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1.
Cytological observations on the cytotoxic interaction between lymphocytes and antibody-coated monolayer cells 总被引:5,自引:0,他引:5
Chang cells in monolayers were killed when exposed to highly purified human peripheral blood lymphocytes in the absence of complement and in the presence of a concentration of heat inactivated rabbit anti-Chang cell serum (ACS) chosen for inability by itself to cause cytolysis. As evident by plaque formation, the cytotoxic reaction was confined to areas of the cultures covered with lymphocytes. In the presence of ACS, lymphocytes attached to and infiltrated the monolayers. Uropod formation indicated activation of the lymphocytes. Close contact was established between effector and target cells. The cytotoxic reaction was not accompanied by protracted cytopathological changes but led to detachment and rapid lysis of the target cells. Horse anti-thymocyte globulin, added to the cultures, completely inhibited plaque formation and lymphocyte infiltration. 相似文献
2.
The kinetics and rate of T cell-mediated cytolysis was assessed by measuring the times required for lysis of isolated target cells by single cytotoxic lymphocytes. Single target cell lysis was determined microscopically by observing trypan blue uptake as a function of time of incubation of effector-target conjugates in agarose. Lysis of EL-4 target cells by alloimmune peritoneal exudate lymphocytes was initiated without a lag and was essentially complete at 2 hr. Both zero-order and first-order kinetics equations were analyzed for fit to the 0 to 2 hr lysis values. Statistically, the zero-order kinetic function could be rejected (p greater than 0.05), but the first-order kinetics function (p less than 0.01) could not. This strong evidence for first-order kinetics of T cell-mediated cytolysis implies that within each CTL-target cell population, cytolysis occurs exponentially as a random decay process and that one event in the entire process of cytolysis is rate limiting. The first-order equation was then applied to measurements of the rate of cytolysis in many different individual effector-target cell combinations. Significant differences in the lytic rate were apparent when either the effector or target cell were varied, with the rate constants spanning a 5-fold range. The heterogeneity of lytic rates is consistent with the hypothesis that lytic efficiency is a function of both the effector and target cells used. 相似文献
3.
A thymic lymphoblastoid cell line derived from a New Zealand Black mouse produces murine leukemia virus (MuLV) and was used as a target in model systems for the in vitro study of antibody-dependent cellular cytotoxicity (ADCC). Several human lymphoblastoid cell lines were investigated as potential effector cells. The most promising (Raji cells) bound to antibody-coated target cells but caused only modest levels of ADCC at 25:1 effector-to-target cell ratio with substantial lysis in the absence of antiserum. Human peripheral lymphocytes were active as effector cells in ADCC at a 5:1 ratio and produced no lysis in the absence of antibody. These cells were used to demonstrate that high dilutions of rabbit antisera to MuLV antigens p30, p15, p12, and p10 were capable of mediating lysis of MuLV-producing target cells but not of a virus-negative murine cell line. A murine antiserum to Thy 1.2 and three caprine antisera to MuLV antigens that were active in complement-mediated cytotoxicity functioned poorly in inducing ADCC; however, rabbit antisera to similar antigens were 16- to 512-fold more efficient in cell-mediated than in complement lysis. The inefficiency of goat antisera was not due to shedding of cell surface antigens or generation of blocking factors but rather to lack of lytic interaction of antibody-coated targets with the effector cells. 相似文献
4.
We have analyzed the effect of various inhibitors of cellular secretion and motility on the cytolytic activity of human natural killer (NK) cells. As effector cells we used highly purified peripheral blood lymphocytes consisting of 75–85% large granular lymphocytes (LGL) that have previously been shown to be responsible for the NK activity in man. Treatment of the effector cells with a carboxylic ionophore monensin inhibited irreversibly the NK-cell-mediated killing. This drug is known to interrupt the vesicular traffic of Golgi-derived vesicles and thus the results strongly suggested that secretory processes are required in the cytolytic activity of human NK cells. In the monensin-treated effector cells large amounts of glycoprotein accumulated in the Golgi area within 24 hr of incubation. The lytic activity did not require intact microtubules since effector cells in which vinblastine-induced tubulin-containing paracrystals were demonstrated still mediated normal NK activity. Energy was required in the human NK-cell-mediated cytolysis. The lethal hit stage of the cytolytic activity was preceded by formation of intimate contacts between effector and target cells and required active cell movement and divalent cations. 相似文献
5.
