Relationship between target cell cycle and susceptibility to natural killer lysis (original) (raw)
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Cellular Immunology, 1986
Natural cell-mediated cytotoxicity (NCMC) against a number of target cells is mediated by at least two distinct effector populations, with natural killer (NK) and natural cytotoxic (NC) cells being the predominant in the murine system. The studies described in this report examine the role that the phase of the mitotic cycle of the target cell has on its susceptibility to lysis by NC and NK cells. We show that neither the kinectics nor the magnitude of NC cell lysis is altered when assayed using target cells which have heen enriched for G 1, S, or G2 + M stages of the cell cycle. Similarly, NK cell lysis by fresh or poly-IC augmented effector cells was not effected by target cell cycle.
Role of target and effector cell structures in natural killer-mediated cytotoxicity
La Ricerca in clinica e in laboratorio
An analysis of target and effector cell structures involved in the in vitro natural killer (NK)-mediated cytotoxicity has been performed. The degree of surface expression of transferrin receptor (TR) was only in part correlated with that of cell lysis. Moreover, the lysis could not be blocked by treating target cells with two anti-TR monoclonal antibodies. Finally, cell lines poorly affected by NK cells express TR only at the cytoplasmic level. As to the effector cells, the integrity of cytoskeleton components (especially microtubules) was found to be essential for the occurrence of cell lysis. In fact, vinblastine, an anti-microtubule agent, was able to significantly reduce the percentage cell lysis. This effect was not due to a selective depletion in NK cells induced by the drug. It is concluded that the mechanisms underlying NK activity are complex and involve both target and effector cell structures.
Cellular Immunology, 1985
Monoclonal antibodies (MoAb) against cell surface determinants were employed to investigate the specificity of natural killer (NK)-like lysis by cloned human effector cells recognizing only K562, only HSB2, or both K562 and HSB2 target cells. MoAb W6/32.HL, m39, YDl/ 48.HLK, and anti-Tat failed to inhibit lysis despite the expression of antigens bound by these MoAb on the effector cell surface. MoAb OKT3 moderately (<50%) blocked lysis of K562 and HSB2 targets, whereas MoAb 13.1, which binds T200 molecules, strongly (up to 95%) blocked lysis of K562, but not HSBZ, targets. MoAb 13.1 inhibited lysis by clones which killed only K562, as well as lysis by those which killed both HSB2 and K562. In the latter case, however, only lysis of K562 was inhibited. Taken together, these results may suggest the existence of multiple receptor specificities on a single NK-active clone. 0 1985 Academic FWS, h.
Kinetics of in vitro natural killer activity against K562 cells as detected by flow cytometry
Cytometry, 1998
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 cytokinestimulated 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. Cytometry 32:280-285, 1998.
Relevance of target cell-induced apoptosis as mechanism of resistance against natural killer cells
Annals of Hematology, 2010
Natural killer (NK) cells contribute to the graftversus-leukemia effect after allogeneic stem cell transplantation. However, the efficacy of NK cell-mediated tumor cell lysis is limited due to target cell resistance, and target cellinduced apoptosis (TiA) was proposed to contribute to differences in susceptibility to NK cells. Here we analyzed the effects of target cells on the apoptosis of cytokineactivated NK cells in vitro. We found no association of target cell susceptibility and TiA of NK cells in an array of human and murine target-effector cell combinations. Incubation of NK cells with caspase inhibitors blocked TiA incompletely, indicating that TiA is partly based on caspase-independent mechanisms. Modulating NK cell susceptibility against TiA by caspase inhibition did not influence cytotoxic efficacy. Furthermore, we found cytotoxic potential of NK cells to be markedly decreased following first target cell contact. Exhaustion of NK cell activity by first target cell contact was, however, not mediated by TiA. In addition, we found no relevant TiA by lymphoma cell lines against activated murine NK cells. We conclude that TiA represents only a minor factor of target cell resistance against NK cell-mediated cytolysis.
