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1.
Recent evidence has demonstrated a protein kinase C (PKC)-dependent step in cytotoxic T lymphocyte activation. Here, we examined the influence of PKC in the lytic response of human NK cells to K562, an NK-sensitive tumor target cell. We used the known protein kinase inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) and HA1004. H-7 caused a dose-related inhibition of NK cell-mediated cytolysis (CMC) when the inhibitor was present throughout the course of the 3-h chromium release assay. The 50% inhibitory concentration for H-7 was 7 microM. In contrast, HA1004, which exerts a greater inhibitory effect on cyclic nucleotide-dependent protein kinases than PKC, had no effect on NK-CMC. The suppression of NK-CMC by H-7 was not due to inhibition of binding of the effector cells to target cells and could be reversed by the addition of PMA. H-7 was most effective in abrogating NK-CMC when added to the assay within the first 30 min and treatment of the effector and target cells with H-7 resulted in no loss of NK-CMC. Because nearly 50% of the normal NK lytic activity had taken place by 30 min, this suggested that H-7 inhibited an early event. H-7 exerted a dose-related suppression of antibody-dependent cell-mediated cytotoxicity (ADCC) suggesting that NK-CMC and ADCC share the utilization of PKC, however, HA1004 did not inhibit ADCC. Treating NK cells with IL-2 or IFN-beta did not overcome the inhibition of NK-CMC by H-7. In this study, we have thus demonstrated the presence of a PKC-dependent step in NK-CMC and ADCC. 相似文献
2.
The functional loss of human natural killer cell activity induced by K562 is reversible via an interleukin-2-dependent mechanism 总被引:1,自引:0,他引:1
In a recent study, we evaluated the functional status of human natural killer (NK) cells after their interaction with the NK-sensitive tumor target cell (TC), K562. We demonstrated that effector cells (EC), after treatment with K562 for 4 hr, lost greater than 90% of their original lytic activity. In this investigation, we examined whether this functional loss of NK cell activity represented an irreversible event in the NK lytic mechanism. Initial studies focused on the ability of K562-inactivated EC (ECi), which had been separated from their TC, to recover cytolytic activity following an 18-hr incubation. Our results indicated that ECi recovered 28% of their lytic activity in complete medium (CM) alone, 64% in CM containing interferon-beta (IFN-beta), and 91% in CM supplemented with interleukin 2 (IL-2). Analysis of the data revealed, however, that neither IFN-beta nor IL-2 simply boosted the lytic capacity of NK cells which initially escaped inactivation, but also, each cytokine affected the lytic capabilities of EC that were either truly inactivated by K562 or precursor NK (pre-NK) cells. Thus, to evaluate further the basis of IFN-beta and IL-2-induced ECi augmentation, we first treated the EC with IFN-beta or IL-2 prior to their interaction with K562 so that pre-NK cell subsets would be promoted to fully competent NK cells. Both pretreated EC preparations, after interacting with K562 for 4 hr, lost greater than 90% of their original lytic activities. NK inactivation did not result from cell death nor reflect alterations in conjugate formation or the percentages of Leu-7- and Leu-11-positive EC. IL-2-pretreated ECi, as did ECi, regained some lytic activity after incubation in CM alone, but recovered significantly more activity in CM containing IFN-beta or IL-2. In contrast to the restimulation profiles obtained for ECi and IL-2-pretreated ECi, IFN-pretreated ECi regained lytic function after incubation with IL-2, but not appreciably with IFN-beta or in CM alone. Overall, these findings suggest that EC, either untreated or pretreated with IFN-beta or IL-2, significantly lose their lytic capabilities following interaction with K562 while retaining their ability to bind to the TC; IFN-beta acts predominantly on pre-NK cells, but not on ECi; and IL-2 appears to play an important role in restoring lytic potential to functionally inactive NK cells. 相似文献
3.
