Cytolysis Of Tumor Cells Research Articles

MHC‐dependent cytolysis of autologous tumor cells by lymphocytes infiltrating urothelial carcinomas

MHC‐dependent cytolysis of autologous tumor cells by lymphocytes infiltrating urothelial carcinomas

Purification and characterisation of soluble intercellular adhesion molecule-1 and its effect on cell-mediated cytolysis of bladder tumour cells.

Intercellular adhesion molecule-1 (ICAM-1) is a transmembrane glycoprotein and a major ligand for the integrin leukocyte function-associated antigen1 (LFA-1). The interaction between ICAM-1 and LFA-1 plays an important role in many lymphocyte activities during inflammatory and immunological reactions. Recently, a soluble form of ICAM-1 (SICAM-1) has been detected in normal serum [ 13 and we have previously demonstrated the secretion of SICAM-1 by cytokine stimulated bladder tumour cell lines as well as its detection in the urine of patients receiving bacillus Calmette-Guerin (BCG) immunotherapy for bladder cancer 121. Reports of a functional role for SICAM-1 are conflicting. While some have suggested that SICAM-1 can inhibit cell-cell adhesion events there are several which have failed to demonstrate this. In an attempt to elucidate a role for SICAM-1 we studied whether highly purified SICAM-1 could compete with membrane bound ICAM-1 for lymphocyte binding, thus allowing tumour cells to evade immunodetection. This was approached by isolating and characterising SICAM-1 from a variety of starting materials and determining their effect on the lymphokine-activated killer (LAK) cell-mediated cytolysis of bladder tumour cells in vitm. Soluble ICAM-1 was isolated by immunoaffinity chromatography from normal human serum, bladder tumour cell culture supernatants and the urine of patients receiving BCG immunotherapy for bladder cancer on two different anti-ICAM-1 monoclonal antibody (MAb) columns. Figure 1 shows that the SICAM-1 isolated from all three sources resolved as a single diffuse band of molecular mass 80 to 90 kDa under both nonreducing and reducing conditions characteristic of a heavily glycosylated glycoprotein and that this was slightly higher than the mass found for recombinant SICAM-1. The molecular mass range obtained is in the expected size range for monomeric SICAM-1 and is in agreement with previous results [1,31. Monomeric SICAM-1 idated from serum retained its ability to inhibit the binding of an anti-ICAM-1 MAb to ICAM-1 positive target cells. In contrast, physiological concentrations of monomeric ICAM1 isolated from serum and urine failed to inhibit the LAK cell mediated cytolysis of four bladder tumour cell lines (Table 1). However, sensitivity to lysis could be effectively blocked by the addition of antibodies to both ICAM-1 and LFA-1 demonstrating the importance of this adhesion pathway (Table 1). It would therefore appear unlikely that the concentrations of SICAM-1 found in the urine following BCG immunotherapy could antagonise LFAl/ICAM1 mediated events in vivo in the bladder.

RIFN-γ-activated rat neutrophils induce tumor cell apoptosis by nitric oxide

We have previously shown that 1) neutrophils activated with various cytokines, including rat recombinant interferon gamma (rIFN-gamma), inhibit tumor cell growth and that 2) nitric oxide (NO) is the effector molecule in tumor inhibition by rIFN-gamma-stimulated rat peritoneal exudate neutrophils. In this study, we examined the nature of tumor cell death induced by rat peritoneal neutrophils activated by rIFN-gamma in order to clarify the mechanism of apoptosis in neoplastic tumor cell death. DNA of 3 syngeneic rat tumor cell lines was significantly fragmented within 3 hr of incubation in the presence of rIFN-gamma-activated neutrophils, and this effect was dependent on both the concentration of rIFN-gamma and the number of neutrophils. This DNA fragmentation was inhibited by L-N-(I-iminoethyl)-ornithine (L-NIO), a NO synthase inhibitor, but not by superoxide dismutase (SOD). Tumor cells treated with the activated neutrophils were shown by electron microscopy to be apoptotic, exhibiting necrotic features with a longer incubation. On the other hand, cytolysis of tumor cells, as determined by a [3H]-uridine release assay, was first observed only at 24 hr of incubation with the rIFN- gamma-activated neutrophils. Taken together, our results suggest that tumor cell apoptosis by activated neutrophils is NO-dependent and that apoptotic tumor cells undergo necrosis as a secondary process. We suggest that tumor cell apoptosis induced by activated neutrophils plays an important role in regulation of neoplastic tumor cell growth and death in vivo.

