Regulation of NK92-MI cell cytotoxicity by substance P

The neuropeptide substance P (SP) can regulate a number of immunological functions in vitro and in vivo and may regulate natural killer (NK) cell activity. Here, we investigated whether SP has a role in regulating NK92-MI cell function in vitro, and how it influences NK cell activity. We found that SP dose dependently increased the cytotoxicity of NK92-MI cells and had a maximal effect at a concentration of 10(-12) and 10(-10) m. Furthermore, the expression of cytotoxic-associated molecules (perforin, granzyme) and activating receptor NKp46 [a member of natural cytotoxicity receptors (NCRs)] was observed to be upregulated by SP at optimal concentration, at which SP enhanced the cytotoxicity of NK92-MI cells. Neurokinin-1 receptor (NK-1R), a functional receptor of SP, was found on NK92-MI cells, and the observed effects of SP on NK92-MI cells could be more partially blocked by an NK-1R antagonist. Our data suggest that SP induces NK92-MI cell cytotoxicity by directly increasing the expression of cytotoxic granules and upregulates NK92-MI cell receptor-mediated functions indirectly. Thus, SP may regulate NK cell function mainly through NK-1R.     

Functional hybrids between human cytotoxic T and mouse myeloma cells

The establishment of functional human cytotoxic T lymphocyte (CTL) hybrids was investigated. Human CTL, generated in a seven-day, one-way mixed lymphocyte-tumor cell interaction (MLTI) against an allogeneic melanoma cell line (DW) in the presence of a third-party helper cell line and crude interleukin 2 (IL2), were fused with a mouse myeloma cell line (P3-X63 Ag8). Following fusion in polyethylene glycol, the hybrids were examined for cytotoxic potential against the sensitizing target cells DW. Hybrids with detectable levels of cytotoxicity were cloned in soft agar. Two clones demonstrating stable activity were selected for analysis of lineage and specificity of cytotoxicity. Both clones expressed cytotoxicity in a reasonable stable manner without dependence on IL2 for growth or function. Interferon had no effect on the cytotoxicity of the hybrids against the natural killer (NK)-sensitive target cells K562 or the DW cells. The cytotoxic activities of the hybrids against the sensitizing target cells DW, however, could be markedly facilitated in the presence of IL2-containing supernatants in the assay medium and less so in the presence of lectin. The range of the cytotoxic activities of the two clones was identical and restricted to the DW cells and another melanoma cell line, suggesting the possibility of a shared target molecule(s) between these two target cells for these cytotoxic hybrids. These observations indicate that the hybrids might require a mediator present in IL2 supernatant for optimum expression of cytotoxicity and suggest that the hybrids express the cytotoxic specificity of the hybridized CTL. These hybrids offer unique opportunities for critical examination of the molecular mechanisms of cellular cytotoxicity and specificities exhibited by activated human CTL.     

体外培养系统中各细胞组分在IL-18诱导的肿瘤特异性CTL中的作用

应用rhIL 18在体外培养系统 (coculturesysteminvitro ,CCS )中诱导肿瘤特异性细胞毒性T淋巴细胞 (cytotoxicTlympho cyte ,CTL ) ,探索不同细胞在IL 18起动和促进肿瘤特异性免疫应答方面的作用。采用StemSepTM免疫磁性细胞分离法分离人外周血NK细胞、T细胞及树突细胞 (DC ) ,流式细胞仪分析细胞表型 ,12 5I UdR标记的细胞毒实验检测杀伤活性。结果表明 ,在肿瘤抗原存在的条件下 ,CCS中rhIL 18能够诱导并促进CTL介导的肿瘤特异性杀伤效应 ;并且 ,在rhIL 18诱导肿瘤特异性CTL的过程中 ,rhIL 18、NK细胞、DC及T细胞均起着十分重要的作用 ,缺少任一组份 ,均对诱导肿瘤特异性CTL有明显差异 (P <0 0 1)。提示在CCS中 ,rhIL 18诱导的NK细胞快速杀伤效应及DC的抗原提呈作用 ,在肿瘤特异性CTL产生过程中 ,均起着决定性的作用。     