Inhibition of the calpain-mediated proteolysis of protein kinase C enhances lytic activity in human NK cells 总被引:1,自引:0,他引:1
Recent evidence from our laboratory has demonstrated that NK/LAK cell activation of human lymphocytes is protein kinase C (PKC)-dependent. Here, we have investigated the translocation of PKC in human NK cells exposed to sensitive targets or to PMA, a phorbol ester. In NK cells exposed to K562 for 6 hr, we observed a weak translocation of PKC whereas in NK cells exposed to PMA more than 90% of cytosolic PKC was translocated to the membrane in less than 5 min. Stimulation of NK cells with an NK-resistant target, however, did not translocate PKC even after 6 hr. Translocation of PKC to the membrane was followed by the appearance of PKM, the cytosolic calcium/phospholipid (Ca2+/PL)-independent form of PKC. The conversion of PKC to PKM was mediated by calpain, an intracellular calcium-dependent thiol proteinase. When we used two inhibitors of calpain, calpain inhibitor I (CI-I) and calpain inhibitor II (CI-II), both caused a dose-related enhancement of NK-CMC when the inhibitors were present throughout the 3-hr chromium release assay. This enhancement could be circumvented by PMA or by the PKC inhibitor H-7. CI-I and CI-II added together caused a greater increase in NK-CMC than when each was added alone. CI-I and CI-II also enhanced antibody-dependent cell-mediated cytotoxicity (ADCC), substantiating further our previous contention that the activation of both NK-CMC and ADCC may involve a common lytic pathway. Activation of NK cells with IL-2 for 18 hr at 37 degrees C was inhibited in the presence of CI-I. To investigate a possible feedback inhibition mechanism due to the buildup of PKC, we examined phosphatidylinositol (PI) metabolism in NK cells activated by IL-2 in either the presence or the absence of CI-I. We observed a significant decrease in PI turnover when NK cells, activated in the presence of IL-2 and CI-I, were stimulated with K562 as compared to NK cells activated by IL-2 alone, then stimulated with K562. 相似文献
4.
J Powell J Stone W C Chan Z D Yang A Leatherbury K W Sell W Wiktor-Jedrzejczak A Ahmed-Ansari 《Cellular immunology》1989,118(2):250-264
In vitro incubation of the erythroleukemic cell line K562 with interferon-gamma (IFN-gamma) renders these cells relatively resistant to natural killer (NK) cell lysis. However, such treatment does not alter their sensitivity to LAK cell lysis. Thus, the lytic susceptibility of interferon-gamma-treated K562 (I-K562) cells to LAK cells as opposed to its relative resistance to NK cell lysis provides a functional assay to help distinguish these two types of effector cells. The relative resistance of I-K562 for NK cell-mediated lysis was not secondary to the release of soluble factors or the frequency of Leu-19+, CD3+ T cells, residual IFN-gamma, or expression of MHC Class I molecules. Coincubation of I-K562 cells with NK or LAK cells overnight did not appreciably change the pattern of lytic responses against K562 and I-K562 target cells. However, incubation of PBMC in vitro with I-K562 but not native K562 in the presence of r-IL-2 leads to a marked decrease in the generation of LAK cells. The inhibition of LAK cell generation was not secondary to differences in the consumption of bioactive levels of IL-2. Differences in the lytic capability of NK and LAK effector cells suggest heterogeneity among cells that mediate such non-MHC-restricted lysis. Use was made of cells from a patient with a large granular lymphocyte lymphoproliferative disease (greater than 85% Leu-19+) to determine if such cells could be used to distinguish clonal population of cells which would represent NK or LAK cell function. Of interest was the finding that such cells, even after incubation in vitro with IL-2, showed lytic function representative of NK cells but not LAK cells. Data concerning the inhibition of LAK cell generation by I-K562 cells have important implications for future therapeutic trials of IFN-gamma and IL-2 in the treatment of human malignancies. 相似文献
5.
