Lymphokine-activated Killer Cell-mediated Cytotoxicity Research Articles

Cannabinoids increase lung cancer cell lysis by lymphokine-activated killer cells via upregulation of ICAM-1

Cannabinoids have been shown to promote the expression of the intercellular adhesion molecule 1 (ICAM-1) on lung cancer cells as part of their anti-invasive and antimetastatic action. Using lung cancer cell lines (A549, H460) and metastatic cells derived from a lung cancer patient, the present study addressed the impact of cannabinoid-induced ICAM-1 on cancer cell adhesion to lymphokine-activated killer (LAK) cells and LAK cell-mediated cytotoxicity. Cannabidiol (CBD), a non-psychoactive cannabinoid, enhanced the susceptibility of cancer cells to adhere to and subsequently be lysed by LAK cells, with both effects being reversed by a neutralizing ICAM-1 antibody. Increased cancer cell lysis by CBD was likewise abrogated when CBD-induced ICAM-1 expression was blocked by specific siRNA or by antagonists to cannabinoid receptors (CB1, CB2) and to transient receptor potential vanilloid 1. In addition, enhanced killing of CBD-treated cancer cells was reversed by preincubation of LAK cells with an antibody to lymphocyte function associated antigen-1 (LFA-1) suggesting intercellular ICAM-1/LFA-1 crosslink as crucial event within this process. ICAM-1-dependent pro-killing effects were further confirmed for the phytocannabinoid Δ9-tetrahydrocannabinol (THC) and R(+)-methanandamide (MA), a hydrolysis-stable endocannabinoid analogue. Finally, each cannabinoid elicited no significant increase of LAK cell-mediated lysis of non-tumor bronchial epithelial cells, BEAS-2B, associated with a far less pronounced (CBD, THC) or absent (MA) ICAM-1 induction as compared to cancer cells. Altogether, our data demonstrate cannabinoid-induced upregulation of ICAM-1 on lung cancer cells to be responsible for increased cancer cell lysis by LAK cells. These findings provide proof for a novel antitumorigenic mechanism of cannabinoids.

FADD cleavage by NK cell granzyme M enhances its self-association to facilitate procaspase-8 recruitment for auto-processing leading to caspase cascade

Granzyme M (GzmM), an orphan Gzm, is constitutively and abundantly expressed in innate effector natural killer cells. We previously demonstrated that GzmM induces caspase (casp)-dependent apoptosis and cytochrome c release from mitochondria. We also resolved the crystal structure for GzmM and generated its specific inhibitor. However, how GzmM causes casp activation has not been defined. Here we found that casp-8 is an initiator caspase in GzmM-induced casp cascade, which causes other casp activation and Bid cleavage. GzmM does not directly cleave procaspase-3 and Bid, whose processing is casp dependent. Casp-8 knockdown or deficient cells attenuate or abolish GzmM-induced proteolysis of procaspase-3 and Bid. Extrinsic death receptor pathway adaptor Fas-associated protein with death domain (FADD) contributes to GzmM-induced casp-8 activation. GzmM specifically cleaves FADD after Met 196 to generate truncated FADD (tFADD) that enhances its self-association for oligomerization. The oligomerized tFADD facilitates procaspase-8 recruitment to promote its auto-processing leading to casp activation cascade. FADD-deficient cells abrogate GzmM-induced activation of casp-8 and apoptosis as well as significantly inhibit lymphokine-activated killer cell-mediated cytotoxicity. FADD processing by GzmM can potentiate killing efficacy against tumor cells and intracellular pathogens.

Transforming growth factor‐beta 2 gene silencing with trabedersen (AP 12009) in pancreatic cancer