Using the adhering property of certain types of cells to glass or plastic in human peripheral blood lymphocyte preparations, two populations of effector cells can be identified in the ABCIL system, each with a selective killing directed toward a different antibody coated target. The adherent cells which are radioresistant functionally, with the morphology of macrophages, exhibit strong cytotoxicity against the antibody-coated erythroid target while lacking any killing effect toward the antibodycoated target lymphocytes. The nonadherent cells which are radiosensitive and with the morphology of small lymphocytes, selectively kill the antibody-coated lymphocyte target without possessing any cytotoxicity against the antibody-coated target erythrocytes. 相似文献
6.
The ability of human peripheral blood lymphocytes to kill antibody-coated Chang liver cells in antibody-dependent cell-mediated cytotoxicity (ADCC) can be blocked with aggregated IgG (agg-IgG) or by soluble immune complexes. Dissociation of aggregates of IgG or immune complexes from the cell surface, however, resulted in partial recovery of the ability both to bind agg-IgG and to kill in the ADCC assay. Our results indicate that “unblocking” of effector cells could occur in vivo when the concentration of circulating immune complexes is lowered. 相似文献
7.
In a previous study, we demonstrated that human natural killer cells (NK) lost their lytic activity after interaction with a sensitive target. The loss of NK activity also led to the loss of antibody-dependent cellular cytotoxicity (ADCC), prompting us to postulate that NK and ADCC activities may result from a common lytic mechanism. In this study, we examined whether nonadherent lymphocytes cultured 7 days in the presence of IL-2 (lymphokine-activated killer (LAK) cells) could also be inactivated and, subsequently, be reactivated in the presence of IL-2. We tested three populations of effector cells (EC): cells isolated from freshly drawn blood and tested immediately, cells cultured with IL-2 for 18 hr, and LAK cells. Once they have interacted with K562, all three cell populations lost greater than 90% of their NK-like lytic activity (NK-CMC) but only 80% of ADCC. However, when we treated the three cell types with antibody-coated K562, they lost 90-99% of NK-CMC and 90-97% of ADCC. In these inactivated effector cells we also observed: (i) a reduction in membrane expression of C-reactive protein; and (ii) a decrease in the expression of Leu-11a when EC were inactivated with antibody-coated K562. The loss of lytic activity against K562 was accompanied by a concomitant loss of activity against other LAK-sensitive targets as well as against antibody-coated targets (ADCC). In competitive inhibition experiments the inactivated effector cells failed to inhibit normal NK-CMC and ADCC activities mediated by fresh NK cells. As we have shown previously, this target-directed inactivation was not due to cell death or to lack of conjugate formation. Inactivated LAK cells regained their lytic potential when cultured with IL-2 and this effect was time dependent. By 72 hr, LAK cells inactivated with K562 regained 99% NK-CMC and 82% ADCC, whereas LAK cells inactivated with antibody-coated K562 regained only 80% NK-CMC and 70% ADCC. When we treated the effector cells with emetine, a potent inhibitor of protein synthesis, we could still inactivate the effector cells with K562 and with antibody-coated K562 but could not reactivate them with IL-2. 相似文献
8.
N Matthews B P Maclaurin G N Clarke 《The Australian journal of experimental biology and medical science》1976,53(5):389-398
The normal human lymphocyte population which exhibits "spontaneous" cytolysis of EB2 Burkitt's lymphoma cells has been characterized. The effector cell has EA and EAC' receptors but lacks E receptors and probably surface Ig. "Spontaneous" anti-EB2 cytotoxicity was not reduced by preincubation of the effector cells with plastic or iron carbonyl or by passage through cotton wool or agarose columns but was reduced by passage through nylon wool columns. Thymocytes were not cytotoxic to EB2 cells, and chronic lymphocytic leukaemia cells (of B cell characteristics) had reduced cytotoxicity compared with normal lymphocytes. Cells from various lymphoid organs of rats and guinea-pigs were also cytotoxic to EB2 cells with reactivity in spleen greater than or equal to blood greater than lymph nodes. Spleen cells from neonatally thymectomized rats had increased cytotoxicity compared with normal rat spleen cells, suggesting that T lymphocytes are not essential. The effector cell in rat spleen did not adhere to cotton wool or agarose columns, indicating some resemblance to its counterpart in human peripheral blood. 相似文献
9.