Purification and characterisation of soluble intercellular adhesion molecule-1 (sICAM-1) and its effect on cell-mediated cytolysis of tumour cells

The aim of this study was to determine the effects of highly purified soluble ICAM-1 (sICAM-1) from a variety of sources on lymphokine-activated killer (LAK) cell-mediated cytolysis of bladder tumour cells. Soluble ICAM-1 was isolated by immunoaffinity chromatography using two different anti-ICAM-1 monoclonal antibody (mAb) columns from normal human serum, bladder tumour cell culture supernatants and the urine of patients receiving bacillus Calmette-Guerin (BCG) immunotherapy for bladder cancer. Soluble ICAM-1 from all sources resolved as a single diffuse band with a molecular weight of 80 to 90 kilodaltons (kDa) on Western blots under both non-reducing and reducing conditions, consistent with the predicted molecular weight for monomeric sICAM-1. Monomeric sICAM-1 isolated from serum retained its ability to inhibit the binding of an anti-ICAM-1 mAb to ICAM-1 positive target cells. In contrast, monomeric sICAM-1 isolated from serum and urine failed to inhibit LAK cell-mediated cytolysis of four bladder tumour cell lines. These results are in agreement with recent observations that the monomeric form of sICAM-1 binds to its receptor LFA-1 with extremely low affinity, indicating that at physiological concentrations sICAM-1 does not interfere in ICAM-1/LFA-1 mediated cellular adhesion events.

Tumor Cell Cytolysis Mediated by Valorphin, an Opioid-Like Fragment of Hemoglobin β-Chain

Blishchenko, E. Yu., O. A. Mernenko, I. I. Mirkina, D. K. Satpaev, V. S. Ivanov, L. D. Tchikin, A. G. Ostrovsky, A. A. Karelin and V. T. Ivanov. Tumor cell cytolysis mediated by valorphin, an opioid-like fragment of hemoglobin β-chain. Peptides 18(1) 79–85, 1997.—Valorphin, an endogenous opioid-like hemoglobin fragment, is cytotoxic for L929 and K562 tumor cells in 10 −7–10 −13 M concentration range. Because cytolytic effects induced by valorphin in K562 cells are inhibited by naloxone, opioid receptors should be involved in induction of valorphin-mediated tumor cell death. Three distinct cytolytic processes, differing in the onset time and the development time, take place with K562 cells within 10–18 h of incubation with valorphin. All three processes are not associated with apoptotic mechanism of cell death.

The Immune Response to Class I-Associated Tumor-Specific Cutaneous T-Cell Lymphoma Antigens

In order to determine whether the neoplastic T cells from patients with cutaneous T-cell lymphoma express tumor-specific antigens that can serve as the targets of an immune response, we took advantage of family-specific monoclonal antibodies, magnetic bead technology, and recombinant cytokines, which provided the previously precluded ability to isolate and expand populations of purified tumor and autologous CD8 cytotoxic T cells. Four patients with advanced cutaneous T-cell lymphoma had CD8 cells that specifically killed autologous tumor in a class I limited fashion. Tumor cell cytolysis could be specifically enhanced by pre-culture with autologous gamma-irradiated tumor. The cytolytic T cells produced tumor necrosis factor-alpha in response to stimulation with autologous tumor. The presence of tumor-specific cytotoxic T cells recognizing distinctive class I associated molecules on cutaneous T-cell lymphoma tumor cells suggests that infiltration of early lesions by CD8 cells reflects host immunity to the neoplasm. These studies provide the foundation for the development of tumor vaccines through the use of cytotoxic T cells to isolate and characterize tumor-associated cutaneous T-cell lymphoma peptides.

