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Surface Features of Human Natural Killer Cells and Antibody-Dependent Cytotoxic Cells

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Surface Features of Human Natural Killer Cells and Antibody-Dependent Cytotoxic Cells J. Cell Sd. 77, 27-46 (1985) 27 Printed in Great Britain © The Company of Biologists Limited 1985 SURFACE FEATURES OF HUMAN NATURAL KILLER CELLS AND ANTIBODY-DEPENDENT CYTOTOXIC CELLS CLAIRE M. PAYNE1*, ALEC LINDE1, RUTH KIBLER2, BONNIE POULOS2, LEWIS GLASSER1 AND ROGER FIEDERLEIN1 'Department of Pathology and 2 Department of Microbiology and Immunology, University of Arizona, College of Medicine, 1501 N. Campbell Avenue, Tucson, Arizona 85724, U.SA. SUMMARY The purpose of the present study was to examine the surface features of purified large granular lymphocytes (LGLs) (natural killer (NK) cells, antibody-dependent cytotoxic lymphoid (ADCL) cells, K-cells, Fcy( + ) third population (non-T, non-B) lymphoid cells, Tr cells) by scanning electron microscopy (SEM) and to compare their surface features with granulocytes, monocytes and Fcy(—) lymphoid cells that were all fixed for SEM under identical conditions. We have determined that 72-80 % of LGLs enriched by rosette formation with sensitized erythrocytes or using Percoll gradients, have a complex microvillous surface (CMS) pattern identical to that of lymphocytes. The LGL fraction appears by SEM to represent a morphologically homogeneous population of cells. Monocytes prepared for SEM under identical conditions had distinct surface folds and granulocytes displayed numerous broad-based ridge-like profiles. The majority of lymphoid cells in an unfrac- tioned population have a CMS pattern when incubated at room temperature (25 °C) before fixation, and a sparse microvillous surface (SMS) pattern when incubated at body temperature (37°C). Ficoll-Hypaque (FH) also had a direct effect on the cell surface pattern. Over half of the unfrac- tionated lymphoid cells displayed a CMS pattern after cells were washed free of FH and incubated at 37 CC before fixation. The CMS pattern is therefore not unique to LGLs but can be produced by the surface alteration of non-LGLs found in unfractionated buffy coat and mononuclear fractions. The interactions between LGLs and sensitized erythrocytes in an antibody-dependent cytotoxic assay system, and LGLs and K562 target cells in an NK assay system, were also examined. This is the first report that describes the surface features of human LGLs interacting with K562 target cells in an NK assay system. The LGL populations studied by SEM were determined to have a high percentage ofLeu-ll( + )and Leu-7 ( + ) cells. These same populations were also shown to have high antibody-dependent cytotoxicity and NK activity using the Cr release assay. INTRODUCTION Human natural killer (NK) cells (Herberman & Ortaldo, 1981), antibody- dependent cytotoxic lymphoid cells (K cells) (Perlmann, 1976), Fcy ( + ) third popula- tion (non-T, non-B) lymphoid cells (Winchester, Fu, Hoffman & Kunkel, 1975) and Ty cells (Ferrarini et al. 1980) appear to comprise a unique leucocyte population that is characterized at the light-microscopic level by the presence of azurophilic granules (Timonen, Ortaldo & Herberman, 1981) and at the ultrastructural level by the presence of parallel tubular arrays (PTAs) (Burns, Zucker-FrankJin & Valentine, • Author for correspondence. Key words: SEM, NK cells. 28 C. M. Payne and others 1982; Henkart & Henkart, 1981; Payne & Glasser, 1981; Payne, Glasser, Fiederlein & Lindberg, 1983; Smit, Blom, van Luyn & Halie, 1983). This cell type is an important part of the immune system and is believed to function in tumour sur- veillance, resistance to infection by viruses and other microbes, and rejection of bone marrow grafts (Herberman & Ortaldo, 1981). Although they are referred to as large granular lymphocytes or LGLs (Timonen ef a/. 1981), recent evidence indicates that LGLs may have a myeloid origin (Gallief al. 1982; Kay & Horwitz, 1980; Lohmann- Mattes, Domzig & Roder, 1979; Neighbour, Huberman & Kress, 1982; Oehl et al. 1977). (The term 'myeloid' refers in this paper to non-lymphoid cells that include granulocytes and monocytes). Since LGLs represent a functionally heterogeneous population of cells with different surface antigens (Fitzgerald, Evans, Kirkpatrick & Lopez, 1983), it is conceivable that the LGL population may be mixed, with some subpopulations lymphoid in origin and others myeloid in origin (Zarling, Clouse, Biddison & Kung, 1981). On the other hand, other investigators believe that they may represent a unique population of cells with a separate haematopoietic lineage (Fitz- gerald et al. 1983; Horwitz et al. 1978; Ortaldo, Sharrow, Timonen & Herberman, 1981). Combined surface-marker and transmission electron microscopic studies from our laboratory have indicated that the Fcy( + ) lymphoid cells that characterize