Xenogeneic antiserum (RH1) was prepared in Lewis rats by hyperimmunization with concanavalin A- (Con A) activated alloimmune human lymphocytes. The antiserum RH1 effectively inhibited human antibody-dependent cellular cytotoxicity (ADCC), cell-mediated cytotoxicity (CMC), and natural killing (NK) in the absence of complement (C). Inhibition by RH1 was dependent on the dilution of antiserum employed and the number of cytotoxic lymphocytes present during cytolysis. Pretreatment of lymphocytes with RH1 or the presence of RH1 in culture did not inhibit lymphocyte proliferation stimulated by Con A, phytohemagglutinin, or allogeneic cells; lymphokine production as measured by leukocyte-inhibiting factor production; antibody-dependent C lysis; or CMC mediated by murine cytotoxic T lymphocytes. Analysis of the mechanism of inhibition of cytotoxicity by RH1 revealed that 1) RH1 was not cytotoxic for human lymphocytes at 37 degrees C in the absence of C; 2) purified F(ab')2 fragments were equally inhibitory as whole serum; 3) pretreatment of lymphocytes with RH1 effectively inhibited their capacity to mediate ADCC, CMC, or NK, and this effect was reversible by culturing the cells overnight at 37 degrees C; 4) RH1 did not inhibit target cell binding by K cells, effector cells of ADCC, or alloimmune T cells, but did inhibit binding by NK cells; and finally, 5) the addition of RH1 to preformed lymphocyte-target conjugates in a single cell cytotoxicity assay inhibited killing of the bound target cells in all three systems without disrupting the conjugates. Collectively, these findings suggest that RH1 antiserum interacts with structures present on the surfaces of cytotoxic lymphocytes that are involved in the activation of the lytic mechanism(s) or with the actual lytic molecule or molecules themselves. Furthermore, the ability of RH1 to inhibit ADCC, CMC, and NK during the post-binding cytolytic phase of these reactions indicates that binding and cytolysis are distinct and separate events in all types of cell-mediated cytolysis. 相似文献
10.
G. Masucci P. Wersäll P. Ragnhammar H. Mellstedt 《Cancer immunology, immunotherapy : CII》1989,29(4):288-292
Summary Human peripheral blood mononuclear cells (lymphocytes and monocytes) were preincubated for 0–24 h with human recombinant granulocyte-monocyte-colony-stimulating factor (GM-CSF) and used as effector cells in an 18 h antibody-dependent cellular cytotoxicity (ADCC) assay with SW948 (a human colorectal carcinoma cell line) as target cells and mAb 17-1A. A significant increase in the lytic capability was noted after 0.5–2 h of preactivation while longer preincubation times did not significantly increase the lytic potential. GM-CSF at 0.01 g/ml induced the best tumor cell lysis while higher concentrations were inhibitory. GM-CSF pretreatment induced a statistically significant increase in the lytic capacity of both monocytes and lymphocytes in ADCC as well as in the spontaneous cytotoxicity. 相似文献
11.
The effect of human interferons on different types of lymphocyte-mediated killer assays was explored. Killing by T cells generated through mixed lymphocyte cultures as well as antibody-dependent lymphocyte-mediated cytotoxicity was not influenced by the addition of interferon. Enhancement of cytolysis produced by natural killer cells was observed when interferon was added during the assay, but enhancement could also be induced if the effector cells were pretreated with interferon for 2 hr prior to the lytic reaction. Killing of a cell line susceptible to natural killing was increased and a cell line which is normally relatively resistant to this type of killing became a susceptible target. 相似文献
12.
Assays of H-Y-specific, cell-mediated cytolysis (CMC) in vitro were carried out with B6 female effector cells and B6 male target cells. Monoclonal H-Y antibody was added to the lytic assay to test whether the antigenic determinant(s) involved in H-Y-specific CMC was distinct from the serologically detected H-Y antigen. Significant blocking was observed, suggesting that the H-Y antigen detectable serologically is similar to H-Y antigen recognized by cytotoxic T cells.Abbreviations used in this paper B6
C57BL/6
- BALB
BALB/c
- CMC
cell-mediated cytolysis
- E
effector cells
- T
target cells 相似文献
13.
R J Hogan W R Taylor M A Cuchens J P Naftel L W Clem N W Miller V G Chinchar 《Cellular immunology》1999,195(2):110-118
This study examines cytotoxic mechanisms used by channel catfish peripheral blood-derived effector cells. Transmission electron microscopy (TEM), coupled with [(3)H]thymidine DNA fragmentation (JAM) and terminal deoxynucleotidyl nick-end labeling (TUNEL) assays, provided the first evidence that catfish peripheral blood cytotoxic effectors killed allogeneic targets via an apoptotic pathway. TEM demonstrated that the effector cell population present within peripheral blood leukocytes (PBLs) was composed of agranular lymphocytes that formed conjugates with, and induced apoptosis in, allogeneic target cells. Both JAM and TUNEL assays showed that PBLs induced target cell DNA fragmentation within 1 h of coculture. In addition, fixed effectors did not induce target cell necrosis or apoptosis, and target cell lysis was completely inhibited by chelation of free Ca(2+) by EGTA. These results suggest that catfish peripheral blood-derived effector cells utilize a secretory mechanism rather than a ligand-based mechanism to trigger apoptosis. 相似文献
14.