Role of lipoxygenation in human natural killer cell activation 总被引:1,自引:0,他引:1
Nordihydroguaiaretic acid (NDGA), quercetin, eicosatetraynoic acid (ETYA), phenidone, and esculetin, agents known to inhibit cellular lipoxygenase (LO) activity, also inhibit human natural killer cell-mediated cytotoxicity (NK-CMC) of K562 tumor target cells (TC) in a dose-dependent fashion. Kinetic analysis demonstrated that LO inhibitors blocked an early event in the activation of the lytic mechanism but did not impair conjugate formation. LO inhibitors also did not affect subsequent chromium release, indicating that their site of inhibition was the NK cell and not the TC. The lipoxygenase products 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene-B4 significantly enhanced NK activity, with 5-HPETE being the more effective. Other LO products tested included 15-HPETE and the hydroxy derivatives 15-hydroxyeicosatetraenoic acid (15-HETE) and 5-HETE. These LO metabolites were either without effect on NK-CMC or inhibitory, depending upon the concentration. Additionally, we examined the ability of 5-HPETE to circumvent the effects of LO inhibitors and found that, in the presence of NDGA, ETYA or quercetin, 5-HPETE significantly (p less than 0.001) restored lytic activity. Inhibitors of LTB4 and LTC4 synthesis, diethylcarbamazine and U-60,257 respectively, produced no inhibition of NK activity. In fact, U-60,257 significantly (p less than 0.05) enhanced NK-CMC. Previous studies in our laboratory, with a new technique which allows for the separation of NK cells from K562 cells, have shown that K562-treated effector cells are greater than 90% inactivated when retested against fresh K562 in the standard chromium release assay. Lipids were extracted from K562-treated, Percoll-purified LGL and evaluated by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). No significant increases were seen in the arachidonic acid-derived LO products evaluated. Thus, our studies indicate that lipoxygenation may be required in the activation of NK-CMC, possibly as a means to generate oxygen radicals which have been previously implicated in NK-CMC. 相似文献
6.
We investigated the inactivation of human NK cells, a population of large granular lymphocytes (LGL), with K562, an NK-sensitive target cell (TC) and KLCL, an NK-resistant TC, but which can be lysed by NK cells via antibody (Ab)-dependent cellular cytotoxicity. NK-enriched effector cells (ECc) were first treated with either K562 or Ab-coated KLCL (Ab-KLCL). After incubation, ECc were separated from their TC then examined for residual NK and ADCC activities, phenotypic changes, and changes in LGL morphology. K562-treated ECc and Ab-KLCL-treated ECc, when retested against the inactivating TC, respectively, lost greater than 90% of their lytic activities. However, K562-treated ECc lost 60 to 70% of their activity against Ab-KLCL, whereas Ab-KLCL-treated ECc lost less than 10% of their activity against K562. In contrast to what we observed with K562-treated ECc, we detected significant reductions in plasma membrane expression of Leu-11a and Leu-11b on Ab-KLCL-treated ECc. Although the proportion of OKM1+ cells remained unchanged after the inactivation process, the density of OKM1 on both K562-treated ECc and Ab-KLCL-treated ECc increased significantly. Morphologic analysis revealed no apparent differences in the percentages of LGL before and after treatment with K562 or Ab-KLCL. Finally, IL-2 restored lytic potential to both K562-treated ECc and Ab-KLCL-treated ECc and, in addition, IL-2-induced enhancement of Ab-KLCL-treated ECc was accompanied by a partial reexpression of Leu-11a. These data support the hypothesis that NK-cell-mediated cytotoxicity and antibody-dependent cellular cytotoxicity may result from a common lytic mechanism, although the initiation steps and regulation of the pathway are distinct. 相似文献
7.
In this study, we examined the functional status of human natural killer (NK) cells after their direct interaction with the NK-sensitive tumor target cell (TC), K562. Human peripheral blood lymphocytes depleted of adherent cells were incubated for 4 hr with unlabeled K562 cells at an effector cell (EC) to TC ratio of 2:1. After incubation, the EC were separated from the TC via centrifugation over a single-step Percoll gradient. K562-treated and separated EC were subsequently shown to be unable to lyse fresh K562 TC when retested in the standard chromium-release assay. Kinetic studies revealed that greater than 90% inactivation of NK cell-mediated cytotoxicity (CMC) could be achieved within 2 hr. Inactivation of NK-CMC by K562 was not caused by a specific loss of NK cells, as detected by changes in the expression of two NK cell-associated markers, Leu-7 and Leu-11, or to alterations in EC viability and target binding cell capacity. Interestingly, NK inactivation also occurred in medium devoid of extracellular calcium, although parallel testing of NK-CMC in the same medium resulted in no chromium release. NK inactivation, however, was significantly prevented when the EC and TC were co-incubated at 4 degrees C, or in medium without magnesium. Additional studies revealed that inactivation of NK-CMC could be achieved with another NK-sensitive, but not with an NK-resistant TC. Overall, we demonstrated that NK cells rapidly lost their lytic potential after direct interaction with a sensitive TC, although the cells remained viable, expressed the same percentage of Leu-7 and Leu-11, and could still bind the TC; and NK inactivation occurred in the absence of extracellular calcium, but not when EC and TC were incubated in medium without magnesium. These latter results provide evidence for an early event in the activation of human NK cells that is binding dependent, temperature sensitive, and independent of extracellular calcium. 相似文献
8.