Pancreatic cancer is one of the most aggressive human cancers with a 5-year survival rate of <5%. Overexpression of transforming growth factor-beta 2 (TGF-β2) in pancreatic malignancies is suggested to be a pivotal factor for malignant progression by inducing immunosuppression, metastasis, angiogenesis and proliferation. Trabedersen (AP 12009) is a phosphorothioate antisense oligodeoxynucleotide specific for human TGF-β2 mRNA and was successfully tested in a randomized, active-controlled phase IIb clinical study in patients with high-grade glioma. Here, we report on the antitumor activity of trabedersen in human pancreatic cancer cells and in an orthotopic xenograft mouse model of human metastatic pancreatic cancer. Trabedersen reduced TGF-β2 secretion in human pancreatic cell lines with an IC50 in the low μM range without transfection reagent, clearly inhibited cell proliferation, and completely blocked migration of pancreatic cancer cells. Additionally, trabedersen reversed TGF-β2-mediated immunosuppression of pancreatic cancer cells targeted by lymphokine activated killer (LAK) cells, resulting in considerably increased LAK cell-mediated cytotoxicity. Moreover, in an orthotopic mouse model of metastatic pancreatic cancer, intraperitoneal (i.p.) treatment with trabedersen significantly reduced tumor growth, lymph node metastasis and angiogenesis. These promising results warrant further clinical development of trabedersen.

Aerolysin Cytotoxicity Can Serve as a Surrogate Marker for Immune Sensitivity of Tumor Cells to Granule-Exocytosis Pathway in Vitro.

Background: Aerolysin is a pore-forming toxin produced by Aeromonas hydrophila and interacts with target mammalian cells through binding to glycosylphosphatidyl inositol (GPI)-anchored proteins, subsequently forming a pore in the plasma membrane. Both osmotic lysis of the cell secondary to fluid influx thru pores and a rapid increase in intracellular calcium, which may have been the signal for apoptosis, when a small number of channels were opened appear to be operational in aerolysin cytotoxicity. Perforin (PFP), a prototype of pore-forming proteins is closely linked to granzyme B effect and the central element of natural killer (NK) and T cell mediated cytotoxicity, and induces apoptosis of target cells through granule-exocytosis pathway. Since perforin/granzyme B pathway is pivotal to immune mediated elimination and due to similarities between PFP and aerolysin action, we investigated the relationship between aerolysin cytotoxicity and lymphokine activated killer (LAK) cell-mediated elimination of human tumor cells.Material and Methods: We studied the effect of active aerolysin (2x10−10M) against eight tumor cell lines and five acute myeloid leukemia patient samples. Aerolysin cytotoxicity was assessed at eight different time points: first at ½ to 4 hours of incubation consistent with the known optimal cell kill mediated by PFP in NK cytotoxicity, then lastly at 18 hours to be able see overall effect. Cytotoxicity was measured by flow cytometric detection of annexin V/PI-positive cells, DiOC6 (3) /PI staining and further quantification of fluorosphere-adjusted events. Tumor cell type selection was based on their sensitivity against LAK cell-mediated cytotoxicity. Acute myeloid leukemia cells K562, U937, CMK, Meg-01, HL-60 and resistant central nervous system tumor cells (HTB-11, HTB-12 and HTB-14) were included in this study. LAK cell-mediated cytotoxicity against tumor cell lines and patient samples was measured by flow cytometric cell mediated cytotoxicity assay. Drug cytotoxicity was also evaluated in all cell lines and patient samples.Results: In timing studies, aerolysin treatment reached peak cytotoxicity around 2 hours of incubation. U937 was the most sensitive cell type to both LAK cells and aerolysin. Some cells were very resistant to LAK killing and aerolysin at the concentration used. There was significant correlation between aerolysin cytotoxicity and LAK cell-mediated kill at 2 hours in cell lines and patient samples (r = 0.99; p < 0.001, r = 0.85; p = 0.035, respectively). There was also a statistically significant correlation between optimal dose aerolysin killing at 18 hours of incubation and LAK cell-mediated cytotoxicity (r = 0.94; p = 0.04). However, there was not any correlation between drug cytotoxicity and LAK cell-mediated cytotoxicity or aerolysin-induced cell kill.Conclusion: Aerolysin sensitivity may be used as a surrogate marker for the assessment of in vitro LAK cell-mediated tumor cell kill through granule/exocytosis pathway.

Syngeneic pregnancy induces overexpression of natural killer T cells in maternal liver.

