MEASURE OF NATURAL KILLER CELL ACTIVITY

NK Cells – Its role in the immune system

Natural killer cells (NK cells for short) are a group of lymphocytes that is able to respond to microorganism living inside the host’s cells by killing the infected cells and by secreting Interferon Gamma (IFNγ), a cytokine that activates macrophages. (Figure 1).

Figure 1: The function of NK cells.

Killing cells infected with microorganisms and activates macrophages

NK cells account for about 5 – 10% of the total number of lymphocytes in the blood and peripheral lymphoid organs. These cells contain large particles in the cytoplasm and have special markings on the surface, while on their surface there are no antibody molecules to identify antigens like B lymphocytes and do not have antigen receptors to recognize antigens like T lymphocytes either.

NK cells identify the body’s cells that have been changed by microbial infection or transformed into cancer cells. NK cells have receptors for molecules present on the surface of the host cell. Among those receptors, some activate NK cells, while some are inhibiting NK cells. Activated receptors are receptors that identify molecules on the surface of the cell – usually on the surface of infected host cells and on the surface of virus-infected macrophages or are containing the infecting microorganisms. Other active receptors include receptors that recognize molecules on the surface of normal cells. Theoretically, these molecules have the effect of activating NK cells to kill normal cells of the body. However, this usually does not happen because NK cells also have inhibitory receptors that recognize normal cells of the body and inhibit the activation of NK Cells. Many viruses are able to create mechanisms that block the expression of MHC molecules class I on the surface of the cells they infect, and that way they can evade attack by CD8 toxic cytotoxic T lymphocytes (cytolitic/cytotoxic T lymphocyte) are cells capable of specifically attacking virus-infected cells (see post “cell-mediated immunity response”). However, it is also difficult for these viruses to bypass NK cells because if this happens, when NK cells encounter those infected cells, the inhibitory receptors of NK cells will not have MHC molecules to allow them to attach, and so these receptors become inactive. As a result, there is no cell suppressor so the NK cell becomes activated and attacks to eliminate the infected cell (Figure 3.5). The infection resistance of NK cells is further increased when they are stimulated by cytokine secreted by macrophages when they come in contact with microorganisms. One of the cytokines secreted by macrophages that activates NK cells is interleukin-12 (IL-12). NK cells also have receptors for the Fc portion of some IgG antibodies and NK cells use those receptors to attach to the antibody-coated cells (opsonized by antibodies). The role of this reaction in humoral immunity by antibodies will be addressed in the humoral immune response

Figure 2: Function of inhibitory receptors in NK cells. The inhibitory receptors needs to be attached to the MHC molecules class I to signal the inhibition of the cell activation signal provided by the active receptor. When an inhibitory receptor without a ligand MHC is attached, the inhibitory signal is lost and the activated NK cell attacks the target cell it is exposed to.

When NK cells are activated they respond in two ways (Figure 1). In the first way, the activation process triggers the release of proteins contained in the cytoplasmic particles of the NK cell towards the infected cell. The proteins contained in these particles of the NK cell include molecules capable of making holes in the protoplasmic membrane of the infected cell, while other molecules of these proteins “creep” into them. The infected cell activates the enzymes of the infected cell itself, which triggers programmed cell death (also called apoptosis). The mechanism of dissolving target cells of NK cells is similar to those used by toxic T lymphocytes to kill infected microorganisms (see post: Cell-mediated immunity response). As a result of these reactions, NK cells kill host cells that have been contaminated with microorganisms. By killing host cells infected with microorganisms, NK cells as well as toxic T lymphocytes perform the function of eliminating potential foci of infection within host cells by killing infectious microorganisms and multiply inside the host’s cells like viruses. In addition, activated NK cells synthesize and secrete cytokine IFN-g, a macrophage activating factor that increases the ability to kill microorganisms that have been swallowed by macrophages. NK cells and macrophages work together to eliminate intracellular microorganisms: macrophages swallow microorganisms and produce IL-12, IL-12 activating NK cells, secreting IFN-g, then IFN-g activating macrophages to kill the microorganisms they ingest.

As such, both the host and the microorganisms are involved in the constant and persistent struggle for survival. A host uses toxic T-cells to identify viral antigens presented by MHC molecules. The viruses, in turn, prevent the expression of MHC molecules. NK cells have evolved to cope with the disappearance of MHC molecules. It is not known whether the host or the microorganism will be the winner, the outcome of this battle will determine whether the infection occurs.

Evaluate NK cell activity

• The activity of NK cells can be measured by a classical cytotoxicity assessment (51Chromium assay) or, later, non-radioactive fluorescent cytotoxicity tests (non-radioactive fluorescent dye cytotoxicity assays). Cons: these tests only measure the virulence potential of NK cells and not the ability to secrete and interact with other cell types during adaptive immune response.
• In vitro NK cell activity cannot be measured without activating NK cells because NK cells do not secrete cytokines (ex vivo) without stimulating activation signals suitably. Using a number of stimulating cytokines, one could analyze NK cell activity in whole blood, including assessing both cytotoxic potential of the entire NK cell population (both under CD56^bright and CD56^dim) as well as the ability of these cells to destroy tumors. Both types of NK cells release IFN-γ after stimulation by cytokines.
• The amount of IFN-γ released is an indication of the cytokine-type cytokine-induced cytotoxic and cytokine-induced immunomodulation (IFN-γ release also works to stimulate dendritic cells, thereby supporting anti-tumor via T cell mediated reactions).