Functional consequences of cAMP accumulation in human natural killer cells. Implications for IL-2 and IL-4 signal transduction 总被引:1,自引:0,他引:1
I Vitté-Mony R Stancou J H Bertoglio 《Journal of immunology (Baltimore, Md. : 1950)》1990,145(12):4272-4278
To approach the mechanisms whereby IL-2 activates human NK cells, we have compared the effects of IL-4 and of Bt2cAMP on this activation. Both agents block completely the proliferation induced by IL-2 on highly purified CD3-negative human NK cells. We also report that the net LAK response of PBL is inhibited by IL-4 and cAMP. However, kinetics analysis showed that IL-4 appears to inhibit an early stage of IL-2-induced activation of NK cells. IL-4 does not affect the cytotoxicity of freshly isolated NK cells against the K562 target and is ineffective on IL-2-preactivated cells. In contrast, cAMP primarily blocks the lytic effector phase, whether cells have been cultured in IL-2 or not, and its effect appears independent of time of addition. These differences between the activity of IL-4 and cAMP suggested that cAMP was not directly involved in IL-4 signal transduction in human NK cells. Consistent with this interpretation, we did not observe any variation in the level of intracellular cAMP concentrations when NK cells were stimulated with IL-4, or when they are stimulated with IL-2 or IL-2 plus IL-4. In addition, we also demonstrate that NK cell cytotoxic activation induced by IL-2 is still demonstrable in the presence of Bt2cAMP under conditions in which NK cell proliferation is blocked. These results clearly indicate that the differentiative effect of IL-2 on NK cells is independent of cell proliferation. Furthermore, the p70-75 IL-2R-initiated signal transduction pathway that leads to increased cytotoxicity appears not to be susceptible to inhibition by cAMP in human NK cells. 相似文献
9.
The ability of fetal trophoblast cells in the placenta to resist cell-mediated lysis may be important for successful pregnancy. Previous studies in this laboratory demonstrated that cultured midterm mouse trophoblast cells are not susceptible to allospecific CTL generated by standard in vitro protocols, to antibody-dependent cell-mediated cytotoxicity, or to naive or IFN-activated NK cells, despite expressing the requisite target structures. However, we now report that murine trophoblast can be killed, in a non-MHC-specific manner, by LAK cells. Normal mouse spleen cells cultured for 4 days in IL-2-containing lymphokine preparations characteristically killed both NK-sensitive (YAC-1) and NK-resistant (EL4, P815) target cells, and mediated significant lysis of both cultured and freshly isolated trophoblast cells (35 to 55%, E/T 100/1). Pretreatment of the LAK cells with anti-ASGM1 antibody and C markedly reduced the lysis of trophoblast and YAC-1 targets, suggesting that the responsible cells belonged to the NK lineage. The ability of IL-2-activated NK cells to kill midterm murine trophoblast cells was confirmed using a population of highly lytic NK cells generated by culturing spleen cells from severe combined immunodeficiency mice in 500 U/ml rIL-2 for 5 days. These effector cells killed YAC-1, EL4 and P815 target cells at much lower E/T ratios than was achieved with the normal splenic LAK cells, and mediated significant lysis of both freshly isolated (45 to 50%, E/T 20/1) and cultured trophoblast cells (68 to 76%, E/T 20/1). The susceptibility of trophoblast to LAK cells and IL-2-activated NK cells supports the need for suppressor mechanisms regulating IL-2 activity at the maternal-fetal interface. 相似文献
10.