Conditions such as stress, infection, autoimmune disease, etc. elevate the number and function of extrathymic T cells that are generated mainly in the liver. As primitive, self-reactive clones of T cells that coexpress receptors of the natural killer (NK) lineage, they mediate cytotoxicity against altered self, malignant and infected cells and have the unique potential to rapidly secrete large amount of T helper 1 (Th1) or Th2 cytokines. To elucidate whether some of these changes occur even during the syngeneic pregnancy, we made phenotypic and functional characterization of mononuclear lymphatic cells (MNLCs) isolated from the liver and spleen of pregnant C57BL/6 mice, testing their cytotoxicity against syngeneic thymocytes as well as against NK- and lymphokine-activated killer (LAK)-sensitive targets. The data have shown that on the sixteenth day of syngeneic pregnancy TCRint, NK1.1+ and IL-2Rbeta+ cells were accumulated in the liver, while the quantities of CD4+ and CD8+ T cells and total number classical NK (NK1.1+CD3- or IL-2Rbeta+CD3-) cells were increased in the spleen. Pregnancy-activated hepatic and splenic MNLCs were more cytotoxic against syngeneic thymocytes, YAC-1 and P815 targets, suggesting that the maternal liver is a main producer of autoreactive NKT clones, which subsequently augment NK- and LAK cell-mediated cytotoxicity in the liver and spleen.

Immunoregulating properties of peptides related to tumor rejection antigens: effect on human monocytes and natural killer cells.

The authors analyzed the effect of several 15-amino acid peptides with sequences related to tumor-rejection antigens, tyrosinase, and the MAGE family on peripheral blood mononuclear cells from healthy donors cultured for periods of 1 to 7 days. Some of these peptides promoted stimulation of monocytes, manifested by phenotypic changes, release of interleukin (IL)-1a, IL-6, and tumor necrosis factor-alpha, and induction of nitric oxide synthase on differentiated CD14++/+ CD16+ DR++ monocytes. An increase in the percentage of cytotoxic monocytes (CD14+/- CD16+) containing granule-associated DNase activity was also observed. Active peptides induced the release of IL-2 and interferon-gamma. Nonspecific natural killer and lymphokine-activated killer cell-mediated cytotoxicity was also observed against classical target cell lines (K-562 and Daudi) and allogenic melanoma cell lines AC and BB, together with an increase in granule-associated DNase in the natural killer cell-enriched population. Monocytes were needed to enhance this innate response, because peptides failed to induce the release of IL-2 on monocyte-depleted peripheral blood mononuclear cells. Data show an enhancement of the rapid innate immune response by peptides related to tumor rejection antigens and suggest that they could also determine the nature of a slow and more definitive specific immune response against tumor cells.

CYTOTOXICITY OF LAK CELLS AND EBV-SPECIFIC T CELLS AGAINST LYMPHOBLASTOID EBV-POSITIVE B CELLS: A MODEL FOR CELLULAR THERAPY OF PTLD

696 Posttransplant lymphoproliferative disorder (PTLD) is a significant cause of morbidity and mortality in transplant patients. The presence of the Epstein-Barr virus (EBV) in many of these lymphomas has prompted the use of cellular immunotherapy using either EBV-directed T cells or autologous lymphokine-activated killer (LAK) cells. EBV-specific T cells recognize viral epitopes in association with HLA Class I molecules, whereas LAK cell cytotoxicity is enhanced by their depletion. The purpose of this study was to examine the relative levels of in vitro cytotoxicity of these effector cells against either untreated or HLA Class I-depleted EBV-immortalized B lymphocytes. LAK cells, EBV-specific T cell lines and EBV-immortalized lymphoblastoid cell lines were prepared from healthy donors. The degree of cell-mediated lympholysis (CML) was determined by using a standard 4 hour 51Cr release and the results were expressed as lytic units (LU-number of effectors required for 20% lysis of 5×103 targets). In some cases, HLA Class I expression in the target cells was depleted by a brief exposure to pH 3.3 buffer. Cell viability and Class I depletion were confirmed by trypan blue exclusion and flow cytometry, respectively prior to execution of CML assays. LAK cell-mediated cytotoxicity against autologous EBV target B cells was 14.4 LU which increased to 69.6 LU when target cells were HLA-depleted(p<0.05, Wilcoxon test). On the contrary, EBV-specific T cell-mediated cytotoxicity against untreated B cell targets was 765 LU, and this decreased to 316 LU when HLA-Class I depleted cells were used as targets (p<0.05). Additionally, as compared to that of LAK cells, T cell-mediated cytotoxicity against untreated targets was significantly greater (Friedman's test p=0.03, Tukey multiple comparison p<0.05). We conclude that EBV-specific T cells show superior in vitro cytotoxicity against autologous EBV-positive B cell targets, regardless of the level of HLA Class I expression. The results may explain in part the uniform response of PTLD to T cell therapy, and the less frequent responses to that of LAK cell. The level of cytotoxicity for both effector cell types is influenced by the degree of expression of HLA Class I in this model system. This approach may be useful in examining other factors which may enhance or decrease the cytotoxic response against EBV-positive B cells.