Mapping functional epitopes of the human LFA-1 glycoprotein: monoclonal antibody inhibition of NK and CTL effectors

Anti-LFA-1 monoclonal antibody (MoAb) was originally identified by screening antibodies for their ability to inhibit cytolysis in the absence of complement. Anti-LFA-1 MoAb has been shown to inhibit both natural killer (NK) and cytolytic T lymphocyte (CTL) mediated cytolysis. To further define the utilization of this molecule in cell-mediated cytolysis, we used a panel of MoAb to functional epitopes on both the alpha and beta chains of the LFA-1 heterodimer. The panel was used to compare OKT3- NK effectors and OKT3+ CTL clones. As expected, function-associated MoAb to CTL antigens (T3, T8, LFA-2) and target cell antigens (HLA, LFA-3) blocked only CTL clones and not NK effectors. In contrast, anti-LFA-1 MoAb blocked both NK effectors and CTL clones. In addition, the panel of anti-LFA-1 MoAb demonstrated an identical hierarchy of functionally relevant LFA-1 epitopes. Given the similar utilization of LFA-1 in NK and CTL mediated cytotoxicity assays, we explored the ability of MoAb to different epitopes on LFA-1 to inhibit conjugate formation. Anti-LFA-1 MoAb inhibition of NK-target binding paralleled the inhibition of CTL-target binding. Thus, functional epitopes on the LFA-1 molecule have been defined for NK and CTL effectors. The identical hierarchy of functional epitopes indicates that the LFA-1 molecule is similarly utilized in NK and CTL mediated cytotoxicity and that the relevant epitopes are involved in effector-target conjugate formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulatory function of cell surface molecules CD2-, LFA- and beta 2-microglobulin in natural killer cell activity

The functional importance of various cell membrane bound molecules was studied and compared in the NK cytotoxicity and CTL activity. LFA-1 and CD2 participate in both killing functions, while CD3 and CD8/CD4 as well as MHC class I molecules are involved only in CTL activity. Nevertheless CD2- and beta 2-microglobulin are representatives of the NK function. It was demonstrated that CD2-, LFA-1 and beta 2-microglobulin molecules have an additive and complementary function in the killing mechanism. The upregulation of alpha- and gamma-interferons on NK function seems not to be a consequence of the enhanced expression of these molecules on the cell surface induced by IF at the same time.     

Cytotoxic effector cells of the immune system

Summary The organism contains several types of cytotoxic cells which are able to lyse host and foreign cells. Cytotoxic T-lymphocytes (CTL) appear to play the most important role among the killer cells but other lymphatic cells, such as natural killer (NK) cells and lymphokine-activated killer (LAK) cells as well as macrophages are also highly effective in the lysis of appropriate targets. The various cytotoxic effector cells differ distinctly concerning origin, phenotype, morphology and target cell specificity, but they bear the common feature that they destroy the target cells in a contact-dependent non-phagocytotic process.CTL are characterized by typical lysosomal granules and by the expression of a characteristic pattern of surface molecules. They recognize specific antigens which are presented in context with molecules of class I major histocompatibility complex (MHC). NK cells, on the other hand, kill the appropriate targets without prior immunisation and without requiring recognition of MHC molecules at the target cells. They also bear a typical pattern of surface markers which differ in several aspects from that of CTL. Human NK cells are further characterized by peculiar cytoplasmic granules with parallel tubular arrays which are not present in other cytotoxic cells. LAK cells constitute an additional, only recently described, killer cell population which arise from lymphatic cells in the presence of interleukin-2. They appear to represent a functional unique cytotoxic effector cell system with an exceptionally wide target cell spectrum including normal and malignant cells of different origin. LAK cells, however, show a profound heterogeneity concerning the expression of phenotype surface markers and it is not yet clear whether they are a unique cell line. By electron microscopy they display peculiar intranuclear inclusion bodies which may be associated with prolonged stimulation by interleukin-2. CTL, NK and LAK cells appear to possess similar mechanisms for cytolysis including secretion of pore-forming proteins, serine proteases and other proteins. Furthermore, they are able to trigger the cleavage of DNA in the target cell nucleus by a hitherto unknown pathway.Macrophages differ substantially from other cytotoxic effector cells concerning morphology, phenotype, kinetic of activation and target cell spectrum. They perform a variety of functions whereby contact-dependent target cell lysis represents only one of their properties. After target cell binding they release over 20 different molecules such as interleukin-1 and tumor necrosis-factor-alpha as mediators for cytolysis. Thus, macrophages appear like other cytotoxic effector cells to destroy their appropriate targets by different factors.Taken together the data obtained hitherto suggest that cellular cytotoxicity is mediated by various effector cells which may make a major contribution in the defence against infections and malignancies.Abbreviations ADCC antibody dependent cellular cytotoxicity - CD clone of differentiation - CTL cytotoxic T-lymphocyte - HTL helper T-lymphocyte - IL-2 interleukin-2 - LAK lymphokine-activated killer cell - LGL large granular lymphocytes - MHC major histocompatibility complex - NK natural killer cell - P1 perforin 1 - PTA parallel tubular arrays - TCR T-cell receptor complex     