Detection of Soluble Adhesion Molecules in Pleural Effusions

Multiple immune functions such as activation of T cells and monocytes or cytolysis of tumor cells are mediated via the adhesion receptor/ligand pairs CD2/LFA-3 and LFA-1/ICAM-1. Because soluble forms of LFA-3 (sLFA-3) and ICAM-1 (sICAM-1) can interfere with these functions, we asked whether increased levels of sLFA-3 and/or sICAM-1 can be found in malignant or inflammatory effusions compared with transudates. sLFA-3 and sICAM-1 levels were measured by enzyme-linked immunoassays in pleural effusions from 70 patients (6 transudates, 10 inflammatory, 47 malignant, and 7 other effusions). Twenty pleural fluid samples were tested in parallel for the complete sLFA-3 molecule or sLFA-3-domain 1 only. Increased levels of sICAM-1 were found in all types of exudates compared with transudates. Highest levels of sICAM-1 were measured in malignant exudates, particularly in effusions caused by mesotheliomas, non-small lung cancers, and gynecologic malignancies. This was also true for sLFA-3. However, sLFA-3 levels were not increased in inflammatory effusions. sLFA-3 levels correlated significantly with protein, cholesterol, lactate dehydrogenase, and sICAM-1 levels. Comparison of sLFA-3-domain 1 and the complete sLFA-3 molecule revealed identical sLFA-3 levels, suggesting the absence of nonfunctional split products. Elevated levels of the complete sLFA-3 molecule were found in malignant pleural effusions, while sICAM-1 level was elevated in both inflammatory and malignant effusions. Secretion of sICAM-1 and sLFA-3 by tumor cells might block T-cell-mediated immune functions such as tumor cytotoxicity. Alternatively, secretion of soluble adhesion molecules might reflect the generalized inflammation within the pleural space.

Cellular immune function in mice tolerant to or abstinent from l-trans-delta 9-tetrahydrocannabinol.

The effects of tolerance to l-trans-delta 9-tetrahydrocannabinol (THC) following repeated administration and of subsequent abstinence following drug withdrawal on the cellular immune function were determined in female B6C3F1 mice. Mice were injected with THC (10 mg/kg s.c.) twice daily for 4 days. On day 5, analgesic response to various doses of THC was determined using the tail flick test. Similarly, hypothermic response to THC was also determined. Multiple injections of THC resulted in the development of tolerance to both the analgesic and hypothermic effects of THC. Immune function studies were performed on mice rendered tolerant to or abstinent from THC by these procedures. Neither tolerance nor abstinence subsequent to a 4-day THC administration had any effect on either body weight or thymus weight and cellularity, although both spleen weight and cellularity were decreased in THC-abstinent animals. Likewise, no significant effects on B cell proliferation were observed in either tolerant or abstinent mice. The production of the cytokine interleukin-2 by T helper cells was markedly suppressed in both tolerant and abstinent mice, whereas the production of interleukin-4 was significantly suppressed only in THC-abstinent mice. Significant suppression of tumor cell cytolysis mediated by cytotoxic T cells and natural killer cells was only observed in THC-abstinent mice. These results suggest that THC-mediated modulation of the immune response may result from a differential effect on cellular populations.

Differential Inhibition of RAW264.7 Macrophage Tumoricidal Activity by 9Tetrahydrocannabinol

delta 9tetrahydrocannabinol (THC), the major psychoactive component of marijuana, has been shown to inhibit macrophage cell contact-dependent cytolysis of tumor cells. The purpose of this study was to determine whether THC inhibited macrophage cytolytic function by targeting selectively tumor necrosis factor (TNF)-dependent pathways versus L-arginine-dependent reactive nitrogen intermediates. An in vitro system employing RAW264.7 macrophage-like cells as effectors and TNF-sensitive mouse L929 fibroblasts or nitric oxide (NO.)-sensitive P815 mastocytoma cells as targets, was employed to assess the effect of THC on cytolysis. Macrophages were pretreated with THC or vehicle for 48 hr, subjected to multistep activation with 10 U/ml recombinant mouse gamma-interferon (IFN-gamma) plus 100 ng/ml LPS or to direct activation with 1 microgram/ml LPS, and co-cultured with tumor cells in the presence or absence of THC. THC inhibited TNF-dependent killing by macrophages subjected to either multistep or direct activation. Decreased amounts of TNF-alpha were detected in medium of macrophage cultures treated with THC. In contrast, THC inhibited NO.-dependent cell contact killing only for macrophages subjected to direct activation. Decreased levels of NO2-, a stable degradation product of the short-lived and highly toxic effector molecule NO., were produced by these macrophages. In addition, the effect of the enantiomeric pairs (-)CP55,940/(+)CP56,667 or (-)HU-210/(+)HU-211 on macrophage cell contact-dependent killing was assessed. Inhibition of macrophage tumoricidal activity against TNF-sensitive L929 cells was effected by both isomers of THC analogs. In contrast, both of the enantiomeric pairs had an effect on killing of NO.-sensitive P815 mastocytoma cells only for macrophages subjected to direct activation. These data suggest that cannabinoids inhibit macrophage cell contact-dependent killing of tumor cells by a noncannabinoid receptor-mediated mechanism. However, specific cytolytic pathways are inhibited differentially by cannabinoids depending on the activation stimuli to which macrophages are exposed.