抗癌剤耐性悪性腫よう細胞に対するＬＡＫ細胞の抗腫よう活性に関する研究

It has been hypothesized that lymphokine-activated killer (LAK) cells may be effective against antitumor drug resistant (ATDR) squamous cell carcinoma. The objective of this study was to determine 1) the susceptibility of ATDR human tumor cell lines to interleukin-2 (IL-2)-induced LAK cell-mediated cytotoxicity, and to assess 2) the characteristics of the population of LAK cells as effector cells on the tumor cell lines tested and 3) the relationship between the susceptibility of ATDR cells and their expression of surface antigens.Parent strains of CEM (derived from leukemia), KB (derived from oral squamous cell carcinoma), and TCM (derived from metastasized lung cell carcinoma from oral squamous cell carcinoma) cells were used as drug-sensitive cells. The ATDR cell lines were established from the parent cells used; the ATDR cell lines were CEM (resistant to vinblastine, VLB; CEM/VLB0.1 and CEM/VLB1.0), KB (resistant to cisplatin, CDDP, and bleomycin, BLM; KB/CDDP1.0 and KB/BLM1.0, respectively) and TCM (resistant to CDDP; TCM/CDDP1.0). Peripheral blood lymphocytes were obtained from nine healthy volunteers and fractionated; the fractions consisted of unfractionated whole cells, CD 4 + cells, CD 8 + cells, and CD16+ cells. Each fraction was cultured with IL-2, and the induced LAK cells were used as effector cells.Whole-LAK cells were the most cytotoxic against all ATDR cell lines tested (as compared with the parent cells). However, the antitumor activity of the fractionated cells was limited (antitumor activity: CD16+-LAK, 60-80% on CEM/VLB0.1 and 1.0; CD 8+-LAK, about 30% on KB/CDDP1.0 and about 50% on TCM/CDDP1.0). The expressions of P-glycoprotein (Pgp), major histocompatibility complex (MHC) class I, intercellular adhesion molecule-1 (ICAM-1), and lymphocyte function-associated antigen-3 (LFA-3) were respectively high in CEM/VLB; KB/CDDP1.0, KB/BLM1.0 and TCM/CDDP1.0; KB/CDDP1.0 and KB/BLM1.0; and TCM/CDDP1.0. In contrast, the susceptibility of the ATDR cells to LAK killing was not affected by anti-P-gp, anti-MHC class I, anti-ICAM-1, or anti-LFA-3 monoclonal antibodies.These results indicate that the effective population of LAK cells varies according to the tumor cell line, and are consistent with the assumption that the susceptibility of drug resistant cells to LAK cells depends on the property of the parent tumor cells and the type of antitumor drug that induces ATDR cells.

Immunosuppression by D-isomers of HLA class I heavy chain (amino acid 75 to 84)-derived peptides is independent of binding to HSC70.

Peptides derived from the class I heavy chain were shown to modulate immune responses in vitro and in vivo. A peptide derived from HLA-B2702 (2702.75-84) inhibited differentiation of cytotoxic T cells as well as T cell and natural killer cell-mediated cytotoxicity in vitro. Peptide-mediated immunomodulation seemed to be independent of the MHC proteins expressed by responder and stimulator cells. In vivo studies in rodents demonstrated prolongation of heart and skin allograft survival after peptide therapy. Here, the correlation between the peptide's biological activity and its amino acid sequence was analyzed using peptides derived from amino acid 75-84 of several mouse, rat, and human MHC class I proteins as well as peptides with single amino acid substitutions in the 2702.75-84 sequence. Peptides consisting of both L- and D-amino acids were tested for inhibition of murine and human T cell-mediated and lymphokine-activated killer cell-mediated cytotoxicity, binding to hsc70, and prolongation of heart allograft survival in vivo. Replacement of glutamic acid residue (E) at position 75 with valine (V) resulted in a peptide [2702.75-84(E>V)] with increased in vitro and in vivo activity but unchanged affinity for hsc70. Surprisingly, both L- and D-isomers of 2702.75-84 and 2702.75-84(E>V) inhibited cytotoxic cells in vitro and prolonged heart allograft survival in vivo. However, as expected, the peptides consisting of D-amino acids did not bind to hsc70. Assuming that both D- and L-isomers modulate immune responses by similar mechanisms, these results suggest that the peptides' effect is independent of binding to hsc70.