Inhibition of human NK cell-mediated cytotoxicity by exposure to ammonium chloride

Ammonium-chloride-containing solutions (AC) are routinely used to lyse red blood cells during preparation of PBMC. Although exposure to AC has been described to affect the ultrastructural appearance of large granular lymphocytes and to temporarily inhibit cytolytic activity of PBMC preparations, the cellular basis of this phenomenon has not been studied. Here, the inhibitory effect of AC on human CTL and NK-mediated cytotoxicity has been analyzed in 4-h 51Cr-release assays. The results show that NK killing of K562 leukemia cells and xenogeneic endothelial cells is inhibited by AC exposure. The effect is dose-dependent and reversible, because recovery of cytotoxicity is observed within 15 h of re-culturing. AC does not reduce the viability of NK cells and the inhibitory effect is not mediated by the exhaustive release of granzymes upon AC treatment. In contrast, antigen-specific CTL killing of EBV-transformed B-lymphoblastoid cell lines and xenogeneic PHA lymphoblasts was less sensitive to AC and data are presented suggesting that FasL-induced apoptosis is not inhibited by AC. In conclusion, perforin-mediated NK killing is AC-sensitive whereas CTL killing and FasL-mediated killing appear to be AC-resistant. Therefore, AC represents a powerful tool to study different mechanisms of cell-mediated cytotoxicity and may be helpful in assessing antigen-specific CTL cytotoxicity without the influence of NK cell-mediated background killing.     

灵芝孢子粉对荷HAC肝癌小鼠抗肿瘤的实验性研究

本文通过荧光细胞检测技术、体外细胞毒试验以及肿瘤抑瘤率的测定 ,观察了灵芝孢子粉对荷HAC小鼠T细胞表面分化抗原 ,体外细胞杀伤功能以及肿瘤抑瘤率的影响。结果显示灵芝孢子粉治疗组中总T细胞的百分率 (6 3 4% )高于对照组 (5 6 3% ) ,其中对总T细胞、T辅助细胞的上调较为明显。在对HAC、YAC 1和P815肿瘤细胞的杀伤活性中灵芝孢子粉组的杀伤活性分别为 2 7 3%、 2 3 4%和 2 0 0 % ,高于对照组。灵芝孢子粉组的总抑瘤率为 42 2 %。结果表明灵芝孢子粉是一种能激活和提高特异性 (CTL )和非特异性杀伤细胞 (NK、LAK )的抗肿瘤作用 ,抑制肿瘤细胞的增殖以及调控机体免疫功能的中药。     