The Induction and Augmentation of Macrophage Tumoricidal Responses by Platelet-Activating Factor

Platelet-activating factor (PAF) has previously been shown to stimulate intracellular signal transductional events similar to the macrophage tumoricidal activators interferon-γ (IFN-γ) and lipopolysaccharide (LPS). Macrophages stimulated with IFN-γ and LPS utilize various cytolytic mediators in order to kill tumor cells. However, PAF has been shown to only induce and enhance tumor necrosis factor-α (TNFα)-mediated cytolysis by macrophages. Therefore, the purpose of this study was to determine whether PAF, in comparison with IFN-γ and LPS, could stimulate macrophages for both TNFγ- and non-TNFγ-mediated tumoricidal activity. For this, the activation of macrophages for a cytolytic response was characterized by the production of TNFγ and nitric oxide (NO-2), as well as, their ability to kill select tumor cells. PAF together with IFN-γ stimulated macrophage secretion of NO-2. In addition, PAF enhanced IFN-γ- and LPS-stimulated NO-2 production. PAF, together with IFN-γ, also activated macrophages for tumoricidal activity against TNFγ-resistant tumor cells. In assays to determine the temporal sequence of activation, increased tumor cell cytolysis was observed only when macrophages were first treated with IFN-γ. Moreover, PAF enhanced macrophage tumoricidal activity when added with LPS and IFN-γ. With respect to TNFγ production, macrophages activated with high concentrations of PAF stimulated significant levels of TNFγ compared to macrophages without PAF. A similar level was observed following multiple additions of a lower concentration of PAF, Also, PAF induced macrophage cytolytic activity against a TNFγ-sensitive tumor cell. In addition, PAF significantly enhanced LPS-induced TNFα production. Thus, PAF can play a modulatory role in the activation for non-TNFγ-mediated tumoricidal activity of macrophages.

Epidermal growth factor-induced protection of tumour cell susceptibility to cytolysis

Using radiobinding, transfection and colorimetric assays, the biological significance of epidermal growth factor (EGF) and its receptor on established human tumour cell lines was investigated. The intensity of class I major histocompatibility antigen (MHC) and EGF receptor (EGFR) expression on 20 tumour cell lines was investigated and showed no direct correlation (coefficient of correlation r = 0.43 and P = 0.06). Furthermore, transfection of the β2-microglobulin gene into a class I negative bladder tumour cell line, resulting in the re-expression of fully assembled cell surface class I antigens, did not result in alteration of EGFR expression. However, there was an inverse correlation between the intensity of EGFR expression and the stimulatory response of cells to exogenously added EGF. The per cent inhibitions of cell proliferation by EGF at 100 ng/ml for A431 (highest EGFR expressor) and Scaber (lowest EGFR expressor) were 37 and −7%, respectively. The results also showed that cell lines isolated from testis tumours positive for epithelial markers (using pan keratin antibody LP34 as an epithelial marker), expressed significantly lower EGFR levels than cell lines from bladder tumours. The expression of EGFR receptor was not modulated by interferons (IFN-α and -γ and only a minor effect with IFN-β) or active supernatant containing a mixture of cytokines. Whilst the pretreatment of tumour cells with IFNs resulted in a significant increase in the susceptibility of tumour cells to interleukin-2-activated peripheral blood mononuclear cells, EGF treatment resulted in their protection. Thus, the per cent killing at an effector:target ratio of 20:1 for untreated cells and EGF (100 ng/ml), IFN-α (1000 U/ml), -β (2000 U/ml) and -γ (100 U/ml) were 53%, 33% ( P = 0.004), 64% ( P = 0.004), 69% ( P = 0.001) and 66% ( P = 0.001), respectively. These results indicate the complex interactions between EGF and EGFR and their relevance in modifying tumour cell behaviour. The hypothesis that the resistance to cytolysis of tumour cells induced by EGF stimulation may be a factor in the accelerated tumour growth seen in patients after traumatic tissue damage is discussed.