The Role of Phorbol Ester-Sensitive Protein Kinase C Isoforms in Lymphokine-Activated Killer Cell-Mediated Cytotoxicity: Dissociation between Perforin-Dependent and Fas-Dependent Cytotoxicity

Treatment of lymphokine-activated killer (LAK) cells with phorbol ester (PMA) caused the downmodulation of LAK activity concomitantly with the inhibition of serine esterase (SE) release, which has been shown as a marker for perforin-dependent cell-mediated cytotoxicity. The reduction of perforin-dependent LAK activity by PMA-treatment appeared to be due to the disappearance of PMA-sensitive protein kinase C (PKC) isoforms such as PKCα, γ, ϵ, θ. In contrast, Fas-mediated LAK activity was refractory against PMA-induced downregulation. Treatment of LAK cells with PMA caused a disappearance of cytotoxicity against Fas−L5178Y tumor cells, while cytotoxicity against Fas+transfectants was not affected by PMA treatment. Moreover, Fas-mediated LAK activity of perforin-knockout mice was not inhibited by PMA treatment. These results clearly demonstrated that Fas-mediated cytotoxicity could be dissociated from perforin-mediated cytotoxicity by their different requirement of PMA-sensitive PKC isoforms.

Suppressed cell-mediated immunity and monocyte and natural killer cell activity following allogeneic immunization of women with spontaneous recurrent abortion.

Spontaneous recurrent abortion (SRA) has been treated by means of immunization with paternal or third-party white blood cells, yet the immunological basis for SRA and for the role of immunization protocols in pregnancy outcome remains controversial. To elucidate this question, nine women with SRA were immunized with paternal mononuclear cells and studied before and 2 weeks after immunization. Seven women who became pregnant gave birth to live newborns. Secretion of the T helper 1 cytokines IL-2 and interferon-gamma by patients, mononuclear cells decreased, while production of IL-10 increased. The levels of natural killer and lymphokine-activated killer cell-mediated cytotoxicity were markedly decreased. Monocyte functions such as secretion of IL-1 alpha, tumor necrosis factor alpha, IL-6, and cytotoxic activity decreased concurrently with elevations in IL-10 and transforming growth factor beta secretion. Production of IL-12, a pivotal regulatory cytokine, decreased. Furthermore, B7/1 expression on patients' mononuclear cells was downregulated. This resulted in a decrease in monocyte costimulatory activity of purified T cells with soluble anti-CD3, paralleled by a decline in allogeneic proliferative responses. These results suggest that the improved pregnancy success rate in women with SRA following immunization may be partly related to suppression of cell-mediated immunity and monocyte and natural killer cell activity.

Predominance of TH1 response in tumor-bearing mice and cancer patients treated with AS101.

