Katarina Radosevic

Production and characterization of monoclonal antibodies raised against recombinant human granzymes A and B and showing cross reactions with the natural proteins

The human serine proteases granzymes A and B are expressed in cytoplasmic granules of activated cytotoxic T lymphocytes and natural killer cells. Recombinant granzyme A and granzyme B proteins were produced in bacteria, purified and then used to raise specific mouse monoclonal antibodies. Seven monoclonal antibodies (mAb) were raised against granzyme A, which all recognized the same or overlapping epitopes. They reacted specifically in an immunoblot of interleukin-2 (IL-2) stimulated PBMNC with a disulfide-linked homodimer of 43 kDa consisting of 28 kDa subunits. Seven mAb against granzyme B were obtained, which could be divided into two groups, each recognizing a different epitope. On an immunoblot, all mAb reacted with a monomer of 33 kDa protein. By immunohistochemistry, these mAb could be used to detect granzymes A and B expression in activated CTL and NK cells. The availability of these mAb may facilitate studies on the role of human cytotoxic cells in various immune reactions and may contribute to a better understanding of the role of granzymes A and B in the cytotoxic response in vivo.

A simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells

A new, simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells is described. The assay is based on the use of two fluorochromes. The target cell population is stained with one fluorochrome (octadecylamine-fluorescein isothiocyanate, F-18) prior to incubation with the effector cells. F-18 remains in the membrane of the target cells even when they are killed thereby permitting a clear separation between effector and target cells. Dead cells are determined by staining with a second fluorochrome (propidium iodide) after incubation of effector and target cells. staining with a second fluorochrome (propidium iodide) after incubation of effector and target cells. F-18 is not toxic and does not decrease the cytotoxic activity of human natural killer cells. It is also stable (exchange between labeled and non-labeled cells is negligible in a period of at least 4 h at 37 degrees C) and it remains in the membrane of the killed cells. A clear distinction between unlabeled effector and labeled target cells is obtained, even after incubation of target and effector cells for 4 h at 37 degrees C and using a high effector cell-target cell ratio (75:1). A good correlation with the 51Cr release assay was obtained. A potential application of the flow cytometric cytotoxicity assay using whole blood instead of isolated lymphocytes is presented.

Atomic force microscopy combined with confocal laser scanning microscopy: a new look at cells

A stand-alone atomic force microscope (AFM) has been developed, which features a large scan area and which allows operation under liquid. This system was combined with a confocal laser scanning microscope (CLSM). Information about cell structures, obtained by CLSM, can be complemented with images of the cell surface obtained with the AFM. This is illustrated by studying the pseudopodia of cells from a human cell line (K562-cells, predecessor of erythroblasts) and the cytoskeleton of monkey kidney cells (in air and under liquid), both stained with F-actin-specific fluorescent probes. Images of the cytoskeleton during the cytotoxic interaction between a natural killer and a K562 target cell are presented. Our results show that combination of these techniques can provide new information about cells and cellular structures.

A flow cytometric study of the membrane potential of natural killer and K562 cells during the cytotoxic process

This study demonstrates that it is possible to investigate the membrane potential of interacting cells during the cytotoxic process using flow cytometry. Changes in the membrane potential of NK and K562 cells, involved in a cell-mediated cytotoxic process, were studied by standard and slit-scan flow cytometry, using the membrane potential sensitive fluorescent probe DiBAC4(3). The NK cells were labeled with a membrane marker (TR-18 or DiI) prior to incubation with K562 cells and the conjugates that were formed could be identified on the basis of the membrane marker fluorescence and light scattering signals. With a slit-scan technique we measured the membrane potential of each cell in a conjugate separately. The results show that depolarization of the K562 cell occurs as a consequence of the cytotoxic activity of the NK cell. This depolarization appears to be an early sign of cell damage because the cell membrane still remains impermeable to propidium iodide. Our data also indicate that depolarization of the NK cell occurs as a result of its cytotoxic activity.

Use of Different Cytometric Techniques to Study the Cytotoxic Interaction between Human Natural Killer Cells and k562 Target cells

The studies described in this manuscript have been aimed to obtain a better insight into the cytotoxic interaction between human Natural Killer (NK) cells and K562 target cells1. In order to obtain information about crucial processes leading to target cell death, the processes taking place in the target cell during a killer cell attack were studied using different optical techniques. Furthermore, flow cytometric methods for determination of the phenotype and the cytotoxic activity of killer cells were developed.

Assessment of Lymphokine-Activated Killer Activity Against Myeloid Leukemic Blasts and the Myeloid Cell Line K562 by Flow Cytometry

It is well known that recombinant interleukin-2 (rIL-2) and other lymphokines can be used to increase cell mediated cytotoxicity of PBL against non-HLA restricted targets. Futhermore rIL-2 is able to induce cell mediated cytotoxicity of PBL against tumor cells which were first insensitive to cell mediated cytotoxicity. These activated lymphocytes are called lymphokine-activated-killer (LAK) cells. It is of great importance to determine the functional capacities of these activated cells for killing specific target cells like leukemic blasts. In this study we determined the functional capacity of activated killer cells from healthy donors for killing blasts from acute myeloid leukemia (AML) patients and compared it with the capacity for killing K562 cells. We use newly developed flow cytometric assays for determination of cytotoxicity [1] and conjugate formation. In this way we obtain information on whether leukemia related defects in the killing mechanism occur and to what extent it is possible to overcome these defects by using specific cytokines.