Natural killer and lymphokine-activated killer cell activities from human marrow precursors. II. The effects of IL-3 and IL-4 总被引:1,自引:0,他引:1
C A Keever K Pekle M V Gazzola N H Collins J H Bourhis A Gillio 《Journal of immunology (Baltimore, Md. : 1950)》1989,143(10):3241-3249
Both IL-3 and IL-4 have multi-CSF activity on early marrow progenitors. We have examined the effect of IL-3 and IL-4 on the differentiation of NK cells from their marrow-derived precursors and have further examined the interactions of these cytokines with IL-2 and IL-1. We tested marrow which had been depleted of mature cells and of E rosette-positive cells (including NK cells) by treatment with soybean lectin and SRBC (SBA-E-BM). The cytolytic activities of the SBA-E-BM samples were tested in 51Cr-release assays after 7 days of liquid culture. K562 targets were used as a measure of NK activity and NK-resistant Daudi targets were used to measure lymphokine-activated killer (LAK) cell activity. Neither NK nor LAK activity was detectable in marrow cultured in medium without cytokines, or in medium containing IL-3, or IL-4 alone. Both of these cytokines were shown to be inhibitory to the IL-2-induced generation of NK and LAK activity from SBA-E-BM at concentrations as low as 1 U/ml. The inhibitory activity of both IL-3 and IL-4 was found to occur early in the marrow cultures, with little or no inhibitory effects seen if added 48 h after IL-2. IL-3 appeared to be specifically inhibitory to NK cell precursors since addition of IL-3 to cultures of PBMC did not inhibit IL-2-induced lytic activities. In contrast, IL-4 was equally inhibitory to the activation of marrow and peripheral blood NK cells by IL-2. Mixing experiments demonstrated that the reduced lytic activity in IL-3 or IL-4 containing marrow cultures were not due to suppression of the NK effectors, nor could marrow cultured in IL-3 or IL-4 serve as targets for IL-2-activated NK cells. Phenotype analysis of the lymphoid cells in marrow cultures containing IL-2 combined with IL-3 or IL-4 revealed fewer cells expressing Leu-11 (CD16), or Leu-19 (CD56) and fewer CD16, CD56 coexpressing cells compared with marrow cultured in medium containing IL-2 alone. The inhibitory activity of IL-4, but not IL-3, could be partially reversed if IL-1 was added to the cultures, suggesting that IL-1 and IL-4 have opposing activities on NK cells responsiveness to IL-2. These interactions between cytokines might be important in the regulation of NK cell differentiation and on the functional activity of mature NK cells. 相似文献
11.
M K Heiskala O Carpén E Saksela T Timonen 《Journal of immunology (Baltimore, Md. : 1950)》1987,139(5):1414-1418
Natural killer cell activity is inhibited by primary cultures of monolayer cells. In this study, we analyzed the mechanism of the inhibition. Inhibited NK cells showed unaltered binding capacity to NK sensitive K562 cells. The orientation of the effector cells' actin-containing microfilaments, an event known to occur during the programming for the lysis stage in lytic conjugates, was unaffected by the inhibition. In single cell cytotoxicity experiments, the number of killer cells among conjugate-forming cells was reduced. The capacity of the inactivated NK cells to secrete cytotoxic factors upon stimulation with Con A was also impaired. Both NK-resistant inactivating target cells and NK-sensitive K562 cells were sensitive to the toxic factors secreted by NK cells. Thus, the results indicate that the target cell-mediated inactivation of NK cell is based on a block in the lethal hit stage, possibly due to reduced release of toxic factor(s) from the effector cells. The capacity of inactivated effector cells to mediate antibody-dependent cellular cytotoxicity was unimpaired, suggesting that the contact-mediated inhibition of cytotoxicity selectively affects NK cells. 相似文献
12.