Several studies have recently suggested that the immune response to malignant growths is regulated by distinct patterns of type 2 cytokine production. These cytokines, regulating the cytotoxic T-lymphocyte response in patients with advanced cancers, may be associated with disease progression. Evidence suggests that the T Helper 1 (TH1) and T Helper 2 (TH2) types of reaction are reciprocally regulated in vivo. The immunomodulator AS101 (ammonium trichloro[dioxoethylene-O,O']tellurate) was found to stimulate mouse and human cells to proliferate and secrete a variety of cytokines. Clinical trials using AS101 on cancer patients are now in progress. The aim of this study was to evaluate the ability of AS101 to modulate TH1 and TH2 responses in tumor-bearing mice and in patients with advanced cancer. In addition, we investigated the association between the predominance of each type of response with the antitumoral effects of AS101. Mice into which Lewis lung carcinoma (3LL) had been transplanted (n = 221) and cancer patients (n = 13) were treated with AS101 on alternate days, at 10 micrograms/mouse intraperitoneally, or for the patients, at 3 mg/m2 intravenously. The types were sarcoma, melanoma, and colon, lung, ovarian, and renal cancers. Cytokine levels were determined by immunoassay kits and compared with the paired Student's t test: in mice, they were tested in spleen cell supernatants; in humans, in sera and mononuclear cell supernatants. The chi-squared test was used to compare tumor volumes. All P values represent two-sided tests of statistical significance. Our results show that treatment of mice and patients with AS101 results in a clear predominance in TH1 responses, with a concomitant decrease in the TH2-type response. This was reflected by a significant enhancement in interleukin 2 (IL-2) and interferon gamma (IFN gamma) levels (P < .01) paralleled by a substantial decrease in IL-4 and IL-10 (P < .01). Moreover, the concentration of IL-12 was significantly increased (P < .01) in AS101-treated patients who also showed enhanced levels of natural and lymphokine-activated killer cell-mediated cytotoxicity. The statistically significant increases in IL-2 and IFN gamma levels, paralleled by the pronounced decrease in IL-4 and IL-10 in the AS101-treated mice, were associated with its antitumoral effects. In addition, systemic cotreatment of 3LL-transplanted mice with AS101 and anti-IL-12 antibodies partly abrogated the antitumoral effect of AS101. Immunotherapy with AS101 enhances TH1 function while interfering with the TH2 response. This TH1 trend may be related to the antitumor effects of AS101. Isolation and characterization of a distinct cytokine pattern in patients with advanced cancer treated with AS101 may contribute to the development of intervention strategies using this compound.

Thrombin Modulation of Natural Killer Activity in Human Peripheral Lymphocytes

In addition to its pivotal role in the coagulation cascade, thrombin is mitogenic for fibroblasts and endothelial cells, and activates a number of inflammatory cells including monocytes and T-lymphocytes. To determine if other immune functions are modulated by thrombin and if this modulation is direct or indirect, we investigated whether highly purified human α-thrombin affects natural killer (NK) and lymphokine-activated killer (LAK) cell-mediated cytotoxicity. Thrombin enhanced NK cell-mediated cytotoxicity by more than 60% and enhanced IL-2 production and NK 3.3 cell responsiveness to IL-2. Unexpectedly, thrombin and the receptor activating “tethered ligand” domain of the thrombin receptor (TRP-7:SFLLRNP) inhibited LAK cell-mediated cytotoxicity by 50%. DIP–thrombin (a proteolytically inactive form of α-thrombin) had no inhibitory activity, suggesting that proteolytic activation of thrombin receptor is requisite for inhibition. These results indicate that cell-mediated cytotoxicity may be enhanced by thrombin through a mechanism involving stimulation of cytokine production and NK cell responsiveness, but that activation of thrombin receptor may also inhibit cytotoxic effects of LAK cells. The role of this dual regulation in processes of cell surveillance, wound healing, and inflammation remains to be determined.

Modulation of lymphokine-activated killer cell-mediated cytotoxicity by estradiol and tamoxifen.

The effect of tamoxifen (TX) and estradiol (E2) on interleukin-2 (IL-2 )-activated killer (LAK) cell-mediated cytotoxicity was examined using spleen cells of Fischer 344 rats as the source of effectors and P815 murine mastocytoma cells as targets. Treatment of target cells with either TX or E2 for 4 or 18 hr rendered them highly sensitive to LAK cell-mediated lysis. When TX and E2 were applied jointly, cytotoxicity remained at the level of TX alone. The cytotoxic potential of IL-2-primed LAK cells was not modified consistently by TX and E2. When TX-treated target and effector cells were combined, high cytotoxicity characteristic of sensitized target cells was observed. In similar experiments with E2-treated cells, both enhancement and inhibition of cytotoxicity by treated effector cells was seen in some designs. Target cells could be sensitized for LAK cell-mediated destruction by physiological concentrations (1 nM) of E2 and equimolar concentration of TX. Sensitization led to the accelerated release of the nuclear label 3H-thymidine from target cells after cytotoxic insult and could be prevented by treatment with the metabolic inhibitors cycloheximide and actinomycin D. Enhanced 3H-thymidine release from TX-treated targets was also demonstrated after induction of Ca2+ influx by exposure to the ionophore A23187. Neither E2 nor TX exerted a direct cytotoxic effect on P815 cells. P815 cells had no classical receptors for E2 or progesterone.