Pei-Ju Hsu Ko-Jiunn Liu Ying-Yin Chao Huey-Kang Sytwu B. Linju Yen 《Journal of visualized experiments : JoVE》2015,(106)
The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes. 相似文献
15.
The binding and ultrastructural features of antibody dependent cellular cytotoxicity (ADCC) mediated by human peripheral blood lymphocytes were studied in herpes simplex virus type I (HSV-1) infected Chang liver (CL) cells plus human anti-HSV-1 serum, and in uninfected CL cells plus guinea pig anti-CL antiserum. Non-cytolytic controls included target cells treated with normal serum in place of sensitized targets and heat shocked lymphocytes instead of normal lymphocytes. By transmission electron microscopy, target cell membranes were either broadly indented by effector cells or locally invaginated by means of effector cell filopodia. In neither case did the indentation appear to break the plasma membrane of the target. Control preparations showed only non-indented areas of simple membrane contact. By scanning electron microscopy, the effector lymphocytes in both the active ADCC and normal serum control preparations had a sparse distribution of short microvilli over their surfaces. The majority of heat shock control lymphocytes appeared normal, but 12-20% demonstrated surface patches devoid of microvilli. The hypothesis that ADCC may involve a three-step process is discussed. 相似文献
16.
R J van de Griend R L Bolhuis G Stoter R C Roozemond 《Journal of immunology (Baltimore, Md. : 1950)》1987,138(10):3137-3144
Anti-CD3 MAb can inhibit MHC-restricted cytolytic activity of CD3+ mature cytotoxic T cells. In particular effector-target cell combinations, however, anti-CD3 MAb enhance or induce cytolysis by cross-linking CD3+ effector and IgG-FcR+ target cells. Virtually all natural killer (NK) cells or NK cell-derived clones are CD3-4-8- but do express CD2 and CD16 (IgG-FcR) antigens. We have studied how these cell surface molecules are involved in the regulation of cytolytic activities. The addition of anti-CD2 MAb to effector and target cells was found to induce conjugate formation of the IgG-FcR+ target cells with the effector cell and nonspecific cytolysis of, for instance, the P815 mouse mastocytoma cells. Enhancement or induction of conjugate formation and cytolysis of IgG-FcR+, P815, U937, and Daudi cells was also accomplished by using anti-CD16 MAb (e.g., Leu-11c (B73.1) or CLB Fc-gran 1 (VD2) MAb). Some human and mouse tumor cell lines (K562, P815, and U937) appear to express distinct types of IgG-FcR, showing different affinities for distinct subclasses of MAb (e.g., IgG1, IgG2a), but another line (Daudi) expresses only one type of IgG-FcR preferentially binding IgG1 MAb. Here we demonstrate that IgG-FcR on the effector cells can act as activation sites because anti-CD3 as well as anti-CD16 MAb of IgG1 and IgG2a subclasses can induce lytic activity of target cells bearing the relevant IgG-FcR. These data demonstrate that induction of conjugate formation and cytolysis by MAb occur when the target cells bear IgG-FcR with "specificity" for those MAb. Thus, besides via CD3, cytolytic activity by mature T and NK cells also can be induced via the CD2 and CD16 antigens on these cells. 相似文献
17.
A monoclonal antibody (MoAb 11-4) was raised against K562, a human erythroleukemia cell line sensitive to natural killer cell-mediated cytotoxicity (NK-CMC). Immunological analysis revealed MoAb to be IgG2b. Alone, the MoAb was not cytotoxic for K562 and did not bind to the effector cells, but the addition of this antibody to macrophage-depleted human peripheral blood lymphocytes increased killing of K562 in a 4-hr NK-CMC assay. The maximum increase in NK-CMC was observed when MoAb 11-4 was added to target cells prior to the formation of effector/target cell conjugates. This effect was dose dependent, was specific for K562, and, contrary to conventional antisera, occurred at very low concentrations of MoAb. When MoAb was added either to Percoll-purified large granular lymphocytes (LGL) or to LGL-depleted lymphocytes, only the latter demonstrated a significant increase in the killing of K562 in a 4-hr chromium release assay. Kinetics studies revealed that although the overall LGL-mediated lysis was only slightly increased at 4 hr, the maximum lytic activity was reached within 2 hr. These studies suggest that (1) human LGL and LGL-depleted cell populations bear Fc receptors for mouse IgG2b and (2) although the cytotoxic activities of both cell populations are increased by treatment with MoAb 11-4, the kinetics of this increase are different. 相似文献
18.