J D Katz R Mitsuyasu M S Gottlieb L T Lebow B Bonavida 《Journal of immunology (Baltimore, Md. : 1950)》1987,139(1):55-60
Our studies and other investigations have shown that NK effector cells can also mediate antibody-dependent cellular cytotoxicity (ADCC) through the use of the Fc gamma receptor on the NK cell membrane. Peripheral blood lymphocytes (PBL) derived from patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex exhibit a poor NK activity due to a defective "trigger" required for activation in the lethal hit stage of the NK lytic pathway. Consequently, it was important to delineate whether the defect in AIDS NK cells affected the ADCC function. By using the 51Cr-release assay, the ADCC cytotoxic activity of AIDS PBL was found to be within the normal range, despite the absence of significant NK activity. Several experiments corroborated that the same effector cells mediate both NK CMC and ADCC. Depletion of Fc gamma R-bearing cells resulted in elimination of both the ADCC and NK cytotoxic functions. Single cell analyses, using one- and two-target cell conjugates, revealed that the frequency of ADCC effector:target conjugates and the frequency of killer cells from AIDS PBL were comparable to the frequencies seen in the normal controls. However, when mixtures of NK and ADCC targets were used to form mixed two-target conjugates, the AIDS effector cells lysed only the bound ADCC target, whereas the normal effector cells lysed both the bound NK and ADCC targets. These results demonstrate clearly that the same NK/K effector cells from AIDS PBL, defective in NK activity, are not impaired in mediating ADCC activity. These findings were supported by the demonstration that AIDS PBL stimulated with ADCC targets, but not with NK targets, released NK cytotoxic factors, postulated mediators of the NK CMC reaction. These findings indicate that the NK/K cells in AIDS are triggered normally for ADCC activity but are not triggered for NK activity. Furthermore, the results indicate that the lytic machinery is not impaired in the AIDS NK/K cells. 相似文献
13.
O Silvennoinen J Vakkila M Hurme 《Journal of immunology (Baltimore, Md. : 1950)》1988,141(4):1404-1409
In this study we have investigated the role of accessory cells in the development of lymphokine-activated killer cells (LAK) from highly purified human NK and small resting T cell progenitors. As accessory cells we used autologous, as well as allogeneic, monocytes, and dendritic cell enriched cells. Both NK and T cells were able to generate LAK activity, but their activation requirements were different. NK cells were activated merely by IL-2, and accessory cells did not enhance their lytic activity in the presence or absence of IL-2. Conversely, T cells were practically unresponsive to even high concentrations of IL-2 having a strict requirement for accessory cells for the development of lytic activity and proliferation. Accessory cells differed in their ability to activate T cells presumably depending on their ability to induce IL-2 synthesis, allogeneic dendritic cells being the most effective accessory cells and IL-2 synthesis stimulators. Allogeneic accessory cells could induce lytic activity in T cells even in the absence of exogenous IL-2. Thus, accessory cells play a central role in expanding the LAK effector cell population. 相似文献
14.
The protective effects of interferons (IFNs) against NK cell-mediated cytotoxicity (NK-CMC) is well established. We report here that both recombinant tumor necrosis factor-alpha (TNF-alpha) and recombinant interleukin-1 alpha (IL-1 alpha) can also protect some adherent target cells (e.g., the amniotic cells WISH and the cervical epithelial carcinoma cells HeLa-229) from NK-CMC in a dose-dependent manner. Like in the case of IFNs, the level of conjugate formation between target and effector cells (nonadherent peripheral blood lymphocytes) is not affected by pretreatment of the target cells with either TNF-alpha or IL-1 alpha. However, while the main effect of IFNs is to reduce the ability of target cells to stimulate the release of NK cytotoxic factor (NKCF) from effector cells, TNF-alpha and IL-1 alpha do not affect this process but rather reduce the target cell sensitivity to the lytic effect of NKCF. Therefore TNF-alpha and IL-1 alpha induce resistance to NK-CMC by a mechanism that differs from the one attributed to IFNs. The protective effect of TNF-alpha and IL-1 alpha is not mediated by the induction of IFN-beta 2/IL-6. 相似文献
15.
