NKTEST® allows the quantitative determination of the cytotoxic activity of human natural killer (NK) cells. It contains cryopreserved K562 target cells, complete medium and staining reagents. A lipophilic green fluorescent membrane dye is used to label the K562 target cells in order to discriminate effector and target cells. After the incubation period in the cytotoxicity assay, killed target cells are identified by a DNA-stain, which penetrates the dead cells and specifically stains their nuclei. By this way, the percentage of target cells killed by effector NK cells can be determined.

APPLICATIONS

This test is intended to investigate altered NK function found in various disorders and to evaluate the effects of drugs on NK activity.

Depressed NK-cell mediated cytotoxicity is one of the many immunological defects observed in patients with AIDS, AIDS-related complex, or lymphadenopathy syndrome (1) with a concomitant decrease in relative and absolute NK cells. NK activity is also impaired in multiple sclerosis patients (2) and in patients with chronique fatigue syndrome (CFS) (3). Decreased NK-cell mediated cytotoxicity is generally observed in connective tissue disorders, in systemic lupus erythematosus (SLE), rheumatoid arthritis and Sjögren´s syndrome (4). NK activity in Typ I diabetes patients is also lower than in normal controls (5).
A depression of NK cell activity is usually observed in patients with advanced cancer (6). Patients with metastatic disease and patients with advanced metastases displayed significantly lowered NK cell cytotoxic activity. NK cell cytotoxic activity is of prognostic value for the probability of developing metastasis in patients with primary tumors. The recurrence of distant melanoma metastases has been found to be significantly lower in patients with high NK cell cytotoxicity than in those with low activity (7).
NK cells play also a central role in the defence against virus infection. An enhanced NK cell activity has been observed during several acute virus infections (8).

Various immunomodulators (e.g., cytokines (interleukin-2, interferons) seem to augment cytotoxic activity in vitro (9) and in vivo (10). This augmentation of NK cell activity can now be proven in an objective and reproducible manner with this testkit.

PRINCIPLES

The NKTEST® kit contains prestained cryopreserved K562 TARGET CELLS. This cell line derived from a patient with chronic myeloid leukemia in terminal blastic crisis (11) is the most sensitive and widely used target cell for human NK cells. K562 cells lack MHC class I and II antigens.
The basic requirement in any cytotoxicity assay is the ability to discriminate effector and target cell populations. The test kit contains cryopreserved K562 target cells prestained with a green fluorescent membrane dye. This dye remains in the membrane of the target cell thereby permitting a clear separation between effector and target cells. The lytic process is characterized by the following main stages: recognition of target cells by effector cells, binding of effectors to targets, activation of the lytic machinery of effector cells and lytic effects on target cells. NK cells kill the target cells either by a perforin-granzyme-based mechanism, which results in a perforin-mediated loss of membrane integrity, or by the direct or indirect induction of apoptosis in target cells. After incubation of effector and target cells a red fluorescent DNA dye is added to label the target cells permeabilized by NK activity. This dye labels only cells with compromised plasma membranes. In this way, a clear separation between four cell populations can be obtained: live target cells, dead target cells, live effector cells and dead effector cells. Thus, the actual ratio between effector and target cells can be confirmed. Routinely, only FL1 positive events (dead and live target cells) have to be collected for the determination of cytotoxic activity. Since monocytes can suppress NK cell cytotoxic activity, these cells may be removed from the mononuclear cell suspension by plastic adherence.

REAGENTS PROVIDED

• Prestained K562 TARGET CELLS
• INTERLEUKIN-2
• COMPLETE MEDIUM
• DNA STAINING SOLUTION

REFERENCES

(1)	Rosenberg, Z.F. & Fauci, A.S. 1989. The immunopathogenesis of HIV infection. Adv. Immunol. 47: 377-431.
(2)	Neighbour, P.A. 1984. Studies of interferon production and natural killing by lymphocytes from multiple sclerosis patients. Ann. N.Y. Acsad. Sci. 436: 181.
(3)	Caligiuri, M., Murray, C., Buchwald, D., Levine, H., Cheney, P., Peterson, D., Komaroff, A.L. & Ritz, J. 1987. Phenotypic and functional deficiency of natural killer cells in patients with chronic fatigue syndrome.
(4)	Sibbitt, W.L. & Bankhurst, A.D. 1985. Natural killer cells in connective tissue disorders. Clin. Rheum. Dis. 11: 507.
(5)	Negishi, K., Gupta, S., Chandy, K.G., Waldeck, N., Kershnar, A., Buckingham, B. & Charles, M.A. 1988. Interferon responsiveness of natural killer cells in type I human diabetes. Diabetes Res. 7: 49.
(6)	Kadish, A.S., Doyle, A.T., Steinhauer, E.H. & Ghossein, N.A. 1981. Natural cytotoxicity and interferon production in human cancer: Deficient natural killer activity and normal interferon production in patients with advanced disease. J. Immunol. 127: 1817.
(7)	Hersey, P., Edwards, A., McCarthy, W. & Milton, G. 1982. Tumor related changes and prognostic significance of natural killer cell activity in melanoma patients. In: "NK cells and Other Natural Effector Cells" (R.B. Herberman, ed.), p.1167. Academic Press, New York.
(8)	Cauda, R., Prasthofer, E.F., Grossi, C.E., Whitley, R.J. & Pass, R.F. 1987. Congenital cytomegalovirus: Immunological alterations. J. Med. Virol. 23: 41.
(9)	Henney, C.S., Kuribayashi, K., Kern, D.E. & Gillis, S. 1981. Interleukin-2 augments natural killer cell activity. Nature: 291: 335.
(10)	Deehan, D.J., Heys, S.D., Ashby, J. & Eremin, O. 1995. Interleukin-2 (IL-2) augments host cellular immune reactivity in the perioperative period in patients with malignant disease. Eur. J. Surg. Oncol. 21: 16-22.
(11)	Lozzio, C.B. & Lozzio, B,B. 1975. Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome. Blood 45: 321-334.

For further information please contact:

Dr. Werner Hirt
Head of Immunology, Cell Biology and Flow Cytometry
+49 6221 9105-50
e-Mail: w.hirt@orpegen.com