Different forms of cell-mediated cytotoxicity were suppressed in the presence of trypan blue. The systems affected included lysis of antibody-coated tumor cells by normal and C. parvum-stimulated mouse peritoneal cells and lysis of allogeneic targets by immune effector cells. The inhibition, measured in a 4-hr 51Cr release assay, was reversible and did not occur in the presence of 30% fetal calf serum or albumin. Binding between effector and target cells through Fc receptors was not affected, and lysis of allogeneic cells was inhibited at the lytic step rather than at the binding step. In contrast, lysis of sensitized erythrocytes was not inhibited by trypan blue, suggesting that lysis of these targets may not involve the steps required in tumor cell lysis. Trypan blue blocked the function of antibody before binding to target cells and also suppressed complement-induced cytolysis. Most individual complement components were susceptible to the inhibitory action of trypan blue. These results reveal an affinity of trypan blue for proteins in general that may be responsible for many of its biologic actions. 相似文献
19.
Short-term treatment of cloned mouse cytotoxic T lymphocytes (CTL) with interferon (IFN) induces lytic activity for natural killer- (NK) sensitive targets. Extended culture of CTL in high concentrations of interleukin 2 induces promiscuous lytic activity in which state both NK-sensitive and NK-resistant target cells are lysed. Cold-target competition analysis showed that the development of NK activity was associated with the acquisition of binding activity for NK-sensitive but not for NK-resistant targets, whereas the development of promiscuous lytic activity was associated with the acquisition of binding activity for both types of target. Antigen-specific cytolysis was inhibited by antibodies to Ly-2, Ly-5, LFA-1 and to the V region of the T cell antigen receptor (TCR), whereas NK and promiscuous lytic activity in the same cells was resistant to inhibition by anti-Ly-2 and anti-TCR. NK activity was expressed normally against a variant NK-sensitive cell line lacking all MHC antigens. These results show that, in contrast to antigen-specific recognition, the NK and promiscuous lytic activities of CTL are expressed without participation of effector cell Ly-2 and TCR molecules or target cell MHC molecules, and are most likely mediated through novel and distinct receptor systems. 相似文献
20.
Leukocyte rolling velocities and migration are optimized by cooperative L-selectin and intercellular adhesion molecule-1 functions 总被引:5,自引:0,他引:5
Kadono T Venturi GM Steeber DA Tedder TF 《Journal of immunology (Baltimore, Md. : 1950)》2002,169(8):4542-4550
Selectin family members largely mediate initial tethering and rolling of leukocytes on vascular endothelium, whereas integrin and Ig family members are essential for leukocyte firm adhesion. To quantify functional synergy between L-selectin and Ig family members during leukocyte rolling, the EA.hy926 human vascular endothelial line was transfected with either fucosyltransferase VII (926-FtVII) cDNA to generate L-selectin ligands alone or together with ICAM-1 cDNA (926-FtVII/ICAM-1). The ability of transfected 926 cells to support human leukocyte interactions was assessed in vitro using parallel plate flow chamber assays. Lymphocyte rolling on 926-FtVII cells was increased by approximately 70% when ICAM-1 was expressed at physiological levels. Although initial tether formation was similar for both cell types, lymphocyte rolling was 26% slower on 926-FtVII/ICAM-1 cells. Pretreatment of lymphocytes with an anti-CD18 mAb eliminated the increase in rolling, and all rolling was blocked by anti-L-selectin mAb. In addition, rolling velocities of lymphocytes from CD18-hypomorphic mice were 48% faster on 926-FtVII/ICAM-1 cells, with a similar reduction in rolling frequency relative to wild-type lymphocytes. CD18-hypomorphic lymphocytes also showed an approximately 40% decrease in migration to peripheral and mesenteric lymph nodes during in vivo migration assays compared with wild-type lymphocytes. Likewise, wild-type lymphocyte migration to peripheral lymph nodes was reduced by approximately 50% in ICAM-1(-/-) recipient mice. Similar to human lymphocytes, human neutrophils showed enhanced rolling interactions on 926-FtVII/ICAM-1 cells, but also firmly adhered. Thus, in addition to mediating leukocyte firm adhesion, CD18 integrin/ICAM-1 interactions regulate leukocyte rolling velocities and thereby optimize L-selectin-mediated leukocyte rolling. 相似文献