Effects of oxidized LDL on mononuclear phagocytes: inhibition of induction of four inflammatory cytokine gene RNAs, release of NO, and cytolysis of tumor cells.

A critical step in development of atherosclerosis is the interaction of oxidized low-density lipoprotein (LDL) with mononuclear phagocytes. Oxidized LDL, as well as acetyl-LDL, is rapidly taken up into macrophages via a family of scavenger receptors. We report that macrophages treated with oxidized LDL have markedly lower levels of mRNA specific for the genes MCP-1, TNF-alpha, IL-1 alpha, and KC as measured by Northern blot analyses of lipopolysaccharide (LPS)-stimulated macrophages. By contrast, acetyl-LDL does not inhibit these genes at the doses at which oxidized-LDL is effective. Similar effects are observed whether the LDL is oxidized in the presence of Cu2+ or of Fe2+. Such inhibition also occurs when maleylated bovine serum albumin (BSA), which also clears by one or more scavenger receptors on macrophages, is used as the stimulant. Fe2+ or Cu2+ oxidized LDL inhibits release of nitric oxide when triggered by LPS and direct cytolysis of tumor cells when triggered by maleylated BSA or LPS. Taken together, the data presented indicate that oxidized LDL inhibits induction of several important gene RNAs as well as functional markers that characterize the development of inflammatory and fully activated macrophages.

Apoptosis as a mechanism of cytolysis of tumor cells by a pathogenic free-living amoeba

Previous studies have shown that trophozoites of the pathogenic free-living amoeba Acanthamoeba castellanii rapidly lysed a variety of tumor cells in vitro. Tumor cells undergoing parasite-mediated lysis displayed characteristic cell membrane blebbing reminiscent of apoptosis. The present investigation examined the role of apoptosis (programmed cell death) in Acanthamoeba-mediated tumor cell lysis. The results showed that more than 70% of tumor cell DNA was fragmented following exposure to Acanthamoeba cell extracts. By contrast, only 7% of untreated control cells underwent DNA fragmentation. DNA fragmentation increased significantly in a dose-dependent fashion following concentration of the parasite extract. Apoptosis was also confirmed by DNA ladder formation. Characteristic DNA ladders, consisting of multimers of approximately 180 to 200 bp, were produced by tumor cells exposed to Acanthamoeba cell extracts. The morphology of tumor cell lysis was examined by light and scanning electron microscopy. Tumor cells exposed to parasite extract displayed morphological features characteristic of apoptosis including cell shrinkage, cell membrane blebbing, formation of apoptotic bodies, and nuclear condensation. By contrast, similar effects were not found in tumor cells exposed to extract similarly prepared from normal mammalian cells (i.e., human keratocytes). The results suggest that at least one species of pathogenic free-living amoeba is able to lyse tumor cells by a process that culminates in apoptosis.

The LFA‐1/ICAM cell adhesion pathway is involved in tumor‐cell lysis mediated by bispecific monoclonal‐antibody‐targeted T lymphocytes