the LGL population (NK, K and Tr cells) are ultrastructurally unique (Payne & Glasser, 1981; Payne et al. 1983). The cells appear lymphoid in nature by both light microscopy and transmission electron microscopy (TEM), but possess unique cytoplasmic inclusions called PTAs. The PTAs can be used as an ultrastructural marker for the K (Henkart & Henkart, 1981; Payne & Glasser, 1981; Smit etal. 1983), NK (Babcock& Phillips, 1983; Burns et al. 1982; Henkart & Henkart, 1981) and TY (Payne et al. 1983) cells. We have shown, however, that at least some LGLs are capable of phagocytosing complement-coated bacteria (Payne & Nagle, 1980) and in this respect appear more myeloid than lymphoid in nature. Contrary to the enzymic content of myeloid cells, PTA-containing cells lack peroxidase activity (Payne & Nagle, 1980) that is so charac- teristic of granulocytes and monocytes. The scanning electron microscopic (SEM) features of human granulocytes (Hattori, 1972; Polliack, 1978), monocytes (Dantchev & Belpomme, 1977; Polliack, 1978) and lymphocytes (Dantchev & Belpomme, 1977; Hattori, 1972; Kelly & Nockolds, 1977; Newell, Roath & Smith, 1976; Polliack, 1978; Roath et al. 1978) are quite distinctive, but no SEM studies have been done to compare the surface features of these cells with LGL-enriched populations. Previous SEM studies of human LGLs have described the interaction between effector and sensitized target cells (Alexander & Henkart, 1976; Biberfeld, Wahlin, Perlmann & Biberfeld, 1975; Inglisef al. 1975; Kelly & Nockolds, 1977; Perlmann, 1976), but different experimental conditions used in various laboratories make it difficult to compare the surface features of these antibody-dependent cytotoxic effec- tor cells with purified cell populations. The surface features of human LGLs interact- ing with K562 target cells in an NK assay system have not previously been reported. In the present study, the surface features of LGLs were studied and compared with Quantitative SEM study of natural killer cells 29 Fcy(—) lymphoid cells, a granulocyte-enriched fraction and monocytes (that were fixed for SEM under the same experimental conditions) in order to determine if the LGLs have a unique surface appearance or represent a morphologically heterogeneous population of cells. A quantitative SEM evaluation of the effect of several experimental parameters used in the purification of LGLs is reported. MATERIALS AND METHODS Enrichment of LGLs (K, NK cells) by rosette formation ivith sensitized erythrocytes Unfractionated buffy coat fractions. Venous blood was collected by venipuncture from healthy human adults and drawn into heparinized evacuated tubes. Whole blood was spun at 200 gfor 10 min in a swinging-bucket clinical centrifuge. Most of the platelet-rich plasma was removed and discarded. The loose buffy coat layer was removed and mixed 1:1 with phosphate-buffered saline (PBS). A sam- ple of this cell suspension was allowed to stand at room temperature (25 °C) for 1 -5 h before fixing for SEM (fraction 1). A second sample was incubated at 37 °C for 1 -5 h before fixing for SEM (fraction 2). Mononuclear fraction. A PBS-buffy coat cell suspension prepared as above was incubated at 37 °C for 1 -5 h (fraction 2) and then layered over an equal amount of Ficoll-Hypaque (Lymphocyte Separating Medium (LSM), Litton Bionetics, Kensington, MD). The cells were centrifuged at 800gfor 20 min in a swinging-bucket clinical centrifuge and the cells at the interface were removed. One sample was immediately fixed for SEM (fraction 3); another was rinsed with PBS and allowed to incubate at 37CC in PBS for 3 h before fixing for SEM (fraction 4). Monocyte-depleted lymphoid fraction. A mononuclear fraction obtained after Ficoll-Hypaque density-gradient centrifugation was incubated at 37 °C for 30 min with LSR (Technicon Instrument Corp., Tarrytown, NY). The LSR contains /im-sized magnetic particles sensitized with poly-L- lysine. Following incubation, the cells were passed through Tygon tubing wrapped around a magnet that effectively removed iron-laden phagocytes and excess iron filings. LGL/EAiu, rosettes. This procedure selects lymphoid cells that have high-avidity Fcr receptors for cytophilic antibody. Cells that function in an antibody-dependent cytotoxic assay system and in an NK assay system are both enriched by this procedure. The monocyte-depleted fraction (106 cells/ml) was mixed with an equal volume of 1 % sensitized RiRa human erythrocytes (Payne & Glasser, 1981), centrifuged for 5 min at 200 £ and placed upright in tubes at room temperature for 30 min. The cell pellet was gently resuspended and a sample prepared for SEM (fraction 5). LGL(Fcr(+) cells)- and non-LGL(Fcr(—))-enriched fractions. A sample of the rosetted cell preparation from above (fraction 5) was layered over Ficoll-Hypaque and centrifuged at 400 £ for 30 min. The interface cells were removed,
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