Tuomo Timonen

Isolation of human and rat natural killer cells

We describe a method for the purification of human and rat large granular lymphocytes (LGL), which are known to be the mediators of natural killer (NK) activity in these species. Plastic non-adherent and nylon wool passed blood mononuclear cells were separated into 7 fractions by discontinuous density gradient centrifugation on Percoll. Low density cells were highly enriched in LGL (up to 85% purity), whereas high density cells were typical small and medium sized lymphocytes devoid of NK activity. Human LGL could further be enriched by depleting high affinity sheep erythrocyte rosette-forming cells from the LGL-enriched Percoll fractions (resulting in up to greater than 90% purity). One critical variable in the separation technique was osmolarity, since the separation did not work optimally, if 290 mOsmoles/kg H2O in the Percoll solution was exceeded.

Antitumor reactivity in vitro and in vivo of lymphocytes from normal donors and cancer patients propagated in culture with T cell growth factor (TCGF)

Abstract Peripheral blood lymphocytes (PBL) from 38 normal donors and from 27 cancer patients were propagated in bulk cultures for 3–6 weeks using T cell growth factor (TCGF). In addition, cultures derived from lymphocyte preparations enriched for or depleted of natural killer (NK) cells and several clones of cultured cells were studied. The following main observations were made: (a) PBL of both patients and healthy donors could be expanded to large numbers (up to 2500-fold); (b) CLC derived from unfractionated PBL exhibited intermediate levels of cytotoxic activity against autologous and allogeneic fresh lung tumor cells and strong cytotoxicity toward several cultured adherent tumor cells; (c) whereas cultures originated from populations enriched for NK cells were highly cytotoxic against both adherent tumor target cells and against an NK-sensitive leukemic cell line (K562), cultures derived from populations depleted of NK cells were preferentially cytotoxic to adherent target cells; (d) clones of CLC were also strongly cytotoxic, but 2 out of 3 clones tested showed a narrower spectrum of target cytotoxicity than that of uncloned CLC; (e) CLC, when mixed with two carcinoma cell lines, were able to inhibit tumor growth in nude mice.

ASSOCIATION OF HUMAN NATURAL KILLER CELL ACTIVITY AGAINST HUMAN PRIMARY TUMORS WITH LARGE GRANULAR LYMPHOCYTES

Publisher Summary This chapter examines the association of human natural killer (NK) cell activity against human primary tumors with large granular lymphocytes (LGL). It discusses the reactivity of allogeneic peripheral blood leukocytes (PBL) against several solid tumors. Five out of nine tumors were significantly lysed by PBL from 4 of 9 normal donors. As expected, the % cytotoxicity against many of them was not as high as it was against K562 or some adherent cell lines. The reactivity appeared to be restricted to some donors who were able to kill several targets. To know whether the reactivity against these tumors was mediated by NK or other effector cells, Percoll-gradient fractionation of nonadherent PBL was performed and used cells from the different fractions as effectors in the microcytotoxicity assays against spontaneous tumors. Effector cells from the low-density fractions were contained >60% LGL, whereas those from the high-density fractions were usually 80% small lymphocytes. The cytotoxicity against 7/12 primary tumors was mediated by LGL-containing cell populations. Using LGL-enriched fractions, cytotoxic reactions were observed with cells from some donors whose unseparated PBL were not reactive.

PRODUCTION OF INTERFERON BY HUMAN NATURAL KILLER CELLS IN RESPONSE TO MITOGENS, VIRUSES AND BACTERIA

Publisher Summary This chapter discusses the production of interferon (IFN) by human natural (NK) killer cells in response to mitogens, viruses, and bacteria. In a study described in the chapter, adherent cells were rigorously removed by incubation on Petri dishes for 2 h at 37°C and subsequent incubation on nylon wool columns for 30 min at 37°C. The remaining nonadherent cells, enriched in NK and T cells, were then placed on a 7 step discontinuous Percoll density gradient. After centrifugation at 550 g for 30 min, the fractions were collected and checked for large granular lymphocytes (LGL) morphology on cytocentrifuged slides. Cells in Fraction 3, which invariably contained 70–90% LGL, were then incubated overnight with various agents at 37°C. The following day, the supernatants were collected for IFN testing and the cells were tested for NK activity against K562 tumor cells. Removal of T cells with monoclonal OKT3 plus complement from the LGL had no effect on the IFN production or enhancement of NK activity. It was found that incubation of LGL with silica or carrageenan, which inactivates macrophages, did not interfere with the two functions.