Natural Killer Cells Do Not Belong to the Recirculating Lymphocyte Population

Natural Killer (NK) cells constitute a cell type with an as yet undefined lineage, although certain similarities with T lymphocytes have been found in the mouse. Our present results show that NK cells have a significant difference compared with T and B cells in their capacity to traverse the blood-lymph barrier. Thoracic duct lymphocytes from mice or rats are thus devoid of NK activity, when at the same time potential, cytotoxic T lymphocyte (CTL) activity or antibody-dependent cellular cytotoxicity function can be demonstrated. Chronic thoracic duct drainage in the rat also leads to an increase in NK activity per unit cell number in the other lymphoid organs. Thoracic duct lymphocytes from mice and rats may thus serve as convenient sources of CTL and/or killer cells in situations in which it is important to minimize NK cell involvement.     

Triggering receptors involved in natural killer cell-mediated cytotoxicity against choriocarcinoma cell lines

The lack of classical HLA-class I molecules on trophoblast is necessary to prevent allorecognition by maternal CTL, but may induce activation of NK cells. A protective role against NK cells equipped of suitable inhibitory receptors has been proposed for nonclassical HLA-class I molecules including HLA-E and HLA-G. In the present study we show that the NK-mediated killing of two choriocarcinoma cell lines, JAR and JEG3, is induced upon engagement of natural cytotoxicity receptors (NCR) with their specific ligands. In particular, we show that NKp44, a triggering receptor expressed at the NK cell surface only after in vitro culture in the presence of IL-2, plays a central role in triggering NK cytotoxicity against trophoblast cells. Also NKp46 appear to contribute to this function by cooperating with NKp44. On the other hand, other triggering receptors such as NKp30, 2B4, and NKG2D are not involved in killing of choriocarcinoma. Our findings suggest that resistance of trophoblast to NK-mediated cytotoxicity is the result of insufficient activating interactions between the various triggering NK receptors and their target cell ligands. On the other hand, the interaction of nonclassical HLA class I molecules with inhibitory NK receptors appears to play only a marginal role in regulating the susceptibility of choriocarcinoma to NK mediated cytotoxicity.     

The selective downregulation of class I major histocompatibility complex proteins by HIV-1 protects HIV-infected cells from NK cells.

To avoid detection by CTL, HIV encodes mechanisms for removal of class I MHC proteins from the surface of infected cells. However, class I downregulation potentially exposes the virus-infected cell to attack by NK cells. Human lymphoid cells are protected from NK cell cytotoxicity primarily by HLA-C and HLA-E. We present evidence that HIV-1 selectively downregulates HLA-A and HLA-B but does not significantly affect HLA-C or HLA-E. We then identify the residues in HLA-C and HLA-E that protect them from HIV down-regulation. This selective downregulation allows HIV-infected cells to avoid NK cell-mediated lysis and may represent for HIV a balance between escape from CTL and maintenance of protection from NK cells. These results suggest that subpopulations of CTL and NK cells may be uniquely suited for combating HIV.     

A comparison of membrane markers on rat cytotoxic cells.

The antigenic characteristics of cytotoxic T cells (CTL) and cells mediating antibody-dependent cellular cytotoxicity (ADCC) were defined using monoclonal antibodies W3/13, W3/25 and MRC OX8, and compared with the phenotype of natural killer (NK) cells previously defined by these antibodies. CTL, ADCC effector cells and NK cells were also tested for expression of Ia antigen using MRC OX6 monoclonal antibody. The fluorescence-activated cell sorter was used to separate effector populations into antigen-positive and antigen-negative subsets, and the cytotoxicity of the resultant lymphocyte fractions was then assessed using 6 hr 51Cr release assay and a quantitative method of analysis based on consideration of target cell lysis as an enzyme substrate reaction. CTL, ADCC effector cells and NK cells were W3/25 negative and OX6 negative. CTL were positive for W3/13 and OX8 antibody with no cytotoxicity in the W3/13 negative and OX8 negative fractions. In contrast, ADCC effector cells showed heterogeneity of staining with W3/13 and OX8 antibodies, with 50% cytotoxic activity in the W3/13 negative fraction, and 20-30% cytotoxic activity in the OX8 negative fraction. In parallel experiments, NK cells and ADCC effector cells showed identical marker heterogeneity when tested with W3/13 and OX8 antibodies.     