Apoptosis of tumor cells in lectin-dependent lymphokine-activated killer cell-mediated cytotoxicity

By using DNA electrophoresis and propidium iodide (PI) staining flow cytometry (FACS) analysis, we studied the mechanisms of lymphokine-activated killer (LAK) cell-mediated cytotoxicity. In the presence of pokeweed mitogen (PWM), human LAK cells induced DNA fragmentation of two leukemic cell lines (U937 cells and Raji cells) and two solid tumor cell lines (SW1116 cells and Hep-2 cells), a hallmark of apoptosis. The reactions were carried out at the effector/target ratio of 1:1 and in 4 hr coculture. Pretreatment with RNA and protein synthesis inhibitors (actinomycin D and cycloheximide) did not prevent the target cells from apoptosis. As the TNF-resistant tumor cell lines such as SW1116 cells and Raji cells were also triggered to apoptosis, other factors than TNF would play the role. DNA-PI staining FACS analysis also suggested that a part of LAK cells underwent apoptosis to some extent during incubation with target cells. The results provide a new way to investigate the mechanisms of cytotoxicity of LAK cells and to enhance the efficacy of adoptive tumor therapy with LAK cells.

Enhanced human lymphokine-activated killer cell function after brief exposure to granulocyte-macrophage-colony stimulating factor.

Lymphokine-activated killer (LAK) cell function can be generated in peripheral blood mononuclear cells (PBMC) after brief exposure of high dose interleukin-2 (IL-2) over the course of 1 or 2 days' culture in plain culture medium (IL-2-pulsed PBMC). The aim of the present study was to investigate the ability of granulocyte-macrophage-colony stimulating factor (GM-CSF) to augment LAK induction in low dose IL-2-pulsed PBMC derived from patients with cancer undergoing immunotherapy with IL-2. Peripheral blood mononuclear cells were collected from patients with cancer receiving a 5-day cycle of local (intraperitoneal or intrapleural) infusions with IL-2. The cells were incubated with IL-2 in the presence or absence of GM-CSF for 1 hour and then tested as effectors against allogeneic tumor cells and LAK-sensitive cell lines. Granulocyte-macrophage-colony stimulating factor at doses between 10 and 100 ng/ml was synergized with low dose IL-2 (100 IU/ml) in the generation of LAK activity in PBMC. Lymphokine-activated killer cell-mediated cytotoxicity derived from PBMC cultures incubated with IL-2 and GM-CSF was significantly higher (up to three-fold) compared with that generated with IL-2 alone. The GM-CSF-induced enhanced LAK activity was maintained when tested at day 5. GM-CSF increased the percentages of IL-2 receptor (R) positive (+) and CD8+ cells in the IL-2-pulsed PBMC. In contrast to CD56+ cells, highly purified CD8+ cells isolated from PBMC pulsed with IL-2 and GM-CSF responded with increased LAK activity, thus representing the cell-type that mediates the augmenting effect of GM-CSF. Major histocompatibility complex (MHC) molecules or the CD3 surface antigens were not involved in the GM-CSF-mediated enhancement of LAK induction because anti-MHC class I and class II monoclonal antibodies (MoAb) or MoAb against the CD3 molecules remained without any effect in this system. The GM-CSF-mediated LAK-enhancement was IL-2-dependent because MoAb against IL-2 receptor completely inhibited the generation of LAK activity. The use of GM-CSF for the enhancement of IL-2-induced LAK activity in 1 hour cultures may improve clinical results in cancer immunotherapy. In addition, implementation of this procedure could eliminate the high cost of cell culture which usually accompanies IL-2/LAK cell therapy as well as eliminate the known toxic side effects associated with this kind of therapy.