Mechanisms of Tumor Cell Lysis by Natural Killer Cells

Immune reactivity against malignant cells is well-documented for a number of lymphoid cell types (1). The foremost features of anti-timor immune effector cells appears to be their capacity to recognize and subsequently kill tumor cells. Nevertheless, the mechanism(s) by which effector cells of the immune response mediate tumor cell lysis is largely unknown. The lack of precise knowledge concerning lytic pathways is evident for well studied immune killer cells, such as cytotoxic T Ijrmphocytes, as well as for effector cells that have only recently received intense experimental scrutiny, such as natural killer (NK) cells.

[57] Assay of augmentation of natural killer cell activity and antibody-dependent cell-mediated cytotoxicity by interferon

Publisher Summary This chapter presents methods for augmentation of human natural killer (NK) and antibody-dependent cell-mediated cytotoxicity (ADCC) activities by interferon (IF). For detailed studies of the effects of IF on NK and ADCC activities, it is very helpful to work with enriched populations of effector cells. The large granular lymphocytes (LGL), separated on discontinuous Percoll density gradients, have been found to account for virtually all of the NK and ADCC activities. Separation of the LGL, with high cytotoxic activity and almost devoid of other lymphoid cell types, is particularly advantageous for examination of the mechanism of augmentation of function, and the associated biochemical changes, induced in these cells by IF. The percentage of ADCC is calculated by subtracting the percentage of cytotoxicity against the non-antibodycoated target cell from that obtained with the antibody-coated target.

THE ROLE OF NEUTRAL SERINE PROTEASES IN THE MECHANISM OF TUMOR CELL LYSIS BY HUMAN NATURAL KILLER CELLS

Publisher Summary This chapter describes the role of neutral serine proteases in the mechanism of tumor cell lysis by human natural killer (NK) cells. NK cells comprise the key lymphoid effector cell subpopulation involved in natural cell-mediated immunity against primary and metastatic tumors. A number of studies have probed the biochemistry of the NK lytic mechanism, and studied the role of lysosomal enzymes, cellular hydrolases, and phospholipases and phospholipid methylation. The role of neutral serine proteases in tumor cell lysis has also been examined relative to the mechanism of cytolytic, activated macrophages, and it appears that tumor cell lysis mediated by macrophages is dependent, to some extent, on the secretion of neutral serine proteases. It appears that the ability of macrophages to bind to tumor cells and to secrete a cytolytic protease are independent functions, each of which are required for macrophage mediated lysis, and it has also been reported that the macrophage cytolytic protease interacts with hydrogen peroxide in a synergistic fashion in the cytolytic mechanism of this effector cell. A role for neutral serine proteases have also been suggested for the mechanism of K-cell killing in antibody-dependent cell-mediated cytotoxicity.

ANALYSIS OF NATURAL KILLER ACTIVITY OF HUMAN LARGE GRANULAR LYMPHOCYTES AT A SINGLE CELL LEVEL

Publisher Summary This chapter reviews natural killer (NK) activity of human large granular lymphocytes (LGLs) at a single cell level. LGLs comprise approximately 10% of peripheral blood lymphoid cells, and they can be enriched up to ≥ 90% purity by discontinuous density gradient centrifugation and subsequent depletion of high-affinity sheep erythrocyte rosette-forming cells from low density, LGL-enriched populations. In a study described in the chapter, the single-cell cytotoxicity assay in agarose was used to estimate the frequency of LGL capable of binding and lysing NK-sensitive target cells. The results indicate that up to 70% of LGL are NK cells. An additional parameter that affects the lytic potential of killer cells is their recycling capacity. It was demonstrated in another study described in the chapter, by using density gradient-purified LGL-target cell conjugates, that recycling is involved in NK lysis. It was shown that interferon can augment all of the above mentioned phases of NK cell-mediated cytotoxicity. The data obtained do not indicate that LGL are a homogenous subpopulation of lymphoid cells. On the contrary, about 20–30% of them have no detectable NK activity, there is a clear size and density heterogeneity among LGL, and they are antigenically heterogeneous.

PLEIOTROPIC EFFECTS OF INTERFERON (IFN) ON THE AUGMENTATION OF RAT NATURAL KILLER (NK) CELL ACTIVITY

Publisher Summary This chapter discusses the effects of IFN on the ability of large granular lymphocytes (LGL) to bind to target cells and to subsequently lyse them. A model is proposed for augmentation, which includes four stages of LGL activation. In a study described in the chapter, enriched LGL preparations were separated by discontinuous Percoll density gradients and checked for purity by morphological examination of Giemsa stained preparations. The results demonstrated that the vast majority of nylon-wool-passed nude blood and spleen cells that formed conjugates were LGL. Other intermediate forms of LGL and other sites of IFN action may also occur but have not been identified using present techniques. The differentiation of NK-precursor cells from the bone marrow into LGL and the possible involvement of IFN in this process were not examined.