Anti-renal cell carcinoma chimeric antibody G250: cytokine enhancement of in vitro antibody-dependent cellular cytotoxicity 总被引:2,自引:0,他引:2
Liu Z Smyth FE Renner C Lee FT Oosterwijk E Scott AM 《Cancer immunology, immunotherapy : CII》2002,51(3):171-177
The chimeric monoclonal antibody cG250 targets the G250 antigen, a transmembrane protein which is expressed on renal carcinoma cells and is identical to the MN/CAIX antigen. In vitro studies have previously demonstrated that cG250 induces antibody-dependent cellular cytotoxicity (ADCC) of G250-positive targets. In order to investigate the upregulation of ADCC mediated by cG250, ADCC was examined using effector cells cultured in the presence or absence of the cytokines interferon-gamma (IFN-gamma), interferon-alpha isoforms IFN-alpha (2a) and IFN-alpha (2b) and interleukin-2 (IL-2), and the time course of effects over a 7-day period was determined. Renal cell carcinoma lines expressing high (SK-RC-52) and low (SK-RC-09) G250 antigen levels were used as target cells, and freshly isolated peripheral blood mononuclear cells (PBMC) from a healthy donor were used as the effector cells. PBMC were incubated with the respective cytokine at a range of concentrations or with a media alone control for a period of 7 days. The ADCC activity mediated by cG250 or control isotype matched huA33 with the different PBMC treatment groups was assessed in triplicate daily. Corresponding lymphokine activated killing (LAK) activity was measured concurrently for each treatment group. Chimeric G250 specifically recognised G250 antigen on high and low expressing cell lines SK-RC-52 and SK-RC-09, and mediated specific in vitro ADCC of both lines. In the absence of cytokine stimulation, the specific ADCC of cG250 declined rapidly within three days. IL-2 strongly enhanced and maintained cG250-mediated ADCC activity and K562 cytotoxicity when applied to PBMC in culture for seven days. IFN-gamma also enhanced the ADCC of cG250 throughout the study period, but was not as effective as the IL-2 treatment, and the SK-RC-09 line displayed lower specific cytotoxicity than the SK-RC-52 cell line. In contrast, IFN-alpha 2a and 2b increased cG250-mediated ADCC and K562 cytotoxicity for only three days of the study period. The potent and sustained immune effector activity observed with cG250 and cytokines in this in vitro study suggests that the combination immunotherapy of cG250 with cytokines such as IL-2 shows promise in the treatment of renal cell carcinoma (RCC). 相似文献
16.
K562 killing by K, IL 2-responsive NK, and T cells involves different effector cell post-binding trigger mechanisms 总被引:1,自引:0,他引:1
F Shanahan M D Brogan W Newman S R Targan 《Journal of immunology (Baltimore, Md. : 1950)》1986,137(2):723-726
The monoclonal antibody 13.3 specifically blocks the trigger process of the NK-K562 cytolytic sequence at a post-binding effector cell level. This antibody was used to define differences in the lytic trigger processes of NK and other mechanisms of K562 lysis. Monoclonal antibody 13.3 inhibited lysis of K562 target cells by freshly isolated peripheral blood lymphocytes (PBL) and purified large granular lymphocytes (LGL), but had no inhibitory effect on antibody-dependent cell-mediated cytotoxicity to K562 by these effectors. Lectin-dependent cellular cytotoxicity (LDCC) to this target cell was also unresponsive to 13.3. The 13.3-induced inhibition of NK-K562 lytic activity persisted when PBL were activated in culture with interleukin 2 (IL 2) for periods up to 48 hr. After 48 hr of culture, the degree of inhibition diminished progressively in medium containing fetal calf serum but not in medium containing autologous serum. This 13.3-unresponsive lytic activity in cultured PBL could be attributed to more than one cell type and was present in both the LGL and Fc gamma receptor-depleted T cell fraction. Thus, K562 lysis by freshly isolated human lymphocytes via NK, K, and LDCC mechanisms is characterized by heterogeneity of the post-binding effector cell trigger mechanism. K562 lysis by lymphocytes cultured with IL 2 is similarly heterogeneous. 相似文献
17.
