Suppression of Target Cell Proliferation by Natural Killer Cells

Abstract

The cytolytic effects of natural killer (NK) cells have been extensively studied in recent years. In the present study we have investigated the cytostatic effects of NK cells. Human peripheral blood lymphocytes from healthy volunteers were used as a source of effector cells, and the cell lines K562, U937, U1285, and Molt-4 were used as target cells. Effector cells were enriched for NK cells using Percoll gradients and depleted of NK cells on Percoll gradients or by using Leu-19 antibodies and magnetic beads. By monitoring cell numbers during co-culture of effector cells and K562, it was found that after an initial phase of cell killing for 3 h target cell numbers remained stable during the following 24-48 h. In a microcytotoxicity assay measuring inhibition of uptake of [3H]thymidine, the four target cell types were shown to have different NK sensitivity; inhibition of greater than or equal to 80% was obtained for K562 and U937 at an effector to target cell (E/T) ratio of 30:1, 50% for U1285, and 30% for Molt-4. This inhibition was shown to be partly a direct effect on DNA synthesis for all cell lines, as incorporation of [3H]thymidine was decreased in cocultured target cells compared with an equal number of target cells alone. Inhibition of DNA synthesis was thus not directly related to cell death and was also observed for the Molt-4 cell line that was not killed. A cell division assay, with target cells in agarose and effector cells in a liquid upper layer, showed a decline in the rate of target cell divisions. Effects on the cell cycle were studied on latent-phase cells. It was shown that effector cells delayed the onset of DNA synthesis. This anti-proliferative effect was observed for several days, but cell growth then gradually resumed. The effector cells were identified as CD56-positive large granular lymphocytes (LGL). Double-layer cultures and experiments using effector cell supernatants demonstrated that the growth-inhibitory effect could be mediated by soluble factors, and the production of such factors was stimulated by exposure to a small proportion of target cells (50:1). Studies with specific antibodies indicated that growth inhibition was not mediated by alpha interferon (IFN-alpha) but it was partly mediated by tumour necrosis factor alpha (TNF-alpha). It is concluded that NK cells have a growth-inhibitory effect that is distinct from the cytolytic effect and this activity is probably mediated by several soluble factors including TNF-alpha.