We investigated the role of the LFA-1/ICAM, VLA-4/VCAM-1 and CD2/LFA-3 adhesion pathways in the cytolysis of tumor cells mediated by an anti-EGF-R/anti-CD3 bispecific monoclonal antibody (biMAb). The biMAb induced efficient lysis of EGF-R+ tumor cells (A431, HT-29, IGROV-1 and MDA-MB468) by cytotoxic T lymphocytes (CTL) cultured in IL-2. Pretreatment of effector cells by anti-LFA-1 alpha (CD11a) and anti-LFA-1 beta (CD18) MAbs significantly inhibited cytolysis of all types of EGF-R+ tumor cells, while anti-CD2 and anti-VLA-4 MAbs were virtually ineffective. We investigated the expression of adhesion-molecule counter-receptors on tumor target cells by indirect immunofluorescence. HT-29, A431 and MDA-MB 468 tumor cells expressed an ICAM-1+2-3- VCAM-1- LFA-3+ phenotype, while IGROV-1 was ICAM-1-2+3- VCAM-1- LFA-3+. Pre-treatment of A431, HT-29 and MDA-MB468 with anti-ICAM-1 MAb inhibited cytolysis, further supporting the functional involvement of the LFA-1/ICAM adhesion pathway in biMAb-targeted tumor-cell lysis. In addition, treatment of target cells with TNF alpha or IFN gamma for 24 hr increased the expression of ICAM-1 in HT-29, A431 and MDA-MB468 (ICAM-2 was induced on IGROV-1) and also enhanced the sensitivity of these target cells to biMAb-targeted cytotoxicity. These data suggest that up-regulation of ICAM-molecule expression by inflammatory cytokines may increase susceptibility of tumor cells to biMAb-targeted lysis. Anti-LFA-1 MAbs did not significantly inhibit the formation of conjugates between biMAb-coated T lymphocytes and tumor cells. Co-aggregation of LFA-1 molecules with biMAb-bound CD3 molecules resulted in a more sustained and prolonged increase in the intracellular concentration of free Ca++ in CD8+ cultured CTL lines. These findings indicate that in T cells targeted by anti-CD3/anti-TAA biMAb LFA-1 may act as a co-receptor molecule which enhances signal transduction through the CD3/TCR complex.

The biologic window for chimeric l6 radioimmunotherapy

There has been little success in using radioimmunotherapy in patients with adenocarcinoma, partly because of the low tumor uptake of the administered monoclonal antibody (MoAb). The authors recently reported therapeutic response in advanced cancer patients who received 131I chimeric-L6 MoAb. The L6 MoAb identifies abundant, nonshed antigen that is expressed in many human carcinomas, including carcinomas of the lung, breast, colon, and ovary. In vitro both mouse L6 (L6) and chimeric L6 (ChL6) mediate complement-dependent tumor cytolysis with human serum, and antibody-dependent tumor cell cytolysis with normal human peripheral blood mononuclear cells. The authors have used L6 or ChL6 for radioimmunotherapy to exploit their biologic activity to create a "therapeutic window" of increased vascular permeability, allowing more 131I MoAb to reach the tumor. A reactive target is present in the vascular endothelium but can be covered by unlabeled L6 or ChL6. Nine patients with metastatic breast cancer were treated on a therapy protocol and received imaging and therapy doses of 131I ChL6 on two sequential days at 4 week intervals. During each treatment cycle, serum cytokines, complement, albumin, and 131I ChL6 blood clearance were monitored, peripheral blood mononuclear cell activation was assessed, and tumor uptake and response were documented. After L6 or ChL6 was infused, patients demonstrated immediate serum-complement activation, manifested by rapidly decreasing levels of serum complements 3 and 4. Tumor uptake of the second 131I MoAb (therapeutic) injection, given after the second daily injections of 200 mg MoAb, was usually higher than the tumor uptake of the first 131I MoAb (imaging) dose given after a single 200 mg infusion of MoAb. Although serum complement frequently decreased after the first 50-100 mg dose of L6 or ChL6, elevation of soluble interleukin-2 receptor (IL-2R) in serum was seen only in patients who received 150 mg or more of L6 or ChL6. In the nine treated patients, with only one exception, the higher grade of therapeutic tumor response was seen in patients with a greater increase in IL-2R levels. The clinical importance of understanding these mechanisms is emphasized by the occurrence of measurable tumor regressions in five of the first nine advanced metastatic breast cancer patients treated in this manner. Absence of pulmonary edema and delayed release of dose-dependent IL-2R suggest that targeting of the pulmonary endothelium by L6 or ChL6 is not the major cause of the observed biologic effects. This unique response of a solid tumor to radioimmunoconjugate therapy may be secondary to both the increased delivery of the radioimmunoconjugate to tumor cells caused by enhanced vascular permeability, and to synergistic effects of radiation and activated effector cell mechanisms.