International Journal of Immunopharmacology, 1983
The effects of differentiation inducers on the sensitivity of two human myeloid cell lines, K562 and HL-60, to natural killer (NK) cell lysis were examined. K562 cells treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA) and HL-60 ceils treated with either TPA or dimethylsulfoxide (DMSO) not only became less sensitive NK targets, but also became less competitive in cold target inhibition experiments. Target binding cell assays revealed that TPA-treated target ceils bound fewer NK effector cells than did untreated targets. TPA treatment renders K562 and HL-60 target ceils more susceptible to hypotonic lysis, indicating that the decreased NK sensitivity is not due to altered osmotic fragility. The observed reduction in sensitivity to NK lysis was not apparently mediated by interferon (IFN) released by the target cells, as neither alpha nor gamma IFN were detected in culture supernatants. Furthermore, the effects of TPA were additive to the known protective effect of IFN on NK target cells. In contrast to the parent line, a subclone of HL-60 resistant to chemically induced differentiation did not become a less sensitive target after exposure to TPA. These observations with myeloid and erythroid target cells imply that the reduced killing of the TPA-treated cells was secondary to impaired recognition of the target by the effector, and that the state of cellular differentiation is a major determinant of susceptibility to NK cell-mediated lysis.
On the heterogeneity of murine natural killer cells
Journal of Experimental Medicine, 1981
The heterogeneity of cells capable exerting spontaneous cytotoxicity in vitro was explored using antisera to several genetically determined surface markers on mouse lymphocytes. Four phenotypes of cells derived either from fresh or cultured murine lymphoid tissue were found to exert natural killer (NK) activity in vitro. One affector cell subset, termed NKI cells, had the serological phenotype of Thy-1-, Lyt-2-, Qa5+, and lysed measles virus persistently infected target cells (HeLa-Ms) but not P815 mastocytoma cells. It corresponds with the NK cells described in most systems in which lymphoma targets are commonly used. A second subset, with the same target cell specificity, termed NKT is a thymus-independent cell with the phenotype Thy-1+, Lyt-2-, Qa-5+, Ly-5+. A third subset of NK cells, termed T killer (TK) cells deriving from cultures of conventional but not nude mouse spleens, mediated spontaneous cytotoxicity of P815 mastocytoma cells, but not of virus-infected targets. It has ...
Natural killer function in flow cytometryI. Evaluation of NK lytic activity on K562 cell line
Journal of Immunological Methods, 1988
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.
International Journal of Cancer, 1981
Cultured cell lines derived from malignant Epstein-Barr virus (EBVFnegative B cells, and representative of the more common types of naturally occurring non-Hodgkin lymphomas and related leukemias, were found to be sensitive to lysis by human natural killer (NK) cells. The observed lysis of such cell lines was mediated by a population of interferon-augmentable, FcR-positive, non-adherent lymphoid cells, which were also able to kill the "standard" NK targets K562 and Molt-4. When the NK susceptibility of the neoplastic, EBV-negative B cells was compared to that of diploid, EBV-carrying, non-neoplastic B lymphoblastoid cell lines (BLCLC) and of the "standard" NK target K562, several distinct patterns were observed. The killing of the neoplastic B cell lines was significantly less than that of K562, but significantly greater than that of the EBV-derived BLCLS of non-neoplastic origin. An additional finding was a similar NK susceptibility profile for the neoplastic, true histiocytic cell line U-937 (i.e., K562>U937> BLCLC). Furthermore, all cell lines, with the exception of the BLCLC, could effectively compete for the observed killing in cold target inhibition assays. The implications of these findings are discussed in relation to both neoplastic and non-neoplastic targets of NK lysis.
Natural killer cell cytotoxic activity: measurement of the apoptotic inducing mechanisms
Clinical and Experimental Medical Sciences, 2013
Natural Killer cell cytotoxic activity is vital for the clearance of viral and malignantly transformed cells. The aim of this study was to investigate the apoptotic inducing mechanisms of NK cells to propose an additional measurement for NK cell effector function. 19 healthy controls (age=31±7.2 years) participated in this study. Flow cytometric protocols assessed NK cell cytotoxic activity against tumour K562 cells, lytic proteins, degranulation and interferon gamma production. Perforin release was significantly correlated with cytotoxic activity (r=-0.46, p<0.05) and degranulation (r=-0.60, p<0.05). These results suggest that perforin may be an additional measure for NK cell cytotoxic effector function.