人工合成HLA衍生肽对CTL和NK细胞毒功能的影响

目的探讨合成HLA衍生肽对CTL和NK细胞毒功能的影响.方法人工固相合成2种HLA衍生肽(P1HLA-B*0701.75-84和P2HLA-B*2702.75-84),分别用MTT法和LDH释放法,在体外观察HLA衍生肽对CTL和NK细胞毒功能的影响及其等位基因特异性.结果P2可显著抑制CTL的细胞毒效应(P＜0.01),这种抑制作用不具有等位基因特异性,而对NK的细胞毒功能则没有明显影响.结论合成HLA肽对CTL和NK细胞毒功能的影响只与HLA肽的序列有关,P2即HLA-B*2702.75-84,可抑制CTL的细胞毒功能.     

苏木素染色法在细胞介导的细胞毒效应中的应用

目的:建立苏木素染色测定免疫效应细胞介导的细胞毒效应的方法:。方法:采用苏木素染色法观察人贴壁肿瘤细胞系Hep2的增殖活性,测定了人NK细胞系NK92、NKL和YT对SGC7901、Hep2细胞的细胞毒效应,新西兰白兔CTL细胞对VX2细胞的细胞毒效应。同时与MTT比色法及CCK-8比色法进行比较。结果:NK92、NKL和YT三种效应细胞对SGC7901和Hep2细胞杀伤6h或16h后,在效靶比分别为10:1、5:1、2.5:1和1.25:1时,三种方法:所测结果:均具有良好的效靶关系,且杀伤趋势基本一致。6次苏木素染色法测定NK92对SGC7901和Hep2细胞的细胞毒效应结果:显示CV<8%,MTT比色法测定结果:CV<12%,前者具有更好的重复性。苏木素染色法和MTT比色法所测兔CTL杀伤效应结果:一致,线性关系明显。结论:苏木素染色法用于免疫效应细胞对贴壁肿瘤细胞系的细胞毒效应测定,具有特异、稳定、简便、经济等优点。     

Measurement of NK activity in whole blood by the CD69 up-regulation after co-incubation with K562, comparison with NK cytotoxicity assays and CD107a degranulation assay

In present study human peripheral blood NK cell activation after co-incubation with K569 cell line was investigated by CD69 expression. NK lytic activity was studied by two different assays: TDA (2,2':6',2″-terpyridine-6,6″-dicarboxylate) release assay (TRA) and flow cytometry assay (FCA) that display two approach to cytotoxicity measurement. We also investigated NK cell degranulation activity by estimation of CD107a (LAMPa) expression. Comparison of specific lysis value measured by both cytotoxicity assays showed high correlation coefficient between two methods (r=0.94447). Specific lysis value correlated significantly with CD69+ NK frequency and NK degranulation activity. We show that lymphocyte incubation with K562 results to increase CD69 expression on NK and NKT but not on T lymphocytes. Only a part of peripheral blood NK cells became CD69 positive after incubation with excess of K562 cells. CD69+ NK cell frequencies did not increase after elevation of K562/NK ratio or incubation period that confirmed existence of subset of NK cells able to response to K562. CD69 elevation on NK significantly correlated with NK cytotoxicity (r=0.726). CD69 increases were similar when whole blood or isolated PBMC was used in assay. We also found different capacity to activation in NK subsets that express CD62L at various densities. The results demonstrated that K562 induced CD69 expression displays NK lymphocyte functional condition that associated with cytotoxic function.     

A novel method for measuring CTL and NK cell-mediated cytotoxicity using annexin V and two-color flow cytometry.