Targeting of T lymphocytes against egf‐receptor+ tumor cells by bispecific monoclonal antibodies: Requirement of CD3 molecule cross‐linking for t‐cell activation

Targeting of T lymphocytes against epidermal growth-factor-receptor (EGF-R)+ tumor cells was achieved by constructing a hybrid hybridoma which secretes an anti-EGF-R/anti-CD3 bispecific monoclonal antibody (biMAb) of hybrid isotype (IgG1/IgG2a). Purification of biMAb molecules from parental anti-EGF-R and anti-CD3 MAbs was performed by protein-A chromatography. The purified biMAb was able to trigger the lysis of EGF-R+ tumor cell lines (A431, IGROV-1, MDA-468 and U-87) and of NIH-3T3 transfectants expressing human EGF-R by cytolytic T lymphocytes, but it was ineffective in the case of EGF-R-negative tumor targets. Normal EGF-R+ cells (keratinocytes and endometrial cells) were also susceptible to biMAb-targeted cytolysis. However, the amount of biMAb required to induce half-maximal cytolysis of tumor cells over-expressing the EGF-R molecule (A431) was considerably lower than that required to induce lysis of EGF-R+ tumor or normal cells which express EGF-R at considerably lower density. The ability of such biMAbs to deliver activation signals to T cells was evaluated by Ca++ mobilization and lymphokine production experiments. The soluble anti-EGF-R/anti-CD3 biMAb failed to induce intracellular Ca++ increases, which occurred only after cross-linking induced by an anti-mouse IgG antibody. Secretion of lymphokines (IFN-gamma, TNF-alpha and GM-CSF) was induced by contact of the biMAb-coated effector cells with the relevant tumor target, whereas the soluble biMAb was virtually ineffective. In addition, biMAb-coated effector cells retained the ability to recognize and to lyse EGF-R+ tumor cells for a prolonged period of time. Our data indicate that activation of effector cells targeted by biMAbs can only occur at the tumor site, where cross-linking of surface CD3 molecules is induced by contact with the tumor cells.

Adoptive immunotherapy of advanced melanoma patients with interleukin-2 (IL-2) and tumor-infiltrating lymphocytes selected in vitro with low doses of IL-2.

Freshly isolated tumor-infiltrating lymphocytes (TIL) from stage IV melanoma patients were cultured for 2 weeks with low doses of interleukin-2 (IL-2; 120 IU/ml), to select potentially for tumor-specific lymphocytes present in the neoplastic lesion, followed by high doses (6000 IU/ml) to achieve lymphocyte expansion. TIL were serially analyzed for their expansion, phenotype and cytotoxic activity against autologous and allogenic tumor cells. A preferential lysis of autologous melanoma cells was obtained in long-term cultures of 7/13 cases (54%), while the remaining ones showed a major-histocompatibility-complex-unrestricted, lymphokine-activated-killer(LAK)-like activity at the time of in vivo injection. Sixteen patients with metastatic melanoma were infused with TIL (mean number: 6.8 x 10(9), range: 0.35 x 10(9)-20 x 10(9)) and IL-2 (mean dose: 130 x 10(6) IU, range: 28.8 x 10(6)-231 x 10(6) IU); 1 complete and 3 partial responses were observed in 12 evaluable patients (response rate 33%). In all responding patients, injected TIL showed an in vitro preferential lysis of autologous tumor cells, while in no cases were TIL with LAK-like activity associated with a clinical response. The mean autologous tumor cytotoxic activity of TIL at the time of in vivo injection was significantly higher in responding patients in comparison to nonresponding ones, suggesting that a marked and preferential cytolysis of autologous tumor cells is associated with the therapeutic efficacy of TIL.

Apoptosis and DNA fragmentation precede TNF-induced cytolysis in U937 cells.

The hypothesis that activation of apoptosis and DNA fragmentation is involved in TNF-mediated cytolysis of U937 tumor cells was investigated. Morphological, biochemical, and kinetic criteria established that TNF activates apoptosis as opposed to necrosis. Within 2-3 h of exposure to TNF, U937 underwent the morphological alterations characteristic of apoptosis. This was accompanied by cleavage of DNA into multiples of nucleosome size fragments. Both of these events occurred 1-2 h prior to cell death as defined by trypan blue exclusion or 51Cr release. DNA fragmentation was not a non-specific result of cell death since U937 cells lysed under hypotonic conditions did not release DNA fragments. The percentage of cells undergoing apoptosis depended on the concentration of TNF and was augmented by the addition of cycloheximide. A TNF-resistant variant derived from U937 did not undergo apoptosis in response to TNF, even in the presence of cycloheximide. Furthermore, TNF could still activate NFkB in this variant, suggesting that this pathway is not involved in TNF-mediated cytotoxicity. Two agents known to inhibit TNF-mediated cytotoxicity, ZnSO4 and 3-aminobenzamide, were shown to inhibit TNF-induced apoptosis. Taken altogether, these data support the hypothesis that activation of apoptosis is at least one essential step in the TNF lytic pathway in the U937 model system.