Interferon is able to reduce tumor cell susceptibility to human lymphokine-activated killer (LAK) cells 总被引:1,自引:0,他引:1
It is known that IL-2 induces lymphocytes to produce interferon-gamma (IFN-gamma) and this IFN type is particularly efficient in inducing tumor cell resistance to natural killer (NK) cell-mediated lysis. We have investigated the effect of IFN on tumor cell sensitivity to LAK cell-mediated cytotoxicity. Pretreatment of the human K562 leukemia and HHMS melanoma with IFN-gamma and the Daudi lymphoma with IFN-alpha caused a significant reduction in sensitivity to lysis by human LAK cells generated in vitro in the presence of human recombinant IL-2 (100 U/ml). The LAK activity was mediated by cells expressing NK cell markers (CD16,NKH1) as well as by cells with T cell markers (CD3, CD5). IFN-treated K562 cells were protected from lysis mediated by all these populations. Supernatants from LAK cultures containing IFN-gamma were able to induce NK and LAK resistance when used to pretreat K562 overnight. Antibodies to IFN-gamma but not to IFN-alpha were able to neutralize this activity. Taken together, these results indicate that the production of IFN-gamma by LAK cells may be of importance in induction of tumor cell resistance to LAK cell-mediated lysis. 相似文献
18.
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
19.
K H Leung 《Cellular immunology》1989,123(2):384-395
Activation of natural killer (NK) activity K562 target cells from nonadherent (NA) lymphocytes by interleukin 2 (IL-2) was inhibited marginally PGE2 (30-3000 nM). PGE2 did not effectively suppress the NK activity of IL-2-activated cells. The NK activation and acquisition of resistance to PGE2-mediated suppression of NK activity were dependent on protein synthesis. When NA cells were incubated with IL-2 for 3 or more days to generate lymphokine-activated killer (LAK) activity against Raji target cells, PGE2 only partially inhibited the activation of NK/LAK activity by an optimal dose of IL-2 (10 U/ml). The activation of NK/LAK activity by a suboptimal dose of IL-2 (0.1 U/ml) was inhibited by PGE2. When the NK/LAK activity of IL-2-activated cells was assessed in the presence or absence of PGE2, the LAK activity was more sensitive than the NK activity to PGE2-mediated suppression. 相似文献
20.
Natural killer (NK) cells are active in host defence against tumors. In order to determine if NK cells have the capacity to lyse human lung cancer cells, we evaluated blood NK cell activity against human lung carcinoma lines representing each of the commonest histological types of lung cancer, NCI-H157 (large cell), LICM107 and NCI-H146 (small cell), NCI-H226 (squamous cell), and LICM26 (adeno), and compared the results to their activity against a standard NK-sensitive target, K562, using a 16-hr 51Cr-release assay. At effector to target (E:T) ratios up to 50:1, NK activity was very low against each of the lung cancer cell lines compared to the K562 cells (NCI-H157 10 +/- 2%, LICM107 12 +/- 2%, NCI-H146 14 +/- 5%, NCI-H226 8 +/- 5%, and LICM26 7 +/- 3%, compared to K562 60 +/- 3%, P less than 0.001, for each compared to K562 cells). Recombinant interleukin 2 (IL-2) produced a dose-dependent augmentation of NK activity against each of the lung cancer cell lines, with doses as low as 0.25 U/ml being effective. The highest level of boosting was seen against NCI-H157 cells where NK activity (E:T, 50:1, IL-2, 250 U/ml) increased from 9 +/- 2 to 56 +/- 7%, P less than 0.001). Only brief exposure to IL-2 was necessary for augmentation to occur, with as little as 5 min being required for activation, although increased exposure times produced increased levels of augmentation. NK cells appeared to be the IL-2-responsive lytic cell population in these experiments as Leu 11b-depleted lymphocytes expressed little IL-2-mediated augmentation of activity against these target cells, and most of this IL-2-mediated augmentation of activity was located in the large granular lymphocyte-enriched fraction of the lymphocyte population. We conclude that normal blood NK cell activity against human lung cancer cell lines is low but that this activity can be markedly augmented by brief exposure of NK cells to low doses of recombinant IL-2, suggesting a potential role for IL-2 in the immunotherapy of human lung cancer. 相似文献