An assay based on two-color flow cytometry has been developed to measure CTL and NK cell-mediated cytotoxicity. After effector/target cells are incubated together, CTL or NK populations are stained with an effector cell specific PE-conjugated mAb. Subsequently, annexin V-FITC binds to cells expressing phosphatidylserine (an early marker of apoptosis) on the cell surface. Target cells are gated upon as PE-negative and quantified with respect to their annexin V positivity. The shift from annexin Vneg to annexin Vhi is a discrete event such that all target cells fall within discernible populations with respect to annexin V. There is a strong correlation between cytotoxicity measured with our assay and a standard 51Cr release assay (r2 = 0.989). The PE/annexin V assay shows increased sensitivity at early timepoints after target/effector cell mixing. In addition, this method allows for analysis of target cells at the single cell level. Therefore, we have described a promising new technique to measure in vitro cell-mediated cytotoxicity. It avoids the potential difficulties of working with radioactive isotopes, and offers increased sensitivity and versatility.     

The Natural Killer Cell-Like Lytic Activity Expressed by Cytolytic T Lymphocytes is Associated with the Expression of a Novel Function-Associated Molecule

Experiments were performed to analyse the natural killer cell (NK)-like cytotoxicity frequently expressed by human antigen-specific cytolytic T lymphocytes (CTL). To this end, several monoclonal antibodies (MoAbs) previously shown to identify a novel function-associated molecule (FAM) involved in human NK function were utilized. Flow cytometry revealed that these MoAbs reacted with the majority of human NK, but only with a subpopulation of CTL isolated from primary mixed lymphocyte cultures. Preincubation of CTL with the MoAbs inhibited the NK-like lysis of K562 targets. Experiments with anti-CD3 MoAb demonstrated that neither the NK-like cytotoxicity of CTL nor the lytic activity of NK were mediated by the CD3 complex. Expression of the novel FAM was found to develop in T-cell cultures at the time that NK-like cytotoxicity was observed. Repeated in vitro antigenic stimulation of CTL was shown to result in loss of NK-like cytotoxicity, as well as loss of the FAM on the CTL surface. Thus, NK-like cytotoxicity displayed by antigen-specific CTL appears to be mediated by a novel FAM that is identical to that structure found on NK.     

Interferon-alpha (IFN-alpha) enhances cytotoxicity in healthy volunteers and chronic hepatitis C infection mainly by the perforin pathway.

Cell-mediated cytotoxicity is exerted via perforin and Fas ligand (FasL). We have recently shown that IFN-alpha up-regulates FasL expression in T cells isolated from healthy volunteers and augments activation-induced T cell death. Since the Fas/FasL system is implicated in the pathogenesis of hepatic failure and both molecules have been shown to be up-regulated in hepatitis C virus (HCV) infection, we studied whether cytotoxicity via the FasL system is enhanced by IFN-alpha and therefore could contribute to hepatic injury. We investigated FasL and perforin expression in peripheral blood mononuclear cells (PBMC) derived from HCV+ donors by Northern analysis and soluble FasL synthesis by ELISA. Natural killer (NK) cell and cytotoxic T lymphocyte (CTL) cytotoxicity was studied by 51Cr-release assays. IFN-alpha up-regulates FasL mRNA and protein synthesis in mitogen-activated PBMC of HCV+ individuals, as previously found in healthy subjects. Stimulation with IFN-alpha increases perforin mRNA levels in PBMC. In NK cytotoxicity assays, the enhancement of cytotoxicity by IFN-alpha is mainly due to the perforin pathway, while the FasL pathway plays only a minor role. In CTL cytotoxicity experiments neither the FasL nor the perforin pathway is further enhanced by IFN-alpha. Our data suggest that up-regulation of perforin by IFN-alpha results in elevated cytotoxicity, suggesting that IFN-alpha might support elimination of virally infected cells via this pathway. In contrast, the major effect of IFN-alpha on the Fas/FasL system might be the enhanced elimination of activated T cells as a means of finally limiting a T cell response.