Anti-neoplastic action of peritoneal macrophages following oral administration of ether analogues of lysophospholipids

Inflamed lesions of normal and cancerous tissues induce activation of phospholipase A in plasma membranes resulting in the release of various decomposed products of membranous lipids. Oral administration in mice of dodecylglycerol (DDG), a synthetic alkylglycerol, and an alkyl ether analogueoflysophospholipids, 1-0-octadecyl-2-0-methyl-rac-glycero-3-phosphocholine (ET-18-OCH 3-choline) efficiently activated peritoneal macrophages for enhanced Fc-mediated (fragment crystallisable) ingestion of red blood cells and direct cytotoxic action on retinoblastoma tumour cells. The activated macrophages not only inhibited tumour cell growth, but also markedly induced cytolysis of tumour cells. The antitumour capability of the macrophages was substantiated by luminol-enhanced chemiluminescence. These findings suggest that dodecylglycerol and ET-18-OCH 3-choline administered orally retain their ability to induce a high level of macrophage activation and tumour cytotoxicity, just as occurs with intraperitoneal administration. Thus, these compounds have potential practical application in chemotherapy and immunotherapy of the tumour, which could be accomplished by simple oral rather than parenteral administration.

Allospecific recognition of hemic cells in vitro by natural killer cells from athymic rats: Evidence that allodeterminants coded for by single major histocompatibility complex haplotypes are recognized

We have previously shown that large granular lymphocyte (LGL)-enriched cell populations have the capacity to spontaneously recognize and kill allogeneic small lymphocytes and bone marrow cells (BMC) in vitro in certain strain combinations of rats. Here, we have studied the alloreactivity of natural killer (NK) cells from PVG nude (RT1c) rats against a panel of major histocompatibility complex (MHC) incompatible hemic cells. Both lymphocytes and BMC from the AO (RT1u), DA (RT1a), BN (RT1n) as well as the MHC-congenic PVG-RT1u (RT1u) rat strains were efficiently killed in vitro, whereas cells from syngeneic PVG rats were spared. The structures recognized on lymphocytes and BMC were probably similar since the two cell populations inhibited each other in cross-competition experiments. A number of features aligned the alloreactive effector cells with NK cells and not T cells. (a) Only about 5% of the effector cells from nude spleens expressed the T cell antigens CD3, CD5 or T cell receptor (TcR) alpha/beta whereas greater than 50% of the cells expressed markers present on NK cells (CD2, CD8, OX52 and the rat NK cell-specific marker NKR-P1 recognized by the monoclonal antibody 3.2.3). (b) The alloreactive cells were granular since pretreatment of nude spleen cells with the lysosomotropic agent L-leucine methyl ester which eliminated LGL, simultaneously abolished the cytolysis of both allogeneic lymphocytes and YAC-1 tumor cells. (c) Nude spleen cells stimulated with human recombinant interleukin 2 for 1 week in vitro generated large granular proliferating cells which were CD3-, CD5-, TcR alpha/beta-, but greater than 95% 3.2.3+. These cells efficiently killed allogeneic hemic cells from the same rat strains as did freshly isolated effector cells. (d) The cytolysis of allogeneic hemic cells could effectively be inhibited with unlabelled NK-sensitive (YAC-1 and K-562), but not NK-resistant (Roser leukemia) tumor cells. Cross-competition studies showed that PVG nude NK cells discriminated between AO, BN and DA BMC, suggesting that different alloantigens were positively recognized by subsets of NK cells. The mode of inheritance of the allodeterminant specifically recognized on AO BMC was investigated in crosses and backcrosses between AO and BN or DA rats. A gene dosage effect was observed in that this determinant was expressed at a slightly reduced level in F1 hybrids.(ABSTRACT TRUNCATED AT 250 WORDS)