Yuan Chengliang. Research progress on NK cell-associated immune checkpoints and their inhibitors[J].Modern Oncology, 2023, 31(24): 4645-4650
2] shimasaki n, jain a, campana d. nk cells for cancer immunotherapy[j]. nature reviews drug discovery, 2020, 19(3): 200-218.