Interleukin-2 (IL-2) augments host cellular immune reactivity in the perioperative period in patients with malignant disease

Major surgery impairs cellular and humoral immunity, in particular natural cytotoxicity, and this may facilitate the dissemination of tumour cells in the peri-operative period. Recombinant interleukin-2 (rIL-2) has been used to modulate peri-operative immune function. Eighteen patients were randomized to receive either rIL-2 or placebo for three days, as a subcutaneous injection, prior to surgical resection for colorectal cancer. Natural cytotoxicity (natural killer (NK) and lymphokine-activated killer (LAK) cell activity), monocyte phagocytosis and immune cell surface activation marker (CD14+HLA-DR) expression were assessed during therapy and for up to 21 days after surgery. rIL-2 therapy enhanced both NK and LAK cell-mediated cytotoxicity and augmented circulating lymphocyte CD16+ and CD56+ cell subset populations. Circulating monocyte phagocytosis was also increased. Hence, rIL-2 may be used to enhance immune function in the peri-operative period in patients undergoing curative cancer surgery.

Discrepancy between direct and antibody-dependent cytotoxic activities of human LAK cells

Human lymphokine-activated killer (LAK) cells display cytotoxic activity against natural killer (NK)-resistant tumor cells in an antibody-independent and -dependent manner. We compared LAK cell-mediated antibody-independent cytotoxicity (LAK activity) and antibody-dependent cellular cytotoxicity (ADCC) against untreated and antibody-coated Raji cells, respectively. Human lymphocytes showed drastically increased LAK activity after stimulation with interleukin-2 (IL-2) for 3 or 7 days when compared to non-activated cells. The level of ADCC was reduced for 3-day-generated LAK cells and augmented for 7-day-generated LAK cells as compared to non-activated cultured lymphocytes. Phenotypical analysis revealed IL-2-induced up-regulation of the proportion of CD11b+ (but not CD16+) lymphocyte subpopulation in 7-day-generated LAK cells. The data imply that human LAK cells exhibit antibody-dependent and -independent cytotoxic activities via distinct effector pathways at different stages of generation. These stages may be associated with changes in adhesion molecule (CD11b/CD18) expression on the surface of IL-2-activated lymphocytes.

Ability of cell-sized beads bearing tumor cell membrane proteins to stimulate LAK cells to secrete interferon-γ and tumor necrosis factor- α1

We recently reported that lymphokine activated killer (LAK) cells were stimulated to release both interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) when stimulated by a variety of tumor cells. We proposed then that the released cytokines may play a role in mediating tumor cell regression in vivo. In this paper, we provide further information on the nature of the signals, provided by the tumor cells (K562 erythroleukemia), that stimulate LAK cells to secrete IFN-γ and TNF-α. Using a previously published protocol for coating tumor-membrane molecules onto cell-sized hydrophobic beads (also called pseudocytes), we demonstrate that the signal provided by the tumor cell is membrane associated. Beads coated with K562 membranes stimulated LAK cells to release IFN-γ and TNF-α. The pretreatment of these beads with trypsin and sodium periodate eliminated the ability of these pseudocytes to stimulate cytokine release in LAK cells. The glycoproteins that stimulate LAK cells to secrete IFN-γ and TNF-α were further enriched by their ability to bind concanavalin A (Con A, Jack Bean). To determine if the tumor-associated molecules that stimulate LAK cells to release IFN-γ and TNF-α are also the molecules involved in mediating tumor cell lysis, we tested the ability of the Con A binding and nonbinding proteins to inhibit the LAK cell-mediated lysis of K562 cells. Our results demonstrate that molecules that inhibited LAK cell-mediated cytotoxicity were not enriched by Con A. These results are therefore consistent with the conclusion that different sets of tumor-associated molecules are involved in the stimulation of LAK cells to secrete cytokine and in the induction of LAK cells to mediate tumor cell cytolysis.

Down-regulation of LAK cell-mediated cytotoxicity: cancer and ageing

A comparative study was made of the generation of lymphokine-activated killer (LAK) cells in patients with melanoma and healthy donors of different age groups. Significant reduction of effector il cytotoxicity in patients following 72_h culture with 1 000 U/ml or recombinant IL-2 (rIL-2) as well as a decreased ability to generate LAK cells in elderly individuals were shown to be correlated with suppressor cell activation in rIL-2 stimulated cell population. Suppressor effect depends on monocytes and T-lymphocytes: partial abolition of suppression in LAK cells was observed following removal of adherent cells or treatment with OKT8 monoclonal antibodies and complement.