A.R. Mariani

Age-associated changes in CD8+ and CD16+ cell reactivity: clonal analysis.

A cloning technique was used to estimate the frequency of proliferating T cell precursors, the growth capacity of clone‐forming cells and the functional activity of clones established in vitro from peripheral blood lymphocytes of young and old people. The mean frequency of proliferating precursors was lower in the elderly as was the proliferative capacity of CD8+ clones. In contrast, CD4+ and CD16+ clones showed a proliferation similar to that obtained from young subjects. When the clones were examined for their functional activity, CD4+ clones from both groups failed to show any cytolytic activity, while CD8+ clones exerted cytolysis against K562 and in antibody‐dependent cell‐mediated cytotoxicity but this function was reduced in clones derived from old subjects. Similarly, CD16+ clones from the elderly showed a decreased activity at some effector‐to‐target cell ratios. We conclude that the impaired functional activity (T or NK‐dependent) found in the peripheral blood of aged subjects persists after in vitro selection when these cells are analysed at clonal level.

Natural killer function in flow cytometry: I. Evaluation of NK lytic activity on K562 cell line

Flow cytometry has been employed to establish an NK assay using the K562 target cell line. These cells show a perpendicular light scatter (PLS) characteristically different from lymphocytes. Other physical parameters, such as forward light scatter (FLS), do not discriminate between the two populations, since dead K562 cells display similar FLS characteristics as effector cells. Killed cells are stained with propidium iodide and followed by flow analysis. It was advisable to add the dye in the medium so that, as long as the target cells are killed they will also be stained. Moreover the flow cytometric and trypan blue evaluation of target cell death rate showed a stronger correlation than did either test with the conventional 51Cr release assay, the first two methods both being based on the same biological mechanism.

Kinetics of in vitro natural killer activity against K562 cells as detected by flow cytometry

Natural killer (NK) cells bind to K562 tumor target cells in vitro and kill them. The binding and cytotoxic activities of NK cells are tightly related to each other: degranulation of the cytotoxic effector is the basis for target cell damage and a consequence of effector-target recognition and binding. However, the two phases of NK activity, binding and killing, have always been measured separately by various methodologies and under different experimental conditions, because of the lack of a comprehensive methodology able to measure both of them at one time. Here we describe the simultaneous measurement of the binding and killing activities against K562 of resting and cytokine (IL-2 or IL-12)-stimulated NK cells by flow cytometry. NK, K562 and conjugates can be identified and measured by flow cytometry on the basis of NK mAb staining and target cells autofluorescence (Binding Plot). Within each population of the binding plot, killed targets can be identified and measured by their scatter characteristics (Cytotoxicity Plot). We show that i) the conjugate formation is enhanced in cytokine-stimulated cells, even at relatively short co-incubation times; ii) the conjugate release is also accelerated by cytokines; iii) the conjugate release is always quicker than the induction of the morphological changes in the target cell that generate its modified scattering properties.

NK-active cytokines IL-2, IL-12, and IL-15 selectively modulate specific protein kinase C (PKC) isoforms in primary human NK cells

Natural killer (NK) cell function is largely modulated by growth factors and cytokines. In particular, interleukin (IL)‐2, IL‐12, and IL‐15 have major effects on the proliferative and cytotoxic activities of NK cells against tumor and virus‐infected cells. It is thought that the members of the protein kinase C (PKC) family of serine/threonine kinases play an important role in mediating the pleiotropic effects of cytokines on their target cells. We have investigated the downstream effects generated in purified human NK cells by IL‐2, IL‐12, and IL‐15 on PKCα and PKCϵ—a canonical and a novel isoform of PKC, respectively. By means of Western blotting, PKC activity assays, and immunofluorescence performed on highly purified preparations of primary human NK cells, we demonstrate that: 1) the three cytokines have similar effects on PKCα and PKCϵ activities; 2) whereas PKCϵ activity is induced by cytokine stimulation, PKCα activity is inhibited; and 3) both the induction of PKCϵ and the inhibition of PKCα functional activity are relatively early events in NK cells, while longer cytokine stimulations do not generate significant variations in enzyme activity, suggesting that the activation of both the canonical and novel isoforms of PKC are events required in the early phases of cytokine‐induced NK cell stimulation. Anat Rec 266:87–92, 2002.

Morpho-functional changes in human tendon tissue

Insertion tissue biopsies of right arm common extensor tendons from 11 patients with chronic lateral epicondylitis were processed for light and electron microscopy. The subjects were aged between 38 and 54 years (only one was 25). The specimens showed a variety of structural changes such as biochemical and spatial alteration of collagen, hyaline degeneration, loss of tenocytes, fibrocartilage metaplasia, calcifying processes, neovascularization and vessel wall modifications. Tissue alterations were evident in limited zones of the tendon fibrocartilage in which the surgical resection was generally visible. The areas where the degenerative processes were localized, were restricted and in spatial contiguity with morphologically normal ones. The observed cases presented histological and electron microscopic findings that characterize lateral epicondylitis as a degenerative phenomenon involving all tendon components.

Use of poligonal windows for physical discrimination among mononuclear subpopulations in flow cytometry

Peripheral blood mononuclear cells from ten healthy normal donors were analyzed by means of monoclonal antibodies and flow cytometry (FACS IV). In order to apply optimal gating for the identification and exclusion of monocytes from lymphocyte populations, mononuclear cells were analyzed on the scattergram using rectangular or poligonal computer-generated windows. Two main windows on lymphoid population were used which mainly differed for the up-right corner, in the border area between lymphocytes and monocytes. While the number of lymphocytes contained either in the regular or in the poligonal windows was the same, the number of contaminating monocytes decreased by two-fold in the poligonal one. Besides, the use of tight lymphocyte gating, in order to reach lower monocyte contamination, leads to a loss of lymphoid cells which does not appear to be random, but seems to affect mainly the Leu11c+ population with natural killer activity. These cells produce forward and perpendicular scatter signals higher than other lymphocyte subsets, and, therefore, are mainly located in the area of the scattergram which divides lymphocytes from monocytes. These data are in accordance with the large granular morphology of natural killer cells. The use of the poligonal windows seems to be useful to reduce monocyte contamination with no selective loss of natural killer lymphocytes, and may be particularly helpful in the analysis of pathological samples.

Commercial serum-free media : hybridoma growth and monoclonal antibody production

Various growth factors, hormones and proteins present in serum are presumed to be responsible for its growth-stimulating activity in culture media. However, synthetic or serum substitute media supporting cell growth are advantageous when it is necessary to standardize culture conditions, particularly when cell products are used. In this study we have evaluated and compared the effects of some commercially available serum substitute media (10% NU Serum, 10% BMS, 2% Ultroser HY, 1% ITS + Premix, 1% Nutridoma, Ultradoma, FEB100, DCCM1 and DCCM2) on growth and immunoglobulin production in different hybridoma cell lines. Six different hybrids were studied: OKT3, OKT4 and OKT8 (producing monoclonal antibodies against CD3, CD4 and CD8 molecules), HB43 and HB57 (producing monoclonal antibodies against human IgG and IgM), and CRL 8019 (producing monoclonal antibodies against high-molecular weight CEA). Several parameters were evaluated, such as viability, doubling time, DNA synthesis, monoclonal antibody production and cell cycle under different culture conditions. Since not all of the hybridomas grew equally well in the same serum substitute media, one synthetic medium cannot be used for all the lines. We found that the serum-free media that best supported hybridoma growth were Nutridoma, DCCM1, DCCM2, NU Serum and FEB100.

Standardization of a micro-cytotoxicity assay for human natural killer cell lytic activity

Cytotoxicity assays are widely used to evaluate the functional activity of NK and T cells against tumour target cells and the release of radioactive sodium chromate from labelled target cells is still the most commonly used marker of target lysis in culture supernatants. We describe here the standardization of a micro-cytotoxicity test in which the number of cytolytic effector and tumour target cells have been decreased by a factor of 10. The release obtained by 500 tumour target cells was compared with the release obtained by 5000 target cells in the standard cytotoxicity assay for target:effector cell ratios from 1:1 to 1:100. Both gamma and beta emissions of the 51Cr isotope were evaluated to determine the assay release. The results obtained by the micro-cytotoxicity assay (500 target cells) were comparable to those of the standard assay (5000 target cells) and 51Cr release evaluation using the gamma counter was the most sensitive method of determining lytic activity using 500 tumour target cells. beta counter evaluation using solid phase scintillation was found to be a reproducible alternative method, even if the lytic curves cannot be compared with those obtained using the traditional method.

Lineage-related susceptibility of human hemopoietic cell lines to apoptosis.

Apoptosis plays a fundamental role in shaping normal hematopoiesis. We have investigated the relationship existing between susceptibility to apoptosis and lineage commitment in hemopoietic cells.

Evaluation of NK-to-target cell binding and evidence for T cell conjugates by flow cytometry

A flow cytometric methodology was set up to assess the binding capability of peripheral blood NK and T cells to the K562 tumor cell line. Differential side scatter characteristics between effectors and targets were used to analyze conjugated and unconjugated cells. The previous labeling of NK and T cells with anti-Leu 11c and anti-Leu 4 monoclonal antibodies, allowed the distinction between unconjugated non-fluorescent and conjugated fluorescent targets and the percentual evaluation of bound anti-Leu 11c+ and anti